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Controlling membrane protein folding with light illumination and catanionic surfactant systems

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Controlling membrane protein folding with light illumination and catanionic surfactant systems

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CONTROLLING MEMBRANE PROTEIN FOLDING WITH LIGHT
ILLUMINA TION AND CA TANIONIC SURFACTANT SYSTEMS

by

Jing Zhang

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMICAL ENGINEERING)

December 2008

Copyright 2008 Jing Zhang
Acknowledgements

I would like to express my deepest appreciation to those people who give me
guide, collaboration and support to finish the dissertation.
I would like to thank for my advisor Dr. Ted Lee first, who gives me opportunity
to work in his lab and the support for my project.
I would like to thank for my committee members, Dr Katherine Shing and Dr
Ralf Langen, who take time to read my thesis and give me great suggestions.
I would like to thank for my colleagues, Shao-chun Wang, Andrea Hamill,
Anne-Laure M. Le Ny, Yu-chuan Liu and Chia-hao Chang, who give me great help in
experiments and discuss the projects with me.
I would like to thank for Karen, Brendan and Tina, who give me help whenever I
need it.
The most importantly, I would like to thank for my parents, my sister and my
husband, who give me support anytime and anywhere. Without their love, support,
understanding and help, I would not finish the dissertation.

ii
Table of Contents

Acknowledgements ii

List of Tables vii

List of Figures viii

Abstract xii

Chapter 1: Introduction 1
1.1 Membrane proteins 1
1.2 Bacteriorhodopsin 1
1.3 Surfactant-lipid- interactions 7
1.4 Photoresponsive surfactants 8
1.5 Catanionic surfactant systems 11
1.6 Dynamic light scattering (DLS) 17
1.7 Small angle neutron scattering (SANS) 19
1.8 Overview 20
1.9 References 22

Chapter 2: Photoresponsive Surfactants 29
2.1 Abstract 29
2.2 Introduction 29
2.2.1 Surfactant classification 29
2.2.2 Micelles formation by surfactants 30
2.2.3 Photoresponsive surfactants 32
2.3 Materials and methods 33
2.3.1 Photoresponsive surfactant preparation 33
2.3.2 NMR measurements 34
2.3.3 Conductivity measurements (CMC determination) 34
2.3.4 Surface tension measurements (CMC determination) 35
2.3.5 Surfactant photoisomerization 35
2.4 Results and discussion 36
2.4.1 Cationic surfactant preparation 36
2.4.2 Anionic surfactant preparation 39
2.4.3 Non-ionic surfactant preparation 42

iii
2.4.4 Critical micelle concentration (CMC) determination 44
2.4.5 Surfactant photoisomerization 48
2.5 Conclusions 51
2.6 References 52

Chapter 3: Photoreversible Conformational Changes in Membrane Pro-
teins Using Light-responsive Surfactants 55
3.1 Abstract 55
3.2 Introduction 56
3.3 Experimental details 60
3.3.1 Materials 60
3.3.2 Purple membrane preparation 61
3.3.3 UV-vis determination of bR folding 61
3.3.4 FT-IR measurements 62
3.3.5 NMR measurements 64
3.3.6 Dynamic light scattering measurements 65
3.4 Results and Discussion 66
3.4.1 Phase behavior of purple membrane with azoTAB 66
3.4.2 Mixed-micelle formation in azoTAB-PM systems 69
3.4.3 Photoreversible control of bacteriorhodopsin folding inazoTAB
-PM assemblies 75
3.4.4 FT-IR measurements of bR secondary structure in azoTAB-
PM assemblies 82
3.5 Conclusions 89
3.6 Acknowledgement 90
3.7 References 91

Chapter 4: Light Effect on Self-Assembly of Aqueous Mixture of Sodi-
um Dodecyl benzenesulfonate (SDBS) and 4-ethyl-4’-(tri-
methylaminobutoxy) azobenzene Bromide (azoTAB) by
Using DLS and SANS 100
4.1 Abstract 100
4.2 Introduction 101
4.3 Materials and methods 106
4.3.1 Materials 106
4.3.2 Critical aggregation concentration (CAC) measurement 106
4.3.3 UV-vis spectroscopy 107
4.3.4 Dynamics detection of the formation and disruption of the vesi-
cle 107
4.3.5 Dynamics light scattering 108
iv
4.3.6 Small-angle neutron scattering (SANS) 109
4.3.7 SANS analysis 110
4.3.8 Polarized optical microscopy 112
4.4 Results and discussion 112
4.4.1 Critical aggregation concentration (CAC) measurement 112
4.4.2 Dynamics detection of the formation and disruption of the vesi-
cle 116
4.4.3 UV-Vis spectroscopy 119
4.4.4 Dynamics light scattering 124
4.4.5 Small-angle neutron scattering (SANS) 133
4.4.6 Polarized optical microscopy 141
4.5 Conclusions 144
4.6 Acknowledgements 145
4.7 References 146

Chapter 5: Future Work 152
5.1 Phase behavior of photoresponsive catanionic surfactant systems. 153
5.1.1 AzoTAB-4 and AzoTAB-7 with and without sodium bromide as
a function of temperature 153
5.1.2 AzoTAB-1 and SDS with and without sodium bromide as a
function of temperature 154
5.1.3 AzoTAB-1 and AzoTAB-7 with and without sodium bromide
as a function of temperature 156
5.1.4 AzoTAB-1 and AzoTAB-2 with and without sodium bromide
as a function of temperature 157
5.2 Membrane proteins solubilized in catanionic surfactant mixtures. 159
5.2.1 Purple membrane mixed with azoTAB-1/SDBS
catanionic surfactant mixtures (UV-vis measurement) 159
5.2.2 Preliminary results for purple membrane solubilized
with azoTAB-1/SDBS catanionic vesicles 160
5.2.3 Purple membrane solubilized with azoTAB-10/SDBS catanio-
nic surfactant mixtures (dynamic light scattering and UV-vis
measurements) 162
5.2.3.1 Dynamic light scattering 162
5.2.3.2 Preliminary dynamic light scattering results for
azoTAB-10/SDBS catanionic mixture 162
5.2.3.3 Preliminary results for purple membrane with
azoTAB-10/SDBS 164

v
5.3 Conclusions 167
5.4 References 169
Bibliography 170

vi
List of Tables

Table 2.1: Synthesized surfactants and CMC values. 48

Table 3.1: Dynamic light scattering (DLS) measurements of the scattering
count rate and hydrodynamic diameter for pure PM and
azoTAB-PM systems at [azoTAB] = 20 mM. 73

Table 4.1: DLS parameters. 132

Table 4.2: SANS data at all molar ratios and overall surfactant
concentrations, 0.1, 0.25, 0.5 wt%. 135

Table 5.1: The phase behavior of azoTAB-4/azoTAB-7 catanionic
surfactant mixtures. [total surfactant] = 0.1 wt %,
[sodium bromide] = 100 mM,
(H: hazy, P: precipitated, and C: clear) 154

Table 5.2: The phase behavior of azoTAB-1/SDS catanionic surfactant
mixtures. [total surfactant] = 0.1 %.
(H: hazy, P: precipitated, and C: clear) 155

Table 5.3: The phase behavior of azoTAB-1/azoTAB-7 catanionic
surfactant mixtures. [total surfactant] = 0.1 %.
(H: hazy, P: precipitated, and C: clear) 157

Table 5.4: The phase behavior of azoTAB-1/azoTAB-2 catanionic
surfactant mixtures. [total surfactant] = 0.1 %.
(H: hazy, P: precipitated and C: clear) 158

Table 5.5: Dynamic light scattering data for azoTAB-10/SDBS
catanionic mixtures. (V = vesicles, M = micelles) 163

vii
List of Figures

Figure 1.1: (a) The structure of bacteriorhodopsin (bR) within the lipid
bilayer. 3

Figure 1.2: The natural photocycle of bR with retinal photoisomerization. 5

Figure 1.3: The three-stage model of lipid-detergent interactions. 8

Figure 1.4: AzoTAB chemical structure and isomerization. 10

Figure 1.5: UV-vis absorbance spectra of azoTAB. 11

Figure 1.6: The ternary diagram of the SDS- DTAB-water system
determined from the analysis of SANS data. 12

Figure 1.7: The expected surfactant microstructure based on
geometrical reasoning, as calculated through the packing
parameters of surfactant molecules. 13

Figure 1.8: (a) DLS experiment; (b) autocorrelation decay function for
spherical monodisperse molecules. 18

Figure 1.9: SANS experiment. 19

Figure 2.1: Micelles structures in aqueous solution. (a) spherical micelles,
(b), reverse micelles, (c) elongated cylindrical micelles,
(d) flat lamellar micelles, and (e) vesicles. 32

Figure 2.2: NMR measurement for azoTAB 1. 39

Figure 2.3: NMR measurement for azoTAB 2. 41

Figure 2.4: Conductivity changes as a function of surfactant concentration
and light illumination. (a) azoTAB 4 and (b) azoTAB 2. 45

Figure 2.5: The surface tension of azoOG aqueous solutions as a function of
light illumination. 47

viii
Figure 2.6: UV-vis absorbance of (a) azoTAB 1; (b) azoTAB 2; (c) azoOG. 50

Figure 3.1: (a) Miscibility of purple membrane with azoTAB under visible
and UV light. Numbers represent the azoTAB
concentration (mM). Cloudy mixtures can be detected by the
lack of the reflections in the glass test tubes. (b) Optical
microscope images of mixtures of PM with azoTAB. 69

Figure 3.2: (a) Normalized correlation function of PM (0.18 mg/ml) and
azoTAB (20 mM) in 0.1M phosphate buffer as a function of
light illumination. The presence of azoTAB-PM mixed
micelles is evident from the fast relaxation at low values of q
2
τ.
(b)
31
P NMR measurements as a function of azoTAB
concentration and light illumination. 75

Figure 3.3: Relative folding percentage of bR (0.18 mg/ml) as a
function of azoTAB concentration determined from the
protein absorbance at 560 nm (see also Figure 4). The data point
placed at 0.01 mM actually represents pure PM, arbitrarily
assigned due to the log scale. Curves drawn to guide the eye. 76

Figure 3.4: Kinetics of light-induced conformational changes of bR
obtained from in situ absorbance measurements.
[bR] = 0.18 mg/mL, [azoTAB] = 0.10 mM. (a) Selected
individual UV-vis spectrum at various listed time intervals.
Inset: a magnification of the PM peak for the azoTAB-PM
samples (solid curves). (b) the absorbance at 560 nm as a
function of time. The circled data points correspond to the
displayed spectra, with the first spectrum set to a time of 0 sec
in part (a). 77

Figure 3.5: Fourier self-deconvolution IR spectra of PM as a function of
azoTAB concentration under both visible (solid lines) and UV
(dotted lines) illumination.
84

ix
Figure 3.6: (a) Difference spectra of PM in the amide I region as a
function of surfactant concentration, defined as spectra
collected under UV light minus spectra collected under visible
light. (b) Reproducibility in the difference spectra at 8.0 mM
azoTAB during successive visible → UV → visible → UV
light cycles. 88

Figure 4.1: AzoTAB chemical structure and isomerization. 103

Figure 4.2: Fluorescence emission spectra of Nile red in 60/40
(molar) azoTAB/SDBS mixtures under (a) visible and
(b) UV exposure. [Nile red] = 0.1 µM.
The concentration shown is azoTAB concentration, T = 25 ° C. 114

Figure 4.3: CAC of the azoTAB/SDBS catanionic mixtures at given ratios
under visible light and UV illumination. [Nile red]: 0.1 µm,
T = 25 ° C. 115

Figure 4.4: Dynamics of vesicle formation and disruption determination
with time-based fluorescence measurements at an azoTAB/SDBS
ratio of 60/40 and an azoTAB concentration of 30 µM and
nile red at 0.1 µM. 117

Figure 4.5: Illustration of the mechanisms responsible for the reversible
formation and disruption photoresponsive catanionic vesicles. 118

Figure 4.6: UV-Vis absorbance sptectra of azoTAB/SDBS (a) 30/70 and
70/30 in dark, visible illumination, UV illumination;
(b)(c)(d) azoTAB absorbance as a function of SDBS concentration
in dark, visible illumination and UV illumination separately. 123

Figure 4.7: Ternary diagrams showing the regimes of different regimes of
different microstructure found in mixtures of AzoTAB and
SDBS under both visible and UV light. V: Vesicles,
M: Micelles, L: Lamellar structure. 125

x
Figure 4.8: (a) Hydrodynamic diameters (R
H
) of azoTAB/SDBS
catanionic vesicles under visible and UV illumination as a
function of surfactant ratio at a total surfactant concentration
at 0.1 wt%. (b) as a function of the total surfactant concentration
at a given azoTAB/SDBS molar ratio of 30/70 T = 25 ° C. 128

Figure 4.9: SANS data of scattering intensity as a function of scattering
vector Q for samples with surfactant ratios (a) 60/40, 70/30, 80/20
and an overall surfactant concentration 0.1w%; (b) 93/7, 7/93,
60/40 and over all surfactant concentration 0.25 wt% and
0.5wt% as described in figure. 134

Figure 4.10: Igor fitting data with both visible and UV light illumination
for total surfactant concentration
(a) 0.1 wt% azoTAB/SDBS=60/40,
(b) 0.1 wt% azoTAB/SDBS=70/30,
(c) 0.1 wt% azoTAB/SDBS=80/20,
(d) 0.25 wt% azoTAB/SDBS=93/7,
(e) 0.5 wt% azoTAB/SDBS=93/7,
(f) 0.5 wt% azoTAB/SDBS=7/93. 137

Figure 4.11: Polarized optical microscopy images with visible light
illumination. Overall surfactant concentration: 1 wt%.
(Microruler: 5 µm). 143

Figure 5.1: Purple membrane with azoTAB-1/SDBS = 7/93 catanionic
surfactant mixture. (a) Visible light; (b) UV light.
[bR] = 0.17 mg/mL. The arrow show the effect of increasing
SDBS concentration ([SDBS] = 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1
and 2 mM). 160

Figure 5.2: Purple membrane with azoTAB-10/SDBS.
[Protein] = 0.14 mg/mL, [total surfactant] = 0.1 wt %,
cuvette path length = 1 cm. 165

Figure 5.3: Purple membrane with azoTAB-10/SDBS with light
illumination, (a) visible light; (b) UV light.
[protein] = 0.045 mg/mL, azoTAB-10/SDBS = 1/99. 165

xi
Abstract

Membrane proteins are of significant importance, performing a variety of
biological functions including pumps, channels, and receptors. Thus, membrane
proteins represent attractive candidates as drug targets. Bacteriorhodopsin (bR), the
most widely studied membrane protein, consists of seven transmembrane helical
segments and functions which can work as a proton pump in Halobacterium
Salinarium. In the present study, the reversible control of bR conformation with
simple light illumination is examined, providing a protocol to probe membrane
protein folding (a challenge even to this day due to the large, aggregation-prone
hydrophobic regions of membrane proteins compared to soluble proteins). Two
general methodologies are utilized to control membrane protein folding, including (1)
saturation of the natural lipids with a photoresponsive surfactant resulting in
partitioning of the protein into detergent-lipid mixed micelles in the unfolded state,
and (2) the development of artificial bilayers through self-assembly of the
photosurfactant into light-responsive vesicles to solubilize membrane proteins. The
azobenzene-based photosurfactant undergoes a reversible photoisomerization upon
illumination either visible (trans) or UV (cis) light. The trans isomer is relatively
hydrophobic and, thus, readily forms detergent-lipid mixed micelles relative to the cis
form, while the planar trans conformation also enhances the formation of artificial

xii
lamellar structures in vesicle bilayers relative to the bent cis form. Together, these
strategies provide a convenient means to control membrane protein folding with light
illumination.

xiii
Chapter 1: Introduction

1.1 Membrane proteins
Membrane proteins (MPs) constitute 20-30% of all the proteins in a given cell,
1; 2

yet comparatively little is known about the structure and dynamics of membrane
proteins relative to the soluble protein counterparts. This is a consequence of many
factors, including difficulties associated with crystallizing MPs for high-resolution
structural studies due to exposed hydrophobic domains, and naturally low
concentrations of MP in cells and, hence, low yields due to being located primarily
within the bilayers of cell membranes. This is especially unfortunate given that MPs
are responsible for many key biological functions, including serving as mediators
between cells and the surrounding environment,
3; 4
while further acting as molecular
pumps
4
and channel receptors
4; 5
allowing ions and metabolites to be transported into
and out of cells.

1.2 Bacteriorhodopsin
The membrane protein bacteriorhodopsin (bR) has been well-studied in the
literature, primarily a result of the high concentration within, and ease of purification
from, the purple membrane (PM) isolated from Halobacterium Salinarium (PM
contains 75 wt % bR and 25 wt % lipids, or equivalently only 10 lipid molecules per

1
protein).
6; 7
Due to this high protein concentration, PM fragments exhibit a
highly-ordered, paracrystalline lattice
8
that, when dispersed in aqueous solutions,
exists as stiff, planar sheets with an average diameter of ca. 0.5 µm.
6; 8

The high-resolution X-ray crystallographic structure of bR has been known for
over a decade.
9
BR contains 248 amino acids forming seven transmembrane helices
connected by short extramembranous loops,
10
as shown in the Figure 1.1. The
secondary structure of bR has been studied by FT-IR
11
and found to contain about
70% α-helix structure
12
corresponding to the transmembrane helices,
13
with the
remaining 20% β-strands and 10% unordered structures.
14
The folding of bR can be
understood in terms of Popot’s two-step folding model.
15
In the first step, independent
trans-bilayer helices form, principally in response to the hydrophobic effect and the
formation of main-chain hydrogen bonds in the non-aqueous environment. Then in
the second step, intramolecular interactions between these helices lead to the
formation of the helical bundle and the tertiary structure shown in Figure 1.1a. This
two-step folding process is subsequently followed by intermolecular helical
interactions and the formation of bR trimers in PM sheets, as shown in Figure 1.1b.
16

2
(a) (a) (b) (b)

Figure 1.1: (a) The structure of bacteriorhodopsin (bR) within the lipid bilayer
(taken from reference 4), and (b) the crystal structure of bR trimers in PM (taken from
reference 16.).

A retinal chromophore, responsible for the protein function as a proton pump, is
covalently linked within the helix bundle of each bR monomer via a protonated
Schiff-base linkage to Lys-216 on helix G, as shown in Figure 1.2.
17
This
chromophore undergoes a photoisomerization from the all-trans to the 13-cis
conformation following illumination with 560-nm visible light, representing the first
step in the proton-pump photocycle (bR → K state, not shown). A proton is then
transferred from the Schiff base to Asp-85 (L → M reaction) and subsequently
released into the extracellular region, leading to the formation of the N intermediate.
The Schiff base is then reprotonated from the cytoplasmic side of the membrane via
Asp-96 (O state, not shown), after which time the retinal is isomerization back into

3
the all-trans bR ground state. Thus, the net result of this series of bR → K → L → M
→ N → O → bR conformational changes is the transfer of a proton from the
cytoplasmic (inside) to the extracellular (outside) region of the cell.
Changes in bR conformation have been achieved through a variety of traditional
techniques, including unfolding by solubilization from the PM bilayer into sodium
dodecyl sulfate (SDS) micelles,
18; 19
chemical unfolding with urea and guanidine
hydrochloride,
19
and thermal unfolding with increases in temerpature.
20
The
secondary structure of the bR when solubilized in micelles of SDS
21
, Triton X-100, or
n-octyl- β-glucoside(OG)
22
has been studied with FT-IR. In general, solubilization in
SDS and other “harsh” ionic micelles leads to disruption of both the intra- and
inter-molecular helical interactions in bR (i.e., denaturation with loss of
chromophore), while solubilization in “mild” detergents such as the non-ionic Triton
and OG disrupts only the intermolecular helical interactions (i.e., trimers → folded
monomers with intact chromophores).

4

Figure 1.2: The natural photocycle of bR with retinal photoisomerization (take
from reference 17).

The nature of bR folding can, thus, be measured by examining the state of the
retinal chromophore, either bound within the helical bundle and exhibiting an
absorption peak at 560 nm in the folded protein,
19
or released free into the
surrounding solution and absorbing at 380 nm when the protein is transitioned into

5
the unfolding state. Hence, absorption measurements have been used to examine bR
refolding upon going from SDS micelles in the denatured, retinal-free state (i.e.,
bacterioopsin, bO) to non-ionic mixed micelles such as DMPC/CHAPSO,
19

DMPC/CHAPS,
18
and DMPC/DHPC.
23
(DMPC: 1,2-dimyristoyl-sn-glycero-
3-phosphocholine; CHAPSO: 3-[(3-cholamidopropyl) dimethylamino]-2-hydroxyl-1-
propane; CHAPS: 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate;
DHPC: dihexanoylphosphatidylcholine) Furthermore, the dynamics of
conformational changes upon the controlled rapid dilution of SDS with
DMPC/CHAPS micelles has also been examined.
18

All of these methods, while providing insight into the folding mechanisms of bR,
are limited by either irreversible unfolding or the requirement of dialysis to slowly
remove the denaturant to allow refolding. In contrast, in the present study a novel
method to initiate rapid, photoreversible changes membrane protein folding is
achieved through the use of photoresponsive surfactants similar to surfactants used by
our group to control the conformation of BSA,
24
lysozyme,
25
and DNA.
26
These
azobenzene-based surfactants undergo a reversible photoisomerization from the trans
(434-nm visible light) to the cis (350-nm UV light) forms. Since the planar trans
isomer is relatively hydrophobic compared to the bent cis configuration, micelles
more readily form with visible illumination. Thus, it could be envisioned bR folding
could be controlled in a manner similar to exchanging from SDS to CHAPS/DMPC

6
micelles, except in the case of azoTAB simple light illumination could be used to
transition from the “harsh” to the “mild” detergent.

1.3 Surfactant-lipid- interactions
Detergents are commonly used to both separate MPs from lipid components and
to serve as crystallization environments, thus, surfactant-lipid interactions have been
well studied.
27; 28; 29; 30
This interaction generally occurs in three stages with
increasing surfactant concentration, as shown in Figure 1.3: (I) free surfactant is
incorporated into the lipid phase in a non-cooperative manner (i.e., as individual
molecules),
31
gradually leading to saturation of the lipid bilayer, (II) further addition
of surfactant past this saturation point leads to the formation of detergent-lipid mixed
micelles that exist in equilibrium in surfactant-saturated lipid bilayers, and finally (III)
complete dissolution of the bilayers as the lipids are fully solubilized within the
mixed micelles. From the standpoint of MPs imbedded in the original lipid bilayers,
this three-stage model indicates that the MPs will begin to unfold upon partitioning in
the mixed micelles in Stage II, leading to complete unfolding of all of the MPs in
Stage III. This, combined with the ability to tune the hydrophobicity (and, hence,
micellization) of the azoTAB surfactant with light, will form the basis of photocontrol
of MPs.

7

Figure 1.3: The three-stage model of lipid-detergent interactions. Lipids are
denoted by red and surfactant molecules are denoted by green headgroups.

1.4 Photoresponsive surfactants
For photoresponsive surfactants, azobenzene is a common isomerizable unit in
surfactant design.
32
Azobenzene normally exists as the trans isomer, while the cis
form can not generally be made by chemical methods.
32
Instead, the cis form is
obtained through isomerization of the trans isomer. The trans-cis isomerization is
controlled by the exposure wavelength, thus, different light illumination induces
various yields of the cis isomer yield. For example, the azobenzene trans form can be
easily isomerized to the cis conformation by illumination with light wavelengths from
330 to 380 nm,
32; 33
with a yield of the cis isomer ranging from 70-91%.
34
Surfactants
containing this azobenzene group (e.g., “azoTAB” or azobenzene-substituted
phosphate amphiphiles) can be synthesized and applied to biological aspects.
25; 26; 34;
35; 36
For example, azobenzene-substituted phosphate amphiphiles such as phosphoric

8
acid mono-9-((4-phenylazo)-phenoxy)nonyl ester
34
can be synthesized in two steps,
first to synthesize the functionalized tails and then the tail is elongated via an ether
linkage.
34; 37
This kind of surfactant is used to design biological membrane mimics,
while the effects of the tran-cis isomerization on channel proteins has been studied.
34

The photoresponsive cationic surfactant 4-ethyl-4’(trimethylaminobutoxy)
azobenzene bromide (“azoTAB”) shown in Figure 1.4, an analog of the conventional
surfactant dodecyltrimethylammonium bromide (DTAB), can be synthesized
according to the published methods
38; 39
. Generally, azoTAB is prepared by
azocoupling of alkylaniline with phenol, followed by alkylation and quarternization
with dibromoalkane and trimethylamine. The final product purity can be determined
by
1
H-NMR in D
2
O.
The azoTAB surfactant exists as the trans isomer in solution in the dark (100%
trans) or under visible light exposure (~75% trans), while conversion to the cis form
can be achieved upon illumination with UV light (~95% cis). For a typical
isomerization cycle of azoTAB, the absorption spectrum contain three transitions, 248
nm (a π- π* transition), 350 nm (a π- π* transition) and 440 nm (a weak n- π*
transition).
39; 40
Thus, using light at 365 nm from a mercury arc lamp, azoTAB is
isomerized from trans isomer to the cis isomer leading to an increase in absorbance at
440 nm and a decrease at 350 nm. The photoisomeric state can be determined with

9
UV-Vis spectroscopy as shown in Figure 1.5, with the trans isomer exhibiting a
maximum absorbance at 350 nm and the cis isomer at 434 nm.
The dipole moment across the nitrogen double bond is ~0.5D for the trans isomer
compared to ~3.1D for the cis form.
41
As a result, the trans isomer is significantly
more hydrophobic than the cis form, allowing photo-induced changes in a variety of
surfactant properties such as the critical micelle concentration (CMC),
38
surface
tension,
42
and electrical conductivity.
38
Furthermore, this property of azoTAB has also
been used by our group to allow photo-reversible binding of the surfactant to various
biomacromolecules, leading to photoreversible control BSA folding,
24
the enzymatic
activity of the lysozyme,
25
solution structure of an amyloid-forming protein
35
and
DNA condensation.
26

N
+
(CH
3
)
3
Br
N
N O(CH
2
)
4
CH
2
CH
3

N
N
CH
3
CH
2
O(CH
2
)
4
N
+
(CH
3
)
3
Br

350 nm
434 nm
trans form cis form

Figure 1.4: AzoTAB chemical structure and isomerization.

10

0
0.5
1
1.5
2
200 250 300 350 400 450 500 550
trans
cis
Absorbance
Wavelength (nm)
[S] = 0.5 mM
L = 0.2 cm

Figure 1.5: UV-vis absorbance spectra of azoTAB ([azoTAB] = 0.5 mM, path
length = 2 mm).

1.5 Catanionic surfactant systems
So-called “catanionic” surfactant systems, consisting of aqueous mixtures of
cationic and anionic surfactants, potentially provide a novel medium to control MP
folding and gene delivery. As a result of strong electrostatic and hydrophobic
interactions between the oppositely-charged surfactants, a variety of self-assembled
architectures can develop,
43
often at surfactant concentrations orders of magnitude
lower that the pure-component critical micelle concentrations. A range of factors,
including surfactant properties, the cation-anion ratio, temperature, and other
environmental factors determine the formation of ordered structures, with
morphologies determined commonly by techniques such as dynamic light scattering
(DLS)
43
and small-angle neutron scattering (SANS).
44; 45
an example of this behavior

11
is shown in the ternary phase diagram of the SDS- DTAB-water system in Figure 1.6,
where small uni- or oligo-lamellar vesicles (V), rod-shaped micelles (M
R
),
disk-shaped micelles (M
D
), and stacks of lamellar sheets (L) are observed.
44

Figure 1.6: The ternary diagram of the SDS- DTAB-water system determined
from the analysis of SANS data. (taken from reference 44)

The expected type of microstructure that formes in a given catanionic system can
be estimated from the resulting critical packing parameter (p), which is related to the
head groups area (a
H
) and the extended length (l
t
) and the volume (v) of the
hydrophobic part of a surfactant molecules through the equation p = ν/(a
H
l
t
). For
packing parameters p < 1/3 spherical micelles are expected, for 1/3 < p < 1/2
cylindrical or hexagonal structure should form, and for p ~ 1 lamellar structures are
predicted, as shown in Figure 1.7.
42
In comparison, for packing parameters larger than
1 reversed micelles or reversed hexagonal structures are generally favored,

12

Figure 1.7: The expected surfactant microstructure based on geometrical
reasoning, as calculated through the packing parameters of surfactant
molecules.(taken from reference 42).

13
In last ten years, novel catanionic surfactant mixtures containing photoresponsive
surfactants have been investigated, including those containing
4-Butylazobenzene-4’-(oxyethyl)trimethylammonium bromide (AZTMA),
46

bis(trimethylammoniumhexyloxy)azobenzene dibromide (BTHA),
47; 48; 49

2-phenylbenzimidazole-5-sulfonic acid (phBSA),
50
4-hexylphenylazosulfonate
(C6PAS),
51
and a stilbene-containing gemini photosurfactant (E-SGP).
52
For example,
in a previous study of AZTMA/SDBS catanionic mixtures,
46
UV-vis spectra and
transmission electron microscopic by the freeze replica technique were utilized to
investigate the microstructures in aqueous solutions as a function of light illumination.
The results indicated that vesicles were formed under visible light, while a lamellar
structure resulted upon UV illumination because of the decrease of the length of
hydrophobic group when AZTMA/SDBS = 6/4 and total surfactant concentration is
0.05 wt %. Furthermore, the vesicle ↔ lamellar transition was shown to be
photo-reversible. For BHTA/SDS catanionic mixtures, where BHTA is a cationic
surfactant containing two identical photoresponsive azogroup,
47; 48; 49
UV-vis spectra,
quasi-elastic light scattering, small angle neutron scattering, and cyro-TEM
experiments were performed as a function of light illumination. Under certain
conditions, vesicles were found to form, while a vesicle ↔ micelle transition was
observed at a BTHA/SDS ratio of 15/85 and a total surfactant concentration of 0.3 wt
%.

14
Systems containing photo-responsive anionic surfactants have also been
examined, such as 2-phenylbenzimidazole-5-sulfonic acid (phBSA)
50
and
4-hexylphenylazosulfonate (C6PAS).
51
In CTAB/phBSA catanionic mixtures,
time-dependent phase behavior was observed in aqueous solutions.
50
With excess
phBSA, multilamellar vesicles were observed by cryo-TEM and SAXS that were
stable for a couple of days before transitioning into clear hydrogeles. In CTAB-rich
samples, however, threadlike and ribbonlike micelles were observed and found to be
unchanged over time. In this study, light illumination effects were not examined.
Conversely, the CTAB/C
6
PAS catanionic system was termed a “photodestructible
system” in which an irreversible change in the microstructure could be initiated with
light illumination because C
6
PAS reacts with UV light in an irreversible way.
51; 53; 54

UV Illumination of the photoresponsive surfactant C
6
PAS was found to break down
vesicles that were spontaneously formed under visible light, as determined with TEM,
SANS and DLS experiments.
51

Another photoresponsive catanionic mixture studied by Eastoe et al. utilizes a
photoresponsive stilbene group as opposed to the azobenzene group above.
52
Through
SANS and
1
H-NMR studies, different microstructures were observed as a function of
mixture composition and UV exposure, while a vesicles ↔ micelle transition was
observed with light illumination.

15
The unique property of catanionic surfactant mixtures forming a variety of
different microstructures depending on the sample conditions and environment can be
used to study biological systems such as the control of membrane protein structure
and to improve gene delivery. From the point of view of membrane protein
solubilization, catanionic vesicles specifically represent attractive microstructures as
they can be thought of as mimics for the bilayer cellular membrane. The formation of
“traditional” (i.e., non-catanionic) vesicles typically requires the input of energy into
the system due to an increase in interfacial area (A) and, thus, free energy (G) of the
system since dG ~ σdA, where σ is the surface tension. However, in catanionic
systems the aforementioned strong electrostatic and hydrophobic attractions between
the surfactants can be sufficient to offset this energy penalty, resulting in so-called
“spontaneous” or “equilibrium” vesicles at the free energy minimum.
55
For catanionic
surfactant mixtures containing azobenzen-based surfactant, such as azoTAB, through
the use of azoTAB as the catanionic species, novel photoresponsive catanionic
vesicles can be envisioned, where vesicle rupture and re-formation could be
controlled with simple light illumination. Thus, membrane proteins could be
maintained in the folding state in vesicle bilayers and subsequently unfolded through
disruption of the vesicles with light illumination. Furthermore, to improve gene
delivery, vesicles can be formed spontaneously and at relatively low concentrations,

16
thus, avoiding issues of cytotoxicity, while the photo-induced disruption of the
vesicles could be used to increase the rate of DNA delivery into cells.
56

1.6 Dynamic light scattering (DLS)
Dynamic light scattering is a non-destructive, non-intrusive technique used to
measure the size of particles in solution. The method has been applied to polymers,
57

polyelectrolytes,
58; 59
proteins
60
and DNA molecules.
26
In dynamic light scattering, the
light scattering intensity autocorrelation function is described as
61

2
() (0) ( ) Gt I I t =< >, (1)
which can be normalized as

2
22
() () gt G t I = < >. (2)
If in a scattered field the fluctuations have a Gaussian distribution then the
second-order correlation is related to first-order correlation function described as

2
2
() 1 | |( ) gt g t
1
β = +, (3)
where β is a function of the delay time t.
In different microsystems, g
1
(t) has different expression. For example, for
spherical, monodisperse particles, the autocorrelation function is described as a decay
function,

2
1
( ) exp( ) gt qDt = −, (4)

17
where D is the experimental diffusion coefficient and q is the momentum vector
presented as
(4 / )sin( / 2) qn π λ θ = , (5)
where n is the refractive index, λ is the light wavelength and θ is the scattering angle.
For spherical particles in solution, hydrodynamic radius (R
H
) can be calculated from
the Stokes-Einstein equation
/6 HB R kT D π η =, (6)
where k
B
is Boltzmann’s constant, T is the temperature, η is the solvent viscosity, and
D is the experimentally-determined diffusion coefficient. Figure 1.8 shows a dynamic
light scattering experimental setup and the autocorrelation decay function for
spherical monodisperse molecules.

d
θ
detector
632.8nm HeNe Laser
d
θ
detector
632.8nm HeNe Laser
τ
τ
2
2
) (
Dq
e G
−
∝
τ
τ
2
2
) (
Dq
e G
−
∝

Figure 1.8: (a) DLS experiment; (b) autocorrelation decay function for spherical
monodisperse molecules.

18
1.7 Small angle neutron scattering (SANS)
Small angle neutron scattering (SANS) is a technique capable of providing
valuable information on a variety of scientific and technological applications, such as
protein structures,
25; 35; 36
surfactant self-assembly,
44; 62
and polymers and biological
structures.
63
SANS can examine structures from 1 nm to 1 µm.
64
The graph in Figure

1.9 shows a SANS experimental setup. An incident beam is directed into the sample
and a beam scattered with a detector is received.

Figure 1.9: SANS experiment (from NIST:
http://www.ncnr.nist.gov/programs/sans/)

For SANS, the neutrons react with nuclei in the sample and the interactions
depend on the actual isotope. A scattering cross section is described as

1
(/) Ai i
i
N
dd
Nd A
dd
σ Σ
=
Ω Ω
∑
, (7)

19

where N is the number of the atoms i, A
i
is Avogadro’s number, d
i
is the density and
d σ/d Ω
I
is atomic cross section. Because of the distinctive scattering of atoms,
65
it is
possible to define a scattering-length density ρ
j
(j is the distinct phase in solution) as
the scattering length per unit volume expressed as
/ jAjj Ndb Aj ρ =, (8)
where, b
j
is the scattering length. Substituting Eq. (8) and Eq.(7) into a general
expression for atomic cross section, the following equation is given

2
(1/ ) | ( )exp( ) |
V
d
V i
d
ρ
Σ
=
Ω
∫
rQ.rrd. (9)
In different solutions, scattering function is dependent on the properties of the phases
in solution.

1.8 Overview
In the following sections, the photoresponsive surfactant azoTAB will be
discussed and two novel strategies to control membrane protein folding with light
illumination will be outlined. In Chapter 2, azoTAB information including synthesis,
physical properties and applications will be discussed. In Chapter 3, photo-control of
the conformation of the membrane protein bacteriorhodopsin in natural lipids will be
described. A variety of techniques such as UV-vis spectroscopy, optical microscopy,

20
FT-IR spectroscopy, NMR spectroscopy, and DLS measurements will be utilized to
fully examine the membrane protein conformation. The results will show that the
structure of bacteriorhodopsin can be rapidly and reversibly controlled with light
illumination. In Chapter 4, the development of light-responsive catanionic vesicles as
artificial bilayers for membrane protein solubilization will be introduced. DLS,
UV-vis spectroscopy, fluorescence spectroscopy, electron microscopy and SANS will
be used to study the physical properties of the photoresponsive microstructures.
Furthermore, the dynamics of photo-initiated vesicle disruption and re-formation will
be examined. Finally, in Chapter 5, a proposal to extend these two strategies towards
the photo-control of a wide variety of membrane proteins will be offered.

21
1.9 References
1. Valentin I. Gordeliy, R. S., Rouslan Efremov, Georg Buldt, and Joachim
Heberle. (1988). Crystallization in Lipidic Cubic Phases. Methods in
Enzymology 228, 305-316.

2. Kleinschmidt, J. H. (2003). Membrane Proteins-Introduction. CMLS 60,
1527-1528.

3. Findlay, H. E. & Booth, P. J. (2006). The biological significance of
lipid-protein interactions. J. Phys.: Condens. Matter 18, 1281-1291.

4. Berg, J. M., Tymoczko, J. L. & Stryer, L. (2002). Biochemistry (Edition, F.,
Ed.), W. H. Freeman and Company.

5. Oesterhelt, D. & Stoeckenius, W. (1973). Functions of a New Photoreceptor
Membrane. Proc. Nat. Acad. Sci. U.S.A. 70, 2853-2857.

6. Oesterhelt, D. & Stoeckenius, W. (1974). Isolation of Cell Membrane of
Halobacterium Halobium and Its Fractionation into Red and Purple
Membrane. Methods Enzymol. 31, 667-678.

7. Overath, M. H. a. P. (1976). Bacteriorhodopsin depleted of purple membrane
lipids. Biochemical and Biophysical Research Communication 72, 1504-1511.

8. Hwang, S.-B. & Stoeckenius, W. (1977). Purple Membrane Vesicles:
Morphology and Protein Translocation. J. Membr. Biol. 33, 325-350.

9. Henderson, R., Baldwin, J. M., Ceska, T. A., Zemlin, F., Beckmann, E. &
Downing, K. H. (1990). Model for the structure of bacteriorhodopsin based on
high-resolution electron cryo-microscopy. J. Mol. Biol. 213, 899-929.

10. Booth, P. J., Farooq, A. & Flitsch, S. L. (1996). Retinal Binding during
Folding and Assembly of the Membrane Protein Bacteriothodopsin.
Biochemistry 35, 5902-5909.

11. Cladera, J., Galisteo, M. L., Sabes, M., Mateo, P. L. & Padros, E. (1992). The
role of retinal in the thermal stability of the purple membrane. European
Journal of Biochemistry 207, 581-585.

22
12. Krimm, S. & Dwivedi, A. M. (1982). Infrared Spectrum of the Purple
Membrane: Clue to a Proton Conduction Mechanism? Science, New Series
216, 407-408.

13. Kuo-Sen Huang, H. H., and H. Gobind Khorana. (1980). Delipidation of
bacteriorhodopsin and reconstitution with exogenous phospholipid.
Biochemistry 77, 323-327.

14. Cladera, J., Torres, J. & Padros, E. (1996). Analysis of Conformatioal Changes
in Bacteriorhodopsin Upon Retinal Removal. Biophys. J . 70, 2882-2887.

15. Popot, J.-L. & Engelman, D. M. (1990). Membrane Protein Folding and
Oligomerization: The Two-Stage Model. Biochemistry 29, 4031-4037.

16. Mitsuoka, K., Hirai, T., Murata, K., Miyazawa, A., Kidera, A., Kimura, Y. &
Fujiyoshi, Y. (1999). The Structure of Bacteriorhodopsin at 3.0 A Resolution
Based on Electron Crystallography: Implication of the Charge Distribution. J.
Mol. Biol. 1999, 861-882.

17. Kuhlbrandt, W. (2000). Bacteriorhodopsin-the movie. Nature 406, 569-570.

18. Booth, P. J., Flitsch, S. L., Stern, L. J., Greenhalgh, D. A., Kim, P. S. &
Khorana, H. G. (1995). Intermediates in the folding of the membrane protein
bacteriothodopsin. Structural biology 2, 139-143.

19. Gouaux, G. Q. C. a. E. (1999). Probing the Folding and Unfolding of
wild-Type and Mutant Forms of Bacteriorhodopsin in Micellar Solutions:
Evaluation of Reversible Unfolding Conditions. Biochemistry 38,
15380-15387.

20. Kahn, T. W., Sturtevant, J. M. & Engelman, D. M. (1992). Thermodynamic
measurements of the contributions of helix-connecting loops and of retinal to
the stability of bacteriorhodopsin. Biochemistry 31, 8829-8839.

21. Torres, J., Sepulcre, F. & Padros, E. (1995). Conformational Changes in
Bacteriorhodopsin Associated with Protein-Protein Interactions: A Functional
alfa
I
-alfa
II
Helix Switch. Biochemistry 34, 16320-16326.

23
22. Sonoyama, M., Hasegawa, T., Nakano, T. & Mitaku, S. (2004). Isomerization
of retinal chromophore and conformational changes in membrane protein
bacteriorhodopsin by solubilization with a mild non-ionic detergent,
n-octyl-b-glucoside: an FT-Raman and FT-IR spectroscopic study. Vib.
Spectrosc 35, 115-119.

23. Booth, P. J., Riley, M. L., Flitsch, S. L., Templer, R. H., Farooq, A., Curran, A.
R., Chadborn, N. & Wright, P. (1997). Evidence That Bilayer Bending
Rigidity Affects Membrane Protein Folding. Biochemistry 36, 197-203.

24. Wang, S.-c. & Lee, C. T., Jr. (2006). Protein Secondary Structure Controlled
with Light and Photoresponsive Surfactants. J. Phys. Chem. B 110,
16117-16123.

25. Hamill, A. C., Wang, S.-C. & Lee, C. T. J. (2005). Probing Lysozyme
Conformation with Light Reveals a New Folding Intermediate. Biochemisty
44, 15139-15149.

26. Le Ny, A.-L. M. & Lee, C. T. J. (2006). Photoreversible DNA Condensation
Using Light-Responsive Surfactants. J. Am. Chem. Soc. 128, 6400-6408.

27. Helenius, A. & Simons, K. (1975). Solubiliztion of Membranes by Detergents.
Biochim. Biophys. Acta 415, 29-79.

28. Lichtenberg, D. (1985). Characterization of the Solubilization of Lipid
Bilayers by Surfactants. Biochim. Biophys. Acta 821, 470-478.

29. Lichtenberg, D, Robson, R. & Dennis, E. A. (1983). Solubilization of
Phospholipids by Detergents Structural and Kinetic Aspects. Biochimica et
Biophysica Acta 737, 285-304.

30. Maire, M. l., Champeil, P. & Moller, J. V. (2000). Interaction of membrane
proteins and lipids with solubilization detergents. Biochim. Biophys. Acta
1508, 86-111.

31. Ulrich, K.-H., Maire, M. l., Noel, J.-P., Tadeusz, G.-K. & Moller, J. V. (1993).
Transitional Steps in the Solubilization of Protein-Containing Membranes and
Liposomes by Nonionic Detergent. Biochemistry 32, 1648-1656.

24
32. Griffiths, J. (1972). II. Photochemistry of Azobenzene and its Derivatives.
Chem. Soc. Rev. 1, 481-493.

33. Whitten, D. G. (1993). Photochemistry and Photophysics of trans-Stilbene and
Related Alkenes in Surfactant Assemblies. Acc. Chem. Res 26, 502-509.

34. Kuiper, M. (2005). Azobenzene-Substitrted Phosphate Amphiphies. Effect of
Light-induced Trans-cis Isomerization on Vesicular Properties and the
Channel Protein MscL.

35. Hamill, A. C., Wang, S.-c. & Lee, C. T. J. (2007). Solution Structure of an
Amyloid-Forming Protein During Photoinitiated Hexamer-Dodecamer
Transitions Revealed Through Small-Angle Neutron Scattering. Biochemistry
46, 7496-7705.

36. Lee, C. T. J., Smith, K. A. & Hatton, T. A. (2005). Photocontrol of Protein
Folding: The Interaction of Photosensitive Surfactants with Bovine Serum
Albumin. Biochemistry 44, 524-536.

37. Romsted, L. S. & Yoon, C.-O. (1993). Counterion Affinity Orders in Aqueous
Micellar Solutions of Sodim Decyl Phosphate and Sodium Dodecyl Sulfate
Determined by Changes in 23Na NMR Relation Rates: A Surprising
Dependence on Head Group Change. J. Am. Chem. Soc. 115, 989-994.

38. Hayashita, T., Kurosawa, T., Miyata, T., Tanaka, K. & Igawa, M. (1994).
Effect of Structural Variation within Cationic Azo-surfactant Upon
Photoresponsive Function in Aqueous Solution. Colloid. Polym. Sci. 272,
1611-1619.

39. Lee, C. T. J., Smith, K. A. & Hatton, T. A. (2004). Photoreversible Viscosity
Changes and Gelation in Mixtures of Hydrophobically Modified
Polyelectrolytes and Photosensitive Surfactants. Macromolecules 37,
5397-5405.

40. Kuiper, H. M. & Engberts, J. B. F. N. (2004). H-Aggregation of
Azobenzene-Substituted Amphiphiles in Vesicular Membranes. Langmuir 20,
1152-1160.

25
41. Shang, T., Smith, K. A. & Hatton, T. A. (2003). Photoresponsive Surfactants
Exhibiting Unusually Large, Reversible Surface Tension Changes under
Varying Illumination Conditions. Langmuir 19, 10764-10733.

42. Holmberg, K., Jonsson, B., Kronberg, B. & Lindman, B. (2002). Surfactants
and Polymers in Aqueous Solution (edition, n., Ed.), Wiley.

43. Yin, H., Zhou, Z., Huang, J., Zheng, R. & Zhang, Y. (2003).
Temperature-Induced Micelle to Vesicle Transition in the Sodium
Dodecylsulfate/Dodecyltriethylammonium Bromide System. Angew. Chem.
Int. Ed 42, 2188-2191.

44. Bergstrom, M. & Pedersen, J. S. (1998). Small-Angle Neutron Scattering
(SANS) Study of Aggregates Formed from Aqueous Mixtures of Sodium
Dodecyl Sulfate (SDS) and Dodecyltrimethylammonium Bromide (DTAB).
Langmuir 14, 3754-3761.

45. Bergstrom, M. & Pedersen, J. S. (1999). A Small-Angle Neutron Scattering
(SANS) Study of Talet-Shaped and Ribbonlike Micelles Formed From
Mixtures of an Anionic and a Catiionic Surfactant. J. Phys. Chem. B 103,
8502-8513.

46. Sakai, H., Matsumura, A., Yukoyama, S., Saji, T. & Abe, M. (1999).
Photochemical Swiching of Vesicle Formation Using an
Azobenzene-Modified Surfactant. J. Phys. Chem. B 103, 10737-10740.

47. Hubbard, F. P. J. & Abbott, N. L. (2007). Effect of Light on Self-Assembly of
Aqueous Mixtures of Sodium Dodecyl Sulfate and a Cationic , Bolaform
Surfactant Containing Azobenze. Langmuir 23, 4819-4829.

48. Hubbard, F. P. J., Santonicola, G., Kaler, E. W. & Abbott, N. L. (2005).
Small-Angle Neutron Scattering for Mixtures of Sodium Dodecyl Sulphate
and a Cationic, Bolaform Surfactant Containing Azobenzene. Langmuir 21,
6131-6136.

49. Bonini, M., Berti, D., Meglio, J. M. D., Almgren, M., Teixeira, J. & Baglioni,
P. (2005). Surfactant Aggregates Hosting a Photoresponsive Amphiphile:
Structure and Photoinduced Conformational Changes. Soft Matter 1, 444-454.

26
50. Grabner, D., Zhai, L., Talmon, Y., Schimid, J., Freiberger, N., Glatter, O.,
Herzog, B. & Hoffmann, H. (2008). Phase Behavior of Aqueous Mixtures of
2-Phenylbenzimidazole-5-sulfonic Acid and Cetyltrimethylammonium
Bromide: Hydrogels, Vesicles, Tubules, and Ribbons. J. Phys. Chem. B 112,
2901-2908.

51. Eastoe, J., Vesperinas, A., Donnewirth, A.-C., Wyatt, P., Grillo, I., Heenan, R.
K. & Davis, S. (2006). Photoinduced Vesicles. American Chemical Society 22,
851-853.

52. Eastoe, J., Dominguez, M. S., Wyatt, P. & Orr-Ewing, A., J. (2004). UV
Causes Dramatic Changes in Aggregation with Mixtures of Photoactives and
Inert Surfactants. Langmuir 20, 6120-6126.

53. Eastoe, J., Dominguez, M. S., Cumber, H., Burnett, G. & Wyatt, P. (2003).
Photoresponsive Microemulsions. Langmuir 19, 6579-6581.

54. Eastoe, J., Dominguez, M. S., Cumber, H. & Wyatt, P. (2004). Light-Sensitive
Microemulsion. Langmuir 20, 1120-1125.

55. Brasher, L. L., Herrington, K. L. & Kaler, E. W. (1995). Electrostatic Effects
on the Phase Behavior of Aqueous Cetyltrimethylammonium Bromide and
Sodium Octyl Sulfate Mixtures with Added Sodium Bromide. Langmuir 11,
4267-4277.

56. Liu, Y ., Le Ny, A.-L. M., Schmidt, J., Talmon, Y., Chmelka, B. F. & Lee, C. T.
J. (2008). Photo-Assisted Gene Delivery Using Light -Responsive Catanionic
Vesicles. J. Am. Chem. Soc. summited.

57. Delong, L. M. & Russo, P. S. (1991). Thermodynamics and Dynamic
Behavior of Semiflexible Polymers in the Iostropic Phase. Macromolecules 24,
6139-6155.

58. Liu, H., Skibinska, L., Gapinski, J., Patkowski, A., Fisher, E. W. & Pecora, R.
(1998). Effect of Electrostatic Interactions on the Structure and Dynamics of a
Model Polyelectrolyte. I. Diffusion. J. Chem. Phys. 113, 6001-6010.

27
59. Skibinska, L., Gapinski, J., Liu, H., Patkowski, A., Fisher, E. W. & Pecora, R.
(1999). Effect of Electrostatic Interactions on the Structure and Dynamics of a
Model Polylectrolyte. II. Intermolecular Correlations. J. Chem. Phys. 110,
1794-1800.

60. Hamill, A. C., Wang, S.-c. & Lee, C. T. J. (2005). Probing Lysozyme
Conformation with Light Reveals a New Folding Intermediate. Biochemistry
44, 15139-15149.

61. Claire, K. & Pecora, R. (1997). Translational and Rotational Dynamics of
Collagen in Dilure Solution. J. Phys. Chem. B 1997, 746-753.

62. Riley, M. L., Wallace, B. A., Flitsch, S. L. & Booth, P. J. (1997). Slow Alpha
Helix Formation during Folding of a Membrane Protein. Biochemistry 36,
192-196.

63. Jacrot, B. (1976). The Study of Biological Structures by Neutron Scattering
from Solution. Rep. Prog. Phys. 39, 911-953.

64. Windson, C. G. (1988). An Introduction to Small-Angle Neutron Scattering. J.
Appl. Cryst. 21, 582-588.

65. Brasher, L. L. & Kaler, E. W. (1996). A Small-Angle Neutron Scattering
(SANS) Contrast Variation Investigation of Aggregate Composition in
Catanionic Surfactant Mixtures. Langmuir 12, 6270-6276.

28
Chapter 2: Photoresponsive Surfactants

2.1 Abstract
Surfactants containing azobenzene groups have been synthesized by
azocoupling, alkylation and quaternalization steps for cationic surfactants,
azocoupling and alkylation for anionic surfactants and acylation, azocoupling and
hydrosisi reactions for non-ionic surfactants. The critical micelle concentration
(CMC) of each surfactant has been determined by electrical conductivity (charged
surfactant) and surface tension (nonionic surfactant) measurements. Because of the
different hydrophobicity of surfactant isomers, the cis isomer has a higher CMC
values compared to trans isomer, while surfactants containing longer alkyl tails
have lower CMC values. The photoisomerization of each surfactant is further
measured and studied by UV-vis spectroscopy.

2.2 Introduction
2.2.1 Surfactant classification
The term “surfactant” is an abbreviation for a surface active agent, which means
that the surfactant molecule is active at a surface.
1
Surfactants contain head groups,
which are four types, positively, negatively, uncharged or both positively changed and

29
negatively charged under normal conditions. Based on the head groups, they are
classified into four categories, cationic, anionic, non-ionic and zwitterionic
respectively.
1
Carboxylate, sulfate, sulfonate and phosphate groups are the most
popular head groups found in ionic surfactants, while amine- and quaternary
ammonium-base head groups are generally found in cationic surfactants with the
nitrogen atoms carrying the positive charge. For a typical non-ionic surfactant, both
polyether and polyhydroxyl units are commonly used head groups. Head groups can
be a variety of types in zwitterionic surfactants, but the most popular components are
ammonium and carboxylate.
1

2.2.2 Micelles formation by surfactants
Surfactants are amphiphilic, containing a hydrophilic head group and a
hydrophobic tails.
1
Thus, surfactants have a tendency to both absorb at surfaces and
to form aggregates in solutions. Surfactants can form a variety of different aggregates
in aqueous solutions, most commonly micelles. Micelles formation results from the
desire to keep the head group in the aqueous solution, while at the same time
minimizing the interaction between the hydrophobic tails and water.
1
The surfactant
concentration at which micelles first form is called the critical micelle concentration
(CMC).

30
Surfactant micelles have been utilized in many fields, such as analytical
methods,
2; 3
cosmetics and biological aspects. The determination of micelles
formation is of great important and generally is determined through the measurement
of some solution properties,
4
such as a breakpoint of the electrical conductivity,
surface tension, light scattering, or fluorescence spectroscopy upon micelle
formation.
5; 6

Conductivity has been frequently used to determine the onset of micelle
formation of ionic solutions and is the one of the most efficiency methods.
7
Because
of the different physical properties of the micelles and monomer surfactant molecules,
when micelles form the molar conductivity of the solution decreases which induces
a sharp break in the curve, indicating a sharp increase in the mass per unit charge of
the material in solution.
4
Another method to determine the CMC is measurement of
the surface tension, which can determine the onset of micelle formation for non-ionic
surfactants unlike conductivity methods. The surface tension of the solution will have
a notable decrease with increase of surfactant concentration and when micelles are
formed, the surface tension will be independent of surfactant concentration, and the
break points correspond to the micelles formation in aqueous solution.
1

Depending on the surfactant structure, a variety of micelle morphologies can
develop, which can generally be classified into four types: spherical micelles,

31
elongated cylindrical micelles, flat lamellar structure and vesicles,
1; 4
as shown in
Figure 2.1.
8
Micelles structure can be tuned with changes in the temperature or
surfactant concentration, upon addition of a third component within the liquid phase,
or upon changes in the chemical architecture of the surfactant.
9
As mentioned in
Chapter 1, the chemical architecture of the surfactant is estimated by critical packing
parameter (p).
c c

b b

e e

d d

Figure 2.1: Micelles structures in aqueous solution. (a) spherical micelles, (b),
reverse micelles, (c) elongated cylindrical micelles, (d) flat lamellar micelles, and (e)
vesicles (taken from reference 8)

2.2.3 Photoresponsive surfactants
If a surfactant molecule contains a suitable chromophore,
10
light illumination
can result in a change in the chemical structure of the surfactant and thereby induce

32
changes in the physical properties of the surfactant micelles.
11
Azobenzene surfactants
exist as one of two isomers (trans or cis) depending on the light illumination
wavelength. The trans isomer is converted to the cis isomer upon absorption of 365
nm light, while the surfactant is converted back to the trans form upon illumination
with 460 nm light.
10
For each conversion, some irreversible changes are held for the
two isomers, which means the isomerization can not be completely accomplished and
a stationary state exists for the mixture of trans and cis isomers.
10; 12
Different
properties such as hydrophobicity of the azobenzene surfactants with light
illumination are of great important in protein structure control.
13; 14; 15; 16

2.3 Materials and methods
2.3.1 Photoresponsive surfactant preparation
The procedure of photoresponsive surfactant synthesis is described below by
surfactant type. In order to get high quality product, recystallization is applied to the
product from the final step in the azoTAB synthesis. By measurement of
1
H-NMR in
D
2
O, the recystallization might be applied more than once depending on the purity of
the azoTAB by analyzing data with Nuts program (Acorn NMR Inc.). In addition, the
conductivity was measured to ensure removal of all residual salts. All chemicals were
purchased from Sigma-Aldrich and used as received unless otherwise mentioned. The

33
structures and physical properties of all the synthesized surfactants are shown in Table
2.1.

2.3.2 NMR measurements
1
H-NMR measurements are performed with a Bruker AC 250-MHz spectrometer
at room temperature. The data were analyzed using the Nuts program (Acorn NMR
Inc.). 10 mg/ml of crystallized azoTAB was directly dissolved into D
2
O prior to the
NMR measurement.

2.3.3 Conductivity measurements (CMC determination)
2 mL of azoTAB aqueous solutions at various surfactant concentrations were
prepared and measured using a conductivity meter equipped with conductivity cell.
Following the conductivity measurements under room light the identical samples
were then exposed to an 84 W long wave UV lamp-365 nm (Spectroline, Model no.
XX-15A) in the conductivity cell and measured until stable conductivity reading was
reached for the UV-adapted sample. All measurements were taken at 25 °C and
critical micelles concentrations were determined from the break points in plots of
conductivity versus azoTAB concentration.

34
2.3.4 Surface tension measurements (CMC determination)
15 mL of azoOG (see Table 2.1) aqueous solutions diluted from stock solution
were prepared and the surface tension was measured using a Krüss K6 surface tension
apparatus (GmbH Hamburg, Germany). Identical solutions are exposed to an 84 W
long wave UV l lamp-365 nm (Spectroline, Model no. XX-15A) for at least 3 hrs to
convert to cis form. All measurements were taken at 25 °C and the CMC was
determined from the break point of the plots of surface tention versus azoOG
concentration. The reported surface tension values are the average of ten runs for each
azoOG aqueous solution.

2.3.5 Surfactant photoisomerization
Surfactants containing an azobenene group adopt the trans state in the dark, and
at photostationary state with visible and UV illumination which is changed by
surfactants. For example, for azoTAB-1 shown in Table 2.1, surfactant exists as the
trans isomer in solution in the dark (100% trans) or under visible light exposure
(~75% trans), while conversion to the cis form can be achieved upon illumination
with UV light (~95% cis). The measurement of the photoisomerization of azoTAB
surfactants was recorded with an Agilent UV-vis spectrophotometer (model 8453)

35
and the identical trans sample was exposed to an 84 W long wave UV lamp-365 nm
(Spectroline, Model no. XX-15A) for at least 3 hrs to convert to cis form.

2.4 Results and Discussion
2.4.1 Cationic surfactant preparation
Photoresponsive cationic surfactants are generally synthesized in three steps:
azocoupling, alkylation and quaternalization.
11; 17
Choosing
4-Ethyl-4’(trimethylaminobutoxy) azobenzene bromide as an example, the synthesis
process is given in Mechanism 1.

N
N
CH
3
CH
2
OCH
2
CH
2
CH
2
CH
2
N(CH
3
)
3
Br
CH
3
CH
2 NH
2
NaNO
2
HCl
CH
3
CH
2 NN
+
OH
CH
3
CH
2 N=N
OH
BrCH
2
CH
2
CH
2
CH
2
Br
CH
3
CH
2 N=N CH
2
CH
2
CH
2
CH
2
Br
N(CH
3
)

Mechanism 1: Synthesis process for azoTAB-1.

36
(a) Step I: Azocoupling
The synthesis starts from the raw material 4-ethylaniline (0.1 M) dissolved in a
high concentration (5 M) hydrochloric acid solution. A sodium nitrate aqueous
solution (6.67 M) was then added into the reaction system and allowed to react with
4-ethylaniline for 2 hrs in an ice bath. The product from this step was then filtered and
directly added into phenol (0.1 M) containing sodium carbonate (0.1M) and kept in
ice bath for at least 30 mins. Concentrated hydrochloric acid (5 M) was then added to
adjust the pH < 4 and the product (deep orange) was then filtered and dried in a
vacuum oven at room temperature at least 24 hrs. No other purification was applied to
the product of Step I.

(b) Step II: Alkylation
The Step I product (0.04 M) dissolved in THF (40 ml) was drop-wise added into
a mixture of 1,4-dibromide (0.1 M), potassium hydroxide (0.04 M), THF (200 ml)
and DI water (5 ml) . The mixture was then refluxed for 19 hrs in an oil bath with a
condensing column. After the reaction was completed, excess salt was removed by
filtration and the solvent was then removed with a rotary evaporator. The residual
solids were collected and redissolved in a mixture of hexane and THF (80/20
volume ratio), followed by recrystallization in a fridge for 24hrs, and with the Step

37
II product removed by filtration from the cold hexane and THF mixture. The product
with ethanol until the color goes from deep orange to orange followed with water.
This product was dried in vacuum oven at room temperature for further use.

(c) Step III: Quaternalization
The Step II product (1 g) was then dissolved in THF (50 ml) followed by the
addition of trimethylamine liquefied by placing the 400 mL TMA cylinder in an
acetone/liquid nitrogen mixture until the acetone solidified. The liquid TMA (20
pipettes) was then added in the THF solution and allowed to react for at least three
days with reflux in an oil bath with a condensing column. The final product was then
collected via filtration and recystallized from ethanol. Purity measurement is
determined by NMR shown in Figure 2.2 and conductivities (7.68 µM/cm for 1 mM).

38

N
N
CH
3
CH
2
OCH
2
CH
2
CH
2
CH
2
N(CH
3
)
3
Br
(1)
(2)
(3) (4) (5) (6)
(7)
(8)
(9)
(10)
(11)

Figure 2.2: NMR measurement for azoTAB-1. ( δ: 7.289 assigned to (4) and (5);
6.620 assigned to (6); 6.505 assigned to (3); 4.634 assigned to D
2
O; 3.456 assigned to
(7); 3.017 assigned to (10); 2.808 assigned to (11); 2.017 assigned to (2); 1.360
assigned to (8) and (9); 0.692 assigned to (1) and 0.003 assigned to TMS)

2.4.2 Anionic surfactant preparation
The photoresponsive anionic surfactant was synthesized in two steps:
azocoupling and alkylation. Choosing 4-heptyl-4’-sulfate azobenzene sodium as an
example, the synthesis proceeded as described in Mechanism 2.

39

HO
3
S
NaNO
2
HO
3
S
N
3
+
NH
2 NH
2
CH
3
(CH
2
)
6 +
N
N H
3
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
C
SO
3
_
Na
+
Mechanism 2: Synthesis process of azoTAB-2

(a) Step I: Azocoupling
Sulfanilic acid (1.73 g) was added into a sodium hydroxyl aqueous solution (0.5
M) and kept in ice bath until the sulfanilic acid was completely dissolved. Acetic acid
was then drop-wise added into the above solution until the pH = 4. A sodium nitrate
aqueous solution (6.67 M) was then drop-wise added into the reaction system and the
reaction was kept in an ice bath for 1 hr. The Step I product was then filtered and
solution is kept for further use.

(b) Step II: Alkylation
4-Heptylaniline (1 g) was dissolved in acetic acid (5 ml) and the Step I product
was slowly dissolved into this solution and reacted for 3 hrs at ice bath. The orange
solids appear and react for another hour in ice bath. The precipitate was removed by
filtration followed by the addition of a sodium hydroxide aqueous solution (3 M) to

40
the solution until the pH = 10. Orange stuff is suspended in the solution. The
precipitate was filtered and dried it in vacuum oven at room temperature, and then
recrystallized from ethanol in a fridge. The final product was obtained by filtration at
room temperature and dried in a vacuum oven at room temperature. Product purity
was measured by NMR as shown in Figure 2.3 and conductivities (7.7 µM/cm for 1
mM).

N
N H
3
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
C
SO
3
_
Na
+
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8) (9)
(10)
(11)

Figure 2.3: NMR measurement for azoTAB-2. ( δ: 7.437 assigned to (11); 7.117
assigned to (8); 6.756 assigned to (9) and (10); 4.792 assigned to D
2
O; 2.248 assigned
to (7); 1.197 assigned to (2), (3), (4), (5) and (6) and 0.868 assigned to (1).)

41
2.4.3 Non-ionic surfactant preparation
A novel photoresponsive non-ionic surfactants named “azoOG”, an azobenzene
analogue of OG (a non-ionic surfactant) containing glucopyranside moiety.
18
AzoOG
was synthesized in three steps: azocoulping, acylation and hydrolysis reactions. The
mechanism of azoOG synthesis is shown in Mechanism 3, below.

O
O HO
OH
CH
2
OH
OH
O
O
O
O HO
OH
CH
2
OH
OH
Ac
2
O, NaAc
Heat
OAc
CH
2
OAc
AcO
OAc
O
O
OAc
CH
2
OAc
AcO
OAc
N=N
CH
3
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2 N
2
Cl
+
_
Ice bath
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3
N=N
NH
3
, CH
3
OH
(1) (2)
(3)
(4)
(5)
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

Mechanism 3: The synthesis process of azoOG.

42
(a) Step I: Acylation
Phenyl- β-D-glucopyroside (1.28 g) was dissolved in acetic anhydride (60 ml)
and sodium acetate (0.6 g) was then added into the solution. The temperature was
then increased to 60 ° C and the contents were allowed to react for 2 days. The
product was then filtered and dried in a vacuum oven at room temperature.

(b) Step II: Azocoupling
4-Heptylaniline (1.0 g) was dissolved in ethanol (20 ml) and reacted with high
concentration (5 M, 2 ml) hydrochloric acid solution. A sodium nitrate aqueous
solution (6.67 M, 0.5 ml) added into the reaction system and allowed to react with
4-heptylaniline for 2 hrs in an ice bath. After the diazo salt filtration, the Step I
product (1.6 g) in acetic acid (24 ml) and propionic acid (8 ml) mixtures was slowly
added into the diazo salt solution and reacted for another 3 hr. A saturated aqueous
solution of sodium acetate was then added and kept for 3 hrs and the resulting
precipitate was filtered and crystallized from an ethyl ether/n-heptane mixture (80/20
volume ratio).

43
(c) Step III: Hydrolysis
The Step II product (1 g) was hydrolyzed by a 7 N ammonia solution in
methanol. It is needed to be added every twelve hour and reacted for 3 days. After
evaporation of the solvent in air, the residue was dissolved in acetone (20 ml). After
filter the insoluble component in acetone, acetone was removed in air and the final
product was crystallized from an ethonal/acentontrile (50/50 volume ration) mixture.
The azoOG was used without further purification.

2.4.4 Critical micelle concentration (CMC) determination
For charged surfactants, conductivity measurement can be used to readily
determine the onset of micelle formation. At surfactant concentrations below the
CMC, the molar conductivity is independent of surfactant concentration. However,
when the CMC is reached CMC, due to micelle formation the molar conductivity
changes at the concentrations between above and below the CMCs. These changes in
the slope in plots of conductivity vs. concentration are caused by two reasons, one is
the efficiency of the micelles to carry charges inducing the decrease of the effective
charges in aqueous solution and another one is the counter ions would like to bind
with micelles rather than monomer surfactant molecules, inducing the reduce of the
effective charges in aqueous solution.
19
Conductivity vs. surfactant concentration

44
plots are shown in Figure 2.4, and the resulting CMC values of each surfactant are
given in Table 2.1.

0
100
200
300
02 46
Conductivity ( µM/cm)
C oncentration (m M )
azoTAB 2 trans
azoTAB 2 cis
CMC
CMC
0
100
200
300
02 46
Conductivity ( µM/cm)
C oncentration (m M )
azoTAB 2 trans
azoTAB 2 cis
CMC
CMC

cmc
cmc
0
500
1000
1500
0 5 10 15 20
azoTAB 4 trans
azoTAB 4 cis
Conductivity ( µM/cm)
Concentration (mM)
cmc
cmc
0
500
1000
1500
0 5 10 15 20
azoTAB 4 trans
azoTAB 4 cis
Conductivity ( µM/cm)
Concentration (mM)
0
500
1000
1500
0 5 10 15 20
azoTAB 4 trans
azoTAB 4 cis
Conductivity ( µM/cm)
Concentration (mM)
Figure 2.4: Conductivity changes as a function of surfactant concentration
and light illumination. (a) azoTAB-4 and (b) azoTAB-2

Combing Figure 2.2 and Table 2.1, the effects of illumination and the surfactant
structures on micelles formation are determined. For the same head group,
increasing the length of the hydrophobic tail causes micelle formation to occur at
lower concentration, as expected (e.g., the CMCs of azoTAB-1,4 and 10 are 5 mM,
9 mM and 1.6 mM, respectively, under visible light). With longer alkyl chains the
surfactant is more hydrophobic, inducing the formation of aggregates in aqueous
solutions. Similarly, the higher CMC values observed under UV illumination are a
result of differences in surfactant hydrophobicity between the two surfactant isomers.

45
The trans forms of the surfactants are generally more hydrophobic and, thus, more
readily form micelles compared to the relatively hydrophilic cis forms, resulting in
lower CMC values under visible light illumination.
For non-ionic surfactants such as azoOG, the surface tension method can be
applied to report on the onset of micelles formation plots of surface tension vs.
surfactant concentration are displayed in Figure 2.5 the resulting CMC values of the
azoOG isomers listed in Table 2.1. Surfactant is characterized by its tendency to
absorb at the surfaces and interfaces.
1
In an aqueous solution, when surfactant
concentration is low, surfactant molecules favor to arrange on the surface inducing the
decrease of the surface tension. With addition of the surfactant when it reaches CMC,
the free energy of the solution will be decreased by the aggregation of surfactant
molecules to form micelles and the surface tension of the aqueous solution is
independent of the surfactant concentrations then. By measuring the surface tension
of aqueous solution, the break points of the surface tension vs. surfactant
concentrations plots are indicting the formation of the micelles.
4
For azoOG, cmc for
trans surfactant is 0.4 mM while it is 0.7 mM with UV illumination, as shown in
Table 2.1. The CMCs of azoOG under both visible light and UV light are higher to
that of OG, which is 0.025 mM in aqueous solution.
20

46

50
55
60
65
70
75
00.5 1 1.5 2
visible light
UV light
Surface tension (mN/m)
Concentration (mM)
CMC
CMC
50
55
60
65
70
75
00.5 1 1.5 2
visible light
UV light
Surface tension (mN/m)
Concentration (mM)
CMC
50
55
60
65
70
75
00.5 1 1.5 2
visible light
UV light
Surface tension (mN/m)
Concentration (mM)
CMC
CMC

Figure 2.5: The surface tension of azoOG aqueous solutions as a function of
light illumination.

In Table 2.1, the synthesized surfactant structures and CMC values are listed. For
cationic surfactants with the same head groups, longer hydrocarbon tails result in
lower CMC values, while the cis isomers always have higher CMC values relative to
the trans isomer of a given surfactant.

47
Table 2.1: Synthesized surfactants and CMC values (* is from reference 11) and
the data in brackets are taken from reference 11
azoTAB Structure CMC
Visible (mM) UV (mM)
1
N
N CH
3
CH
2
O(CH
2
)
4
N(CH
3
)
3
Br

5 [4.6] 10 [10.5]
2
N
N H
3
C(H
2
C)
6
SO
3
_
Na
+

1.25 2
4
N
N CH
3
CH
2
O(CH
2
)
2
N(CH
3
)
3
Br

9 [9.5] 12 [11.5]
6
N
N H
3
C(H
2
C)
4
SO
3
_
Na
+

Not measured
7
N
N H
3
C(H
2
C)
3
N
SO
3
_
Na
+

2 4
9
N
N CH
3
CH
2
O
O(CH
2
)
2
N(CH
3
)
3
Br

8 9
10
N
N CH
3
CH
2
O(CH
2
)
6
N(CH
3
)
3
Br

1.6 [1.6] 3 [3]*
11.

O
O HO
CH 2 OH
N=N (CH 2 ) 6 CH 3
OH OH
0.4 0.7

2.4.5 Surfactants photoisomerization
AzoTAB exists as one of two isomers depending on the light conditions, while
the absorbance of the two isomers can be measured by UV-vis spectroscopy. For
surfactants with different hydrocarbon tails and the same head group, the typical
absorbance will have slightly changes and the photostationary states are different as
well. For cationic azoTAB-1, 4, 9, 10 and azoOG, the two isomers could be reversibly
controlled with light illumination, while the anionic surfactants azoTAB-2, 6 and 7
appear to be irreversibly converted to cis state. Figure 2.6 shows the absorbance of

48
the azoTAB surfactants in aqueous solution with light illuminations. UV-vis spectra
of azoTAB-4, azoTAB-2 and azoOG, representative spectra of the cationic, anionic
and non-ionic surfactants, are shown in Figure 2.6. For a typical isomerization cycle
of azoTAB, the absorption spectrum contain three transitions, 248 nm (a π- π*
transition), 350 nm (a second intense π- π* transition) and 440 nm (a weak n- π*
transition).
17; 21
For azoOG absorbance, the peak at ~ 260 is the OG peak and it has it
change with light illumination, it is probably the Phenyl- β-D-glucopyroside group
inhibits the transition from trans to cis and the low absorbance of 350 nm peak for
azoOG is determined by low extension coefficient.

49

0
1
2
3
4
200 3 00 400 500 6 00
azoTAB 1 trans
azoTAB 1 cis
Absorbance
w avelength (nm)
0
1
2
3
4
200 3 00 400 500 6 00
azoTAB 2 trans
azoTAB 2 cis
Absorbance
wavelength (nm)
(a) (b)

0
1
2
3
4
266.7 4 00 533.3
azoOG trans
azoOG cis
Absorbance
Wavelength (nm )

(c)

Figure 2.6: UV-vis absorbance of (a) azoTAB-1 ([surfactant] = 0.5 mM, cell
length = 2 mm); (b) azoTAB-2 ([surfactant] = 0.5 mM, cell length = 2 mm); (c)
azoOG ([surfactant] = 3 mM, [OG] = 30 mM, cell length = 1 cm).

50
2.5 Conclusions
Various cationic, anionic, and non-ionic surfactants containing azobenzene
groups have been synthesized. Critical micelle concentrations have been determined
using electrical conductivity (for charged surfactant) or surface tension (for non-ionic
surfactant) measurements. Cis azoTAB isomers have higher CMC values compared to
the trans isomers because the cis isomers are more hydrophilic compared to trans
isomers. With longer alkyl tails, the surfactants have lower CMC values under both
visible and UV illumination. AzoTAB photoisomerization is measured and
investigated by UV-vis spectroscopy.

51
2.6 References
1. Holmberg, K., Jonsson, B., Kronberg, B. & Lindman, B. (2002). Surfactants
and Polymers in Aqueous Solution (edition, n., Ed.), Wiley.

2. Soroka, K., Vithanage, R. S., Phillips, D. A., Walker, B. & Dasgupta, P. K.
(1987). Fluresence Properties of Metal Complexes of
8-Hydroxyquinoline-5-Sulfonic Acid and Chromatographic Applications. Anal.
Chem. 59, 629-636.

3. Hayashita, T. & Bartsch, R. A. (1991). Selective Concentration of Lead(II)
Chloride Complex with Liquid Anion-Exchange Membranes. Anal. Chem. 63,
1023-1027.

4. Rosen, M. J. (2004). Surfactant and Interfacial Phenomena. Third Edition edit,
John Wiley & Sons, Inc.

5. Rhoades, E. & Gafni, A. (2003). Micelle Formation by a Fragment of Human
Islet Amyloid Polypeptide. Biophys. J . 84, 3480-3487.

6. Hiemenz, P. C. & Rajogopalan, R. (1997). Principles of Colloid and Surface
Chemistry, Marcel Dekker, Inc., New Youk.

7. Mukerjee, P. & Mysels, K. J. (1971). Critical Micelles Concentrations of
Aqueous Surfactant Systems., National Bureau of Standards., Washington, DC.

8. Doren, H. A. v., Smits, E., Pestman, J. M., Engberts, J. B. F. N. & Kellogg, R.
M. (2000). Mesogenic Surgars. From Aldoses to Liquid Crystals and
Surfactants. Chem. Soc. Rev. 29, 183-199.

9. Winson, P. A. (1976). Binary and Multicomponent Solutions of Amphiphilic
Compounds. Solubilization and the Formation, Structure, and Theoretical
Significance of Liquid Crystalline Solutions. Chem. Rev. 68, 1-40.

10. Eastoe, J. & Vesperinas, A. (2005). Self-assembly of Light-sensitive
Surfactants. Soft Matter 1, 338-347.

52
11. Hayashita, T., Kurosawa, T., Miyata, T., Tanaka, K. & Igawa, M. (1994).
Effect of Structural Variation within Cationic Azo-surfactant Upon
Photoresponsive Function in Aqueous Solution. Colloid. Polym. Sci. 272,
1611-1619.

12. Frankel, M. & Wolovsky, R. (1955). Wavelength Dependence of
Photoisomerization Equilibria in Azocompounds. J. Chem. Phys 23,
1367-1368.

13. Hamill, A. C., Wang, S.-c. & Lee, C. T. J. (2007). Solution Structure of an
Amyloid-Forming Protein During Photoinitiated Hexamer-Dodecamer
Transitions Revealed Through Small-Angle Neutron Scattering. Biochemistry
46, 7496-7705.

14. Wang, S.-c. & Lee, C. T., Jr. (2006). Protein Secondary Structure Controlled
with Light and Photoresponsive Surfactants. J. Phys. Chem. B 110,
16117-16123.

15. Hamill, A. C., Wang, S.-C. & Lee, C. T. J. (2005). Probing Lysozyme
Conformation with Light Reveals a New Folding Intermediate. Biochemisty
44, 15139-15149.

16. Lee, C. T. J., Smith, K. A. & Hatton, T. A. (2005). Photocontrol of Protein
Folding: The Interaction of Photosensitive Surfactants with Bovine Serum
Albumin. Biochemistry 44, 524-536.

17. Lee, C. T. J., Smith, K. A. & Hatton, T. A. (2004). Photoreversible Viscosity
Changes and Gelation in Mixtures of Hydrophobically Modified
Polyelectrolytes and Photosensitive Surfactants. Macromolecules 37,
5397-5405.

18. Barni, E., Barolo, C., Quagliotto, P., Valldeperas, J. & Viscardi, G. (2000).
Novel Azobenzene Derivatives Containing a Glucopyranoside Moiety. Part I:
Synthesis, Characterisation and Mutagenic Properties. Dyes Pigm. 46, 29-36.

19. Lyklema, J. (2005). Fundamentals of Interface and Colloid Science: Soft
Collids, Academic Press.

53
20. Mukerjee, P. & Chan, C. C. (2002). Effects of High Salt Concentrations on the
Micellization of Octyl Glucoside: Salting-out of Monomers and Electrolyte
Effects on the Micelle-Water Interfacial Tension. Langmuir 18, 5375-5381.

21. Kuiper, H. M. & Engberts, J. B. F. N. (2004). H-Aggregation of
Azobenzene-Substituted Amphiphiles in Vesicular Membranes. Langmuir 20,
1152-1160.

54
Chapter 3: Photoreversible Conformational Changes in
Membrane Proteins Using Light-responsive Surfactants
Jing Zhang, Shao-Chun Wang, and C. Ted Lee, Jr. sumitted

3.1 Abstract
Photoreversible control of the conformation of bacteriorhodopsin in the presence
of a light-responsive surfactant is demonstrated through combined UV-vis, FT-IR, and
31
P NMR spectroscopy and dynamic light scattering (DLS) measurements. The
azobenzene-based surfactant photoisomerizes upon 434-nm visible (trans, relatively
hydrophobic) and 350-nm UV (cis, relatively hydrophilic) illumination, allowing
surfactant micellization to be reversibly controlled. This leads to partitioning of the
membrane protein into micelles in the unfolded state under visible light, while UV
light leads to solubilization of the protein within purple membrane bilayers in the
folded state. A three-stage model of purple membrane-photosurfactant interactions is
proposed and confirmed through NMR and DLS measurements. Photo-triggered
unfolding of bacteriorhodopsin, occurring through α
II
→ α
I
and reverse β-turn →
extended β-strand transitions, requires ~20 sec for completion, while light-induced
refolding requires a somewhat longer 80 sec as the membrane protein repartitions into
the reformed bilayer membrane. Each of these conformational changes can be

55
precisely and reversibly controlled with simple light illumination, providing a novel
technique to probe membrane protein folding.

3.2 Introduction
Membrane proteins, which constitute 20-30% of all cellular proteins, are of
crucial importance for biological function, serving as mediators between the cell and
the surrounding environment, catalyzing the transport of ions and metabolites across
the cell membrane, and can further be responsible for a wide range of diseases (e.g.,
cystic fibrosis, diabetes, and hypertension). Thus, a thorough understanding of
membrane protein structure and function is required to allow for the development of
new classes of drugs capable of specifically targeting membrane proteins.
1

Unfortunately, the fact that membrane proteins simultaneously contain large
hydrophobic and hydrophilic regions, which respond to solvent conditions in different
and complex ways, has made it exceedingly difficult to examine membrane protein
folding pathways as unfolding often leads to irreversible aggregation. Furthermore,
the overexpression of membrane proteins in heterologous host organisms (e.g., E.
coli)
2; 3
typically results in unfolded proteins located in inclusion bodies in the cell.
Thus, the protein needs to not only be recovered from the cell matrix, but also
refolded. A number of techniques for recovery have been developed, including
extraction into organic solvents,
4; 5
urea,
6; 7; 8; 9; 10
sodium dodecyl sulfate (SDS)

56
surfactant,
11; 12; 13; 14; 15
and trifluoroacetic acid,
11
or alternatively affinity tags can be
used in conjunction with an appropriate affinity column.
16; 17; 18
Refolding can then
achieved by transferring the denatured protein into a “mild” detergent solution (i.e.,
non-denaturing, as opposed to SDS) that consists of either micelles or vesicles,
allowing the native-like conformation of the protein to be reconstituted. The choice of
proper extraction and refolding mixtures is by no means trivial, and often must be
tailored for each “notoriously individualistic” membrane protein.
19
Furthermore, the
transfer step mentioned above can bring about unwanted aggregation and other
deleterious effects due to the dual hydrophobic and hydrophilic regions of the protein.
This has led to only a handful of membrane proteins being successfully refolded from
the denatured state,
3; 20
and is often a major hurdle in protein characterization.
Bacteriorhodopsin (bR), one of the most studied membrane proteins, is the major
component of purple membrane (PM) fragments isolated from Halobacterium
Salinarium, and functions as a light-driven proton pump. PM contains ca. 75 wt % bR
and 25 wt % lipids, or equivalently 10 lipid molecules per protein.
21; 22
Due to this
large protein concentration, PM fragments exhibit a highly-ordered, paracrystalline
lattice
23
that, when dispersed in aqueous solutions, exist as stiff, planar sheets with an
average diameter of ca. 500 nm.
22; 23
The high-resolution X-ray crystallographic
structure of bR
24
reveals seven transmembrane helices connected by
extramembranous loops.
25
The secondary structure of the bR has been studied by

57
FT-IR
26
and found to contain about 70% α-helix structure corresponding to the
transmembrane helices,
27; 28
with the remaining 20% β-strands and 10% unordered
structures.
29
A retinal chromophore, covalently linked within the helix bundle of bR
via a protonated Schiff-base linkage to Lys-216 on helix G,
25
undergoes
photoisomerization from the all-trans to the 13-cis conformation following
illumination with 560-nm visible light, representing the first step in the proton-pump
photocycle (bR → K state).
25; 30; 31
A proton is then transferred from the Schiff base to
Asp-85 (L → M reaction)
25; 32
and subsequently released into the extracellular region,
leading to the formation of the N intermediate.
25; 32
The Schiff base is then
reprotonated from the cytoplasmic side of the membrane via Asp-96 (O state),
25
after
which time the retinal is isomerized back into the all-trans bR ground state.
Starting from the unfolded form in SDS micelles, bR can be refolded by rapid
exchange of SDS with a milder nonionic detergent system such as DMPC/CHAPSO,
DMPC/CHAPS, or DMPC/DHPC (DMPC: 1,2-dimyristoyl-sn-glycero-
3-phosphocholine; CHAPSO: 3-[(3-cholamidopropyl) dimethylamino]-2-hydroxyl-1-
propane; CHAPS: 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate;
DHPC: dihexanoylphosphatidylcholine).
33; 34; 35
Similarly, even the highly-ordered
paracrystalline structure of PM sheets could be reformed following removal of the
natural lipids with detergents and then reconstitution through exchange of detergent
with native lipids isolated from PM.
36; 37; 38

58
In the present study, simple light illumination will be used as a novel method of
controlling membrane protein folding. Photoreversible control of bR conformation
will be achieved through the photoisomerization of a light-sensitive surfactant (see
Scheme 3.1, below) that can be switched from the “active” to the “passive” state with
light. Using this unique property, photoreversible control of soluble protein folding
through light-induced molecular binding of the surfactant to the protein has resulted
in the formation of a partially-folding intermediate of lysozyme
39
with enhanced
enzymatic activity
40
as well as photoreversible pre-amyloid oligomization in
α-chymotrypsin.
41
The present study will extend this methodology to membrane
proteins, utilizing the photoreversible partitioning of bR between micellar (unfolded)
and lipid bilayer (folded) environments that form with the active and passive forms of
the photosurfactant, respectively. This concept is similar to the aforementioned rapid
exchange of SDS with a milder detergent system; although in the present case the
process is entirely light activated, greatly simplify the refolding protocol and
promoting refolding versus aggregation as the same surfactant species would be used
throughout the refolding process. Several complementary methods will be used to
study the effect of the photoresponsive surfactant and light conditions on bR folding,
including UV-vis spectroscopy to study absorbance changes at 560-nm of the retinal
chromophore, FT-IR spectroscopy to examine secondary structure changes in the

59
protein, and DLS and
31
P NMR measurements to examine the formation of
PM-azoTAB mixed micelles.

3.3 Experimental Details
3.3.1 Materials
The surfactant 4-ethyl-4’(trimethylaminobutoxy) azobenzene bromide (azoTAB)
shown in Scheme 1 was synthesized as described.
42; 43
All other chemicals were
purchased from Sigma-Aldrich and used as received unless otherwise mentioned. The
dipole moment across the nitrogen double bond is ~0.5D for the trans isomer
compared to ~3.1D for the cis form.
44
As a result, the trans isomer is significantly
more hydrophobic than the cis form, allowing photo-induced changes in a variety of
surfactant properties such as critical micelle concentration,
42
surface tension,
45
and
electrical conductivity.
42

N
N
CH
3
CH
2
O(CH
2
)
4
N
+
(CH
3
)
3
Br N
+
(CH
3
)
3
Br
N
N O(CH
2
)
4
CH
2
CH
3
434 nm
350 nm

Scheme 3.1: Photoisomerization of the azobenzene-based surfactant.

60
3.3.2 Purple membrane preparation
Purple membrane fragments containing both lipids and the membrane protein
bacteriorhodopsin were isolated from Halobacterium salinarium (strain S9, a kind
gift from Professor Dieter Oesterhelt at the Max Plank Institute of Biochemistry in
Martinsried) according to the method of Oesterhelt and Stoeckenius.
22
Briefly,
bacterial cells were grown in 1-L cultures incubated at 37 °C in the dark in a shaking
water bath at 100 rpm for 5-6 days until the absorption at 560 nm was approximately
1.0-1.5.
46
The purification of PM was then achieved as previously described,
47
with
PM isolated from the medium at 4,000 rpm for 40 min. Impurities in PM were
removed by washing with DI water at least 15 times at a speed of 24,000 rpm for 45
min (Beckman Coulter Avanti J-25 I Centrifuge, Beckman rotor JA 25.50) until the
water no longer exhibited a purple color. The obtained PM wet pellet was stored at
–20 °C for future use. The typical yield of the protein was 12-15 mg per liter of
culture determined with an Agilent UV-vis spectrophotometer (model 8453) using an
extinction coefficient of 54,000 M
-1
cm
-1
at 560 nm.
22

3.3.3 UV-vis determination of bR folding
PM suspended in 0.1 M sodium phosphate buffer, pH 6.0, was added directly into
vials with the appropriate amount of crystallized azoTAB. The samples were then
illuminated for 1 hr at 25 °C under gentle stirring with a 200-W mercury arc lamp

61
(Oriel, model no. 6283) equipped with a 400-nm long pass filter (Oriel, model no.
59472), heat absorbing filter (Oriel, model no. 59060), and a fiber-bundle focusing
assembly (Oriel, model no. 77557) to isolate the 436-nm mercury line. After the
UV-vis spectra were recorded, the samples were then illuminated with the arc lamp
equipped with a 320-nm UV filter (Oriel model no. 59980) to isolate the 365-nm
mercury line, with surfactant conversion from the trans to the cis isomer assured with
absorption measurements. The concentration of the protein in each sample was ~0.18
mg/mL, determined spectroscopically. The phase behavior of each sample was
observed visually, while optical microscope images were taken with an Olympus
IX71 inverted microscope equipped with 100× oil-immersion objective lens (UPlanFl,
N.A.=1.3). The dynamics of photo-induced bR conformational changes were studied
by in situ UV-vis spectroscopy, using a surfactant concentration of 0.10 mM to insure
rapid surfactant isomerization. The results were obtained over a period of 20 min with
spectra collected every 0.5 s. Measured spectra were corrected for scattering effects
using a spline routine with a baseline choice of 700-900 nm and 470-480 nm under
visible illumination and 700-900 nm and 380-385 nm under UV exposure.

3.3.4 FT-IR measurements
Approximately 0.4 mL of the purified PM in H
2
O was washed with 5.0 mL D
2
O
at least three times at the speed of 24,000 rpm for 45 min to remove residual H
2
O.

62
The sample was then suspended in 0.1 M sodium phosphate D
2
O buffer (pD = 6.4,
measured with a standard pH electrode and corrected according to pD = pH + 0.4) at
a protein concentration of 7.8 mg/mL detected with UV-vis spectroscopy. Adding the
PM suspension into vials containing crystallized azoTAB generated a series of
surfactant concentrations. The samples were then gently stirred for 24 hr in the dark
prior to the FT-IR measurements.
Infrared spectra were measured with a Genesis II FT-IR spectrometer (Mattson
Instruments). Samples were loaded between a pair of CaF
2
windows using a 50 µm
Teflon spacer with water-jacket circulation used to maintain the temperature at 20 °C.
The FT-IR sample chamber was purged with dry air for 12 hr prior to and during
measurements. The samples were continuously illuminated with either visible or UV
light from the mercury arc lamp using the fiber-bundle focusing assembly as
previously described.
48
For each spectrum, 1000 interferograms were collected with a
2 cm
-1
resolution. The visible and UV filter sets were then alternated every 3 hr to
measure light-induced changes in protein folding. Difference spectra were obtained
by subtracting the spectra measured under visible light from that taken with UV
illumination. Photoreversibility was assured through multiple visible ↔ UV light
cycles. The protein absorbance was obtained by subtracting the spectrum measured
for pure buffer, with the resulting corrected spectra flat in the region between 1900
and 1750 cm
-1
. The technique of Fourier self-deconvolution (FSD) was applied to the

63
original spectra to resolve the overlapping bands in the amide I region using a
band-narrowing factor k = 2.2 and a full width at half-height of 12.5.

3.3.5 NMR measurements
31
P NMR measurements were performed with a Bruker AMX 500-MHz
spectrometer operating at 201.5 MHz and room temperature with continuous
1
H
decoupling and 33K data points in the transformed spectra. The data were analyzed
using the Nuts program (Acorn NMR Inc.). PM suspended in D
2
O at a protein
concentration of 7.5 mM (determined spectroscopically
22
) was added directly into
vials containing the appropriate amount of crystallized azoTAB to achieve the desired
concentrations. The samples were prepared and stirred in the dark for 24 hr before the
measurement. After the NMR measurements were recorded for trans azoTAB (dark
state), the samples were then illuminated with the arc lamp equipped with a 320-nm
UV filter (Oriel model no. 59980) for another 24 hr with gentle stirring. Following
this conversion of azoTAB to the cis state, the samples were maintained in the dark
for no more than 2 hrs prior to and during measurements, resulting in only minimal
thermal conversion of azoTAB back to the trans state (confirmed with UV-vis).

64
3.3.6 Dynamic light scattering measurements
Dynamic light scattering measurements were performed at 25 °C on a
Brookhaven model BI-200SM instrument (Brookhaven Instrument Corp.) equipped
with a BI-9000AT digital correlator (Brookhaven), a 35-mW HeNe (632.8 nm) laser
(Melles Griot, model no. 05-LHP-928), and an BI-APD avalanche photodiode
detector (Brookhaven). PM suspended in 0.1 M D
2
O phosphate buffer, pH 6.0, at a
protein concentration of 0.18 mM (determined spectroscopically) was directly added
into vials with appropriate amounts of crystallized azoTAB. The samples were then
stirred in the dark for 24 hr before the measurement. Samples were measured as made
without filtration to avoid removal of the large purple membrane fragments, with dust
allow to settle in the instrument until the count rate stabilized. After measurements in
the presence of trans azoTAB (dark state), the samples were then exposed to UV light
from the arc lamp for 24 hr with gentle stirring. In order to maintain azoTAB in the
cis state, UV light from the arc lamp was illuminated onto the sample with the fiber
bundle focusing assembly during the entire measurement. The dynamic light
scattering data were analyzed with the nonnegative least-squares (NNLS) routine
supplied by Brookhaven. Typically, at least five independent scattering runs were
conducted for each solution, with the average of all the runs reported. Since the
samples could not be filtered, it was ensured that the scattering count rate remained
approximately constant over time, while flat baselines for each correlation function

65
were maintained over 1-2 decades in relaxation times. For smaller micellar species,
the scattering angle was lowered to 45° in order to observe the full correlation
function (i.e., a flat decade at low relaxation times). Hydrodynamic radii, R
H
, were
calculated from the Stokes-Einstein equation R
H
= k
B
T/6πηD, where k
B
is
Boltzmann’s constant, T is temperature, η is the viscosity of the solvent, and D is the
experimentally determined diffusion coefficient.

3.4 Results and Discussion
3.4.1 Phase behavior of purple membrane with azoTAB
The miscibility of purple membrane fragments (PM) with azoTAB is shown in
the test tube images in Figure 3.1a. At azoTAB concentrations below 0.35 mM,
transparent suspensions are obtained (evident from the reflections in the glass test
tubes), while cloudy samples are observed as the surfactant concentration reached
0.50 mM under both visible and UV light. Under visible light, the mixtures remained
cloudy until trans azoTAB concentrations of 3.0 mM or greater were surpassed, while
with UV exposure cis azoTAB concentrations of 12.0 mM are required for miscibility.
This phase behavior phenomena suggests that the classic three-stage model of
detergent-lipid membrane interactions may be in operation.
49; 50
During Stage I of this
model at low surfactant concentrations (< 0.35 mM azoTAB), azoTAB would be
expected to incorporate non-cooperatively (i.e., as surfactant monomers) into the lipid

66
phase. Introduction of cationic azoTAB into the negatively-charged phospholipid
bilayers
51
would tend to decrease the net charge of the PM sheets and, hence,
decrease the electrostatic stabilization of the PM sheets.
52
Thus, as previously
studied by Oesterhelt,
53
the addition of a cationic surfactant to PM induces
precipitation with the formation of large PM aggregates.
53
This observation is
consistent with the light microscopic images in Figure 3.1b, where PM aggregation is
initially observed at ca. 0.35 mM azoTAB, while further increases in the surfactant
concentration lead to an increased degree of aggregation. The color change from
purple to yellow/orange in Figure 3.1a is a result of mixing PM with surfactant
solutions that are themselves either yellow (trans form) or orange (cis form) due to
the azobenzene absorbance.
In Stage II of the surfactant-lipid interactions, the lipid bilayers eventually
become saturated with detergent with increased surfactant concentrations, resulting in
detergent partitioning into the bulk solution phase. Thus, at sufficient detergent
concentrations, micellization can occur in the bulk solution phase, leading to
surfactant-saturated lipid bilayers existing in equilibrium with surfactant-lipid mixed
micelles. Further increases in detergent concentration then eventually result in
complete dissolution of the lipid bilayers by mixed micelles in Stage III of the
interactions. The images in Figure 3.1 support this mechanism, with solubilization of
PM into azoTAB-lipid mixed micelles suspected from both the phase behavior (a

67
return to transparency) and microscopic (disappearance of PM aggregates, not shown)
observations. It is noteworthy that this apparent PM solubilization occurs at azoTAB
concentrations of approximately 3 mM (visible) and 12 mM (UV), similar to the
critical micelle concentrations obtained for pure azoTAB (5 mM and 10 mM,
respectively
43
). This suggests that the relatively-hydrophobic trans isomer more
readily forms mixed micelles compared to the relatively-hydrophilic cis state, as may
be expected. This potentially provides an avenue to photo-control the partitioning of
bR between the bilayer (folded) and micellar (unfolded) state, as will be discussed
further below.

68

0.35 mM visible
(b)
(a)
UV light
0 .1 .2 .35 .5 .85 .65 1.0 1.5 2 3 4 5 6 7 8 9 10 12 20 18 16 14
visible light
1.0 mM visible
10 µm
1.0 mM UV
7.0 mM UV

0.35 mM UV

Figure 3.1: (a) Miscibility of purple membrane with azoTAB under visible and
UV light. Numbers represent the azoTAB concentration (mM). Cloudy mixtures can
be detected by the lack of the reflections in the glass test tubes. (b) Optical
microscope images of mixtures of PM with azoTAB. [bR] = 0.18 mg/mL.

3.4.2 Mixed-micelle formation in azoTAB-PM systems
In order to confirm the formation of mixed azoTAB-PM micelles, dynamic light
scattering (DLS) and
31
P NMR measurements were performed. Light scattering is
particularly sensitive to the changes in size expected upon solubilization of the ca.
500-nm PM sheets into relatively small micelles. As an example, representative DLS

69
data measured for both pure PM (0.18 mg/mL, same as Figure 3.1) and PM mixed
with azoTAB (20 mM) under visible and UV light are displayed in Figure 3.2a. For
dilute, spherical, and monodisperse particles, the normalized correlation function
would be given by g
(1)
= exp(−Dq
2
τ), where τ is the experimental relaxation time and
the scattering vector is defined as q = (4πn/λ) sin(θ/2) with n the refractive index of
the solvent, λ the wavelength of light, and θ the scattering angle. Thus, the
translational diffusion coefficient D can be estimated from the negative of the slope of
a semi-log plot of the correlation function versus q
2
τ, as in Figure 3.2a. Pure purple
membrane is seen to exhibit a single, relatively small slope in the range in Figure 3.2a,
indicating the presence of only large, slowly diffusing species. In contrast, the
solution with 20 mM azoTAB under both visible and UV light displays two (and in
some cases three) relaxations in this range, notably a relatively fast relaxation at low
values of q
2
τ, indicating the formation of small, fast-diffusing scattering species (i.e.,
micelles).
A series of correlation functions of this type were analyzed with the nonnegative
least-squares (NNLS) routine, capable of resolving the simultaneous relaxations of
multiple scattering species. Hydrodynamic diameters obtained from the diffusion
coefficients through the Stokes-Einstein equation (see Experimental Details) of each
relaxation, assuming that the systems are infinitely dilute and non-interaction, are
given in Table 3.1, averaged over ~10 runs with reproducibility generally within ± 5%,

70
along with the measured scattering count rates in thousands of counts per second
(kcps) at each condition. First, it is noted that the count rates decrease by over an
order of magnitude from pure PM upon addition of 20 mM azoTAB under visible or
UV light, indicating a transition from larger to smaller scattering species as scattering
intensity generally varies as the square of particle volume. However, as these samples
were not filtered to prevent removal of potentially large PM sheets/aggregates,
quantitative interpretation of this decrease in count rate is limited.
The pure PM sample was found to exhibit two relaxations corresponding to
hydrodynamic diameters of ca. 600 nm and 3200 nm, while no relatively small
scatters were detected as in Figure 3.2a. The 600-nm value is consistent with the
known dimensions of the PM disk-like sheet with diameter of ~500 nm and thickness
of ~5 nm.
54
Based on this shape, PM sheets would be expected to undergo
translational diffusion both parallel and perpendicular to the disk plane, along with
rotational diffusion of the entire disk,
55; 56
all of which would combine to give an
effective hydrodynamic diameter in the DLS measurement. In this manner, a
thorough analysis of DLS data by Kubota et al.
55
gave regressed values of the PM
disk diameter and thickness of 590 nm and 4.9 nm, respectively. Using these
tabulated functional values and the diffusion coefficients and dick thickness,
55
the
hydrodynamic diameter of ~600 nm in Table 3.1 would correspond to a disk diameter
of 530 nm, in reasonable agreement with the above reported values. The relatively

71
large hydrodynamic diameter of ~3200 nm in Table 3.1 is likely a result of either a
small portion of aggregated PM sheets, or perhaps dust or other debris due to the lack
of filtration.
Interestingly, in the presence of 20 mM azoTAB only relatively small particle
sizes are detected from the DLS measurements, as shown in Table 3.1. Specifically,
a 3-nm peak is observed under both visible and UV light, suggesting the formation of
relatively small, spherical micelles, consistent with the aforementioned
order-of-magnitude decrease in scattering intensity. Under visible light, two
additional peaks are consistently detected at ca. 43 nm and 310 nm, while under UV
light only a single additional peak at ~185 nm is observed. The larger peaks under
visible and UV light could potentially result from debris from the non-filtered
samples or PM fragments that are not completely solubilized in mixed micelles. Note
that pure azoTAB solutions display a similar single relaxation at 3 nm at 20 mM (not
sown for clarity) is above the critical micelle concentration under both visible and UV
light. However, any residual amount of PM fragments not in micelles would be
expected to be quite small (on a per mass basis) as the NNLS routine ascribes nearly
equal contributions to the overall scattering of these large peaks and the 3-nm peak
(with intensity roughly scaled with the square of particle volume). In other words, the
vast majority of the system exists as micelles under visible and UV light. The
intermediate size of ~43 nm observed for the azoTAB-PM system under visible light

72
could potentially result from the formation of a fraction of larger, non-spherical
micelles. Surfactant-lipid systems often form so-called bicelles in solutions,
essentially small bilayer disks surrounded on the rim by a layer of detergent. For
example, small-angle neutron studies have indicated that DMPC/DHPC bicelles have
a diameter of ~40 nm,
57
consistent with the intermediate peak in Table 3.1. The lack
of this intermediate peak in the UV exposed sample would then suggest that the bent,
cis conformation of azoTAB is unable to effectively pack around the edges of these
potential bicelles. It should be noted, however, that care must be taken when
interpreting DLS data exhibiting bi- and especially tri-modal particle size
distributions, which challenge the regression methods of the DLS technique.

Table 3.1: Dynamic light scattering (DLS) measurements of the scattering count
rate and hydrodynamic diameter for pure PM and azoTAB-PM systems at [azoTAB]
= 20 mM (standard deviations given in parenthesis).
azoTAB-PM
pure PM visible light UV light
scattering angle 90° 90° 45° 90° 45°
count rate (kcps) 118 11 38 7.6 34
D
H
(nm) 605(32) 2.9(0.08) 3.1(0.13)
43(17)
3250(140) 310(13) 185(17)

Overall, the DLS data support the notion of a three-stage model of azoTAB-PM
interactions with the re-solubilization observed in Figure 3.1 at high azoTAB

73
concentrations a result of mixed-micelle formation. Strictly peaking, however, the
DLS results only indicate the formation of micelles, and not necessarily azoTAB-lipid
mixed micelles. Thus,
31
P NMR measurements were performed on the azoTAB-PM
systems as a function of surfactant concentration and light illumination, as shown in
Figure 3.2b. NMR spectra can be quite sensitive to the local microenvironments of
the various chemical species. For example, the
31
P NMR spectrum of the pure PM
suspension in Figure 3.2b gives a broad, asymmetric
31
P peak with a line shape
typical of phospholipids in bilayer structures,
58
a result of reduced mobility of
phospholipids and, thus, heterogeneity of the microenvironments within the rigid PM
structure on the NMR timescale (µsec).
59; 60
In contrast, the
31
P NMR peak is seen to
progressively narrow upon addition of the azoTAB surfactant indicating homogeneity
of the microenvironments on the NMR timescale,
61
a result of relatively rapid
motions of the phosphate lipid groups in the azoTAB/PM mixed micelles. This
evidence further supports the three-stage model of azoTAB-lipid interactions, with the
addition of the surfactant in Stage II and ΙΙΙ resulting in mixed micelles. At 10 mM
azoTAB a shaper NMR peak is observed with the surfactant in the trans relatively to
the cis form, indicating mixed-micelle formation is more pronounced in the presence
of the relatively-hydrophobic, planar trans isomer of the surfactant. At 20 mM
azoTAB, the phospholipids bilayers are apparently totally dissolved in azoTAB-lipid
mixed micelles.

74

0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.002 0.004 0.006 0.008 0.01
pure PM
PM + 20 mM azoTAB (vis)
PM + 20 mM azoTAB (UV)
g
(1)
q
2
τ(10
10
s/cm
2
)
(a) (b)
cis azoTAB trans azoTAB
20 mM
10 mM
0 mM

Figure 3.2: (a) Normalized correlation function of PM (0.18 mg/ml) and azoTAB
(20 mM) in 0.1M phosphate buffer as a function of light illumination. The presence
of azoTAB-PM mixed micelles is evident from the fast relaxation at low values of q
2
τ.
(b)
31
P NMR measurements as a function of azoTAB concentration and light
illumination. [PM] = 7.5 mg/ml.

3.4.3 Photoreversible control of bacteriorhodopsin folding in azoTAB-PM
assemblies
Based on the above evidence of the conversion of PM bilayers into azoTAB-PM
mixed micelles, the effect of surfactant and light on the conformation of the
membrane protein bR was examined with UV-vis spectroscopy, as seen in Figures 3.3.
BR exhibits a convenient measure of protein folding, namely the absorption bands of
the retinal chromophore with bR in the native (560 nm, chromophore covalently
attached to bR) and denatured (380 nm, chromophore free in solution) state.
34; 35; 46

75
Thus, the relative fraction of folded bR molecules in azoTAB-PM assemblies can be
defined as the absorbance measured at 560 nm for a given surfactant concentration
divided by the absorbance at 560 nm for PM in the absence of surfactant (i.e., with
100% of the bR molecules folded). The 560-nm retinal peak is sufficiently removed
from azoTAB absorbance to allow precise measurement (see also Figure 3.4).
Scattering effects due to the presence of PM sheets with dimensions on the order of
the wavelength of light (~500 nm
22; 50; 62; 63
) were removed as described in the
Experimental Details.

0
0.2
0.4
0.6
0.8
1
0.01 0.1 1 10
visible
UV
Folding Fraction
Surfactant Concentration (mM)

Figure 3.3: Relative folding percentage of bR (0.18 mg/ml) as a function of
azoTAB concentration determined from the protein absorbance at 560 nm (see also
Figure 3.4). The data point placed at 0.01 mM actually represents pure PM, arbitrarily
assigned due to the log scale. Curves drawn to guide the eye.

76

0
0.5
1
1.5
2
2.5
300 400 500 600 700 800

Figure 3.4: Kinetics of light-induced conformational changes of bR obtained
from in situ absorbance measurements. [bR] = 0.18 mg/mL, [azoTAB] = 0.10 mM.
(a) Selected individual UV-vis spectrum at various listed time intervals. Inset: a
magnification of the PM peak for the azoTAB-PM samples (solid curves). (b) the
absorbance at 560 nm as a function of time. The circled data points correspond to the
displayed spectra, with the first spectrum set to a time of 0 sec in part (a).

As seen in Figure 3.3, with increasing surfactant concentration the folding
fraction decreases under both visible and UV light, while at a given surfactant
concentration the percentage of folding is observed to be higher with azoTAB in the
cis state (UV illumination) compared to the trans state (visible light). For example, at
a surfactant concentration of 0.10 mM, ~80% of the bR molecules are folded under
visible light, compared to nearly complete folding under the UV light. Furthermore,
bR is completely unfolded when the trans surfactant concentration reaches 2.0 mM,
whereas ~8.0 mM cis azoTAB is required to induce complete unfolding. It is
important to note that the absorption at 560 nm for pure PM does not change upon
Absorb e
Wavelength (nm)
protein
trans
cis
0 s
5 s
18
20 s/80 s
s
0.4
0.5
525 550 575 600
Absorbance
Wavelength (nm)
PM +
azoTAB
0.24
0.26
0.28
0.3
0.32
0 200 400 600 800 1000
A
560
(scattering corrected)
Time (sec)
trans azoTAB
cis azoTAB
anc

77
illumination with either the 365-nm or 436-nm lines from the mercury arc lamp.
Indeed, the natural photocycle of bR involves the absorption of yellow light
64; 65
in the
wavelength range of 500-660 nm to convert retinal from the all-tran to the 13-cis
isomer. Therefore, it is not surprising that the illumination wavelengths used to
control azoTAB isomeric state are insufficient to induce conformational changes in
the protein, indicating that bR unfolding is instead a result of changes occurring to the
morphology of the surfactant assemblies.
Comparing Figures 3.1 and 3.3, the mechanism by which azoTAB induces
protein unfolding can be understood in the context of the three-stage interaction
model. Protein unfolding is seen to begin near the onset of Stage II (~ 0.35 mM
azoTAB), namely where mixed micelles are expected to first form and exist in
equilibrium with surfactant-saturated bilayers. Indeed, membrane proteins solubilized
in charged micelles, as opposed to bilayers, are generally believed to exist in the
unfolded state.
33; 34; 66; 67
For example, bR becomes denatured in micelles formed from
anionic SDS,
33; 35; 46; 68
as well as in cationic detergents.
66
Thus, bR likely partitions
between the bilayers (folded) and micellar (unfolded) states, with increasing azoTAB
concentration leading to enhanced micelle formation and a progressively larger
fraction of the protein residing in micelles in the unfolded state. Note that while
Figure 3.1 estimates the onset of mixed-micelle formation to occur at 0.35 mM
azoTAB under both visible and UV light, the visual methods employed in Figure 3.1

78
are relatively insensitive to the Stage I/II boundary compared to the absorbance
measurements in Figure 3.3. As a result, the onset of mixed-micelle formation likely
occurs as low as 0.1 mM azoTAB under visible light, with approximately 20% of the
protein molecules partitioning into the micellar state at this concentration. In contrast,
micelle formation is delayed until higher cis azoTAB concentrations under UV light
(~0.2 mM) based on the data in Figure 3.3, in agreement with the aforementioned
relative critical micelle concentrations. Furthermore, by comparing Figures 3.1 and
3.3, complete dissolution of the PM bilayers in Stage III (3.0 mM trans and 12.0 mM
cis from Figure 3.1) is accompanied by complete unfolding of bR (2.0 mM trans and
8.0 mM cis from Figure 3.3), at least within the resolution of the techniques, as all of
protein molecules have partitioned into mixed micelles at these concentrations. Note
that direct comparison with the
31
P NMR data in Figure 3.2b is not possible due to the
relatively high PM concentrations required by the NMR technique.
It is noteworthy that bR partitioning from the folded state in bilayers to the
unfolded state in mixed micelles can be photoreversibly controlled. An example of
this behavior is shown in Figure 3.4, where the kinetics of light-induced changes in
membrane protein folding are examined. Figure 3.4a shows representative UV-vis
spectra of PM during the course of converting azoTAB from the trans to the cis
isomer with exposure to 365-nm UV light. The trans and cis absorption peaks of
azoTAB occur at 350 nm and 434 nm, respectively, with isosbestic points clearly

79
evident at 312 nm and 424 nm, consistent with pure azoTAB.
43; 69
In addition, protein
peaks at 280 nm (tryptophan) and 560 nm (retinal) are evident (note that the apparent
increase in A
280
is actually due to overlap with a second cis peak at 320 nm, and not a
change in protein concentration). As seen in the inset of Figure 3.4a, converting
azoTAB to the cis form results in an increase in the absorbance of bR at 560 nm,
indicating that the relative degree of folding of the protein has increased.
After correcting for scattering, the true absorbance of the protein at 560 nm is
plotted as a function of time in Figure 3.4b during the course of several visible ↔ UV
light cycles. For comparison, pure PM exhibits a scattering-corrected A
560
value of
0.3 independent of light conditions, indicating that bR is fully folded in the presence
of 0.1 mM cis azoTAB and ~80% folded in the presence of 0.1 mM trans azoTAB, as
shown in Figure 3.3. While the absorbance changes in the PM peak are fairly small
(~16% of the overall absorbance of PM) the observed changes were reproducible with
both visible ↔ UV light cycles (as seen in Figure 3.4b) and were consistently
measured over a range of surfactant concentrations (as seen in Figure 3.3). The
uncertainty in the measurements is estimated to be approximately ± 0.005 absorbance
units from the scatter in the visible- and UV-equilibrated data.
For comparison, the absorbance spectra obtained upon exposure of pure PM to
436-nm visible and 365-nm UV light is also shown in the inset of Figure 3.4a (blue
and red dotted curves, respectively) as the spectra are indistinguishable except in this

80
narrow region. The absorbance maximum of pure PM is found to change by only
0.005 upon exposure to visible and UV light, an order of magnitude lower than the
typical change seen in the presence of surfactant (see Figures 3.3 and 3.4b) and likely
within the experimental error. This suggests that bR does not undergo any dramatic
unfolding upon light exposure without the presence of surfactant. On the other hand,
the peak position of pure PM is seen to shift from a value of 571 nm under visible
light to a value of 568 nm under UV light, while a similar but opposite shift occurs
with PM in the presence of azoTAB. The all-trans retinal chromophore exhibits an
absorption maximum reported at 568 nm, while the 13-cis conformation of retinal has
a maximum at 548 nm.
70
Thus, the peak shifts observed in the inset of Figure 3.4b
could be a result of modest retinal isomerization. For example, dark-adapted PM
suspensions exhibit an absorption maximum at 558 nm, while exposure to red
(615-660 nm) or yellow (510-660 nm) light results in a shift to higher wavelengths.
70

Nevertheless, any protein reorientation resulting from this low degree of retinal
isomerization is expected to be small compared to the percentage changes in unfolded
observed in Figure 3.3 based on the aforementioned relative orders of magnitude.
Interestingly, while ~20 s of 365-nm UV illumination is sufficient to convert the
surfactant to the cis form in Figure 3.4, as expected from the relatively high
absorbance, an additional 60 s is required for bR to become fully refolded. This
suggests that the rate limiting steps for refolding are the reconstitution of PM bilayers

81
along with conformational rearrangement of the protein. Similarly, conversion of
azoTAB from the cis to the trans form by illumination with 436-nm light takes ~4 s
(not shown), while ~20 s is required to unfold bR in Figure 3.4, again indicating that
either bilayer disruption or protein unfolding are the rate limiting steps. For
comparison, rapid stopped-flow mixing of a solution of bacterioopsin (bO, i.e.,
unfolded bR with the retinal chromophore detached) solubilized in SDS micelles with
a solution of DMPC/CHAPS micelles
35
lead to protein refolding in ~70 s,
35
with
formation of a partially-folded intermediate found to be the rate limiting step.
67

Similar rapid mixing experiments have examined the progression from the
SDS-unfolded to the folded state in
L-α-1,2-Dimyristoylglycerophosphocholine/CHAPS mixed micelles (refolding
within 500 s)
71
or L-α-1,2-dipalmitoleoylphosphatidylcholine vesicles (refolding
within 2 hrs)
72
. In contrast, the data in Figure 3.4 demonstrate photoreversible control
of bR folding without the need for mixing, representing a novel method to examine
membrane protein folding.

3.4.4 FT-IR measurements of bR secondary structure in azoTAB-PM
assemblies
Infrared spectra of PM mixed with a series of azoTAB concentrations are
presented in the Figure 3.5. The pure PM spectrum is found to be independent of

82
either 436-nm visible or 365-nm UV light exposure, as expected based on the
distance from the absorbance of retinal at 560 nm. The main peak occurs at 1666 cm
-1
,
identified with an α
II
helical band in bR
27
that has been correlated with
intermolecular helical interactions,
73
a result of the formation of bR trimers in PM
bilayers.
74; 75
Additional prominent peaks at 1658 cm
-1
( α
I
+ α
II
helical band)
27; 29; 73
as
well as 1656 cm
-1
and 1650 cm
-1
( α
I
-helix)
29; 73; 76
are observed. Similar amide I
spectra are obtained at 2.0 mM azoTAB under visible and UV light, indicating that
the protein structure remains largely unchanged at this surfactant concentration. Due
to the elevated protein concentration required in the FT-IR measurements, 2.0 mM in
Figure 3.5 would be roughly equivalent to 0.05 mM azoTAB in Figure 3.3 based on
the surfactant-to-lipid molar ratio of 2:3, supporting the minimal degree of protein
unfolding observed at 2.0 mM azoTAB in Figure 3.5.

83
1600 1620 1640 1660 1680 1700 1720
0
0.1
0.2
0.3
0.4
0.5
Wavenumber (cm
-1
)
Absorbance
0 mM
2 mM
5 mM
8 mM
10 mM
15 mM
20 mM
[azoTAB]

Figure 3.5: Fourier self-deconvolution IR spectra of PM as a function of azoTAB
concentration under both visible (solid lines) and UV (dotted lines) illumination. [bR]
= 7.8 mg/mL.

Increasing the azoTAB concentration to 5.0 mM and 8.0 mM, however, results in
a reduction of the α
II
band relative to the α
I
bands, with greater reduction observed
with azoTAB in the trans (visible-light) relative to the cis (UV-light) form. The loss
of the 1666 cm
-1
peak indicates an α
II
→ α
I
transition as protein trimers dissociate
into monomers when solubilized in azoTAB-lipid mixed micelles, similar to the
PM-SDS system.
73
Hence, trans azoTAB is again seen to promote mixed-micelle

84
formation and bR solubilization (unfolding) in these mixed micelles relative the cis
isomer. Furthermore, the location of the main band at 8.0 mM azoTAB (1656 cm
-1

assigned to the α
I
band) is similar to that observed in the spectrum of bR solubilized
in SDS micelles,
73
which indicates that in the micelle-solubilized form the primary
secondary structure elements of bR are α
I
-helices.
77

Continuing to increase the azoTAB concentration from 10.0 mM to 20.0 mM,
however, appears to result in an additional unfolding process over that traditionally
seen in the presence of SDS. The 1656 cm
-1
peak disappears, while the band at ~1650
cm
-1
, also assigned to α
I
-helices,
76
becomes the dominant peak. Even more
dramatically, a new peak at 1621 cm
-1
appears that has been assigned to β-extended
strands in bR,
29
reminiscent of the behavior observed during thermal unfolding
26; 78

(note that the band at 1600 cm
-1
results from the presence of the azobenzene moiety
in azoTAB
48
). A similar α-helical to extended β-strand rearrangement was observed
upon the addition of azoTAB to the soluble protein bovine serum albumin, suspected
to result from intramolecular electrostatic repulsion as positive charges develop along
the protein backbone upon surfactant binding.
48
For the case of bR, the helical
segments are made from 15-20 amino acids
79; 80
that are mainly hydrophobic,
81

including tryptophan and tyrosine residues
74
with planar side chains that may interact
with the benzene rings of the azoTAB surfactant, possibly promoting the observed
extended conformations relative to the alkyl-based SDS surfactant. Furthermore, this

85
effect could be accompanied by a reverse turns to β-extended chains transition as the
helical bundle of bR spreads apart upon protein denaturation.
76

To examine the effects of light illumination on the secondary structure of bR in
the presence of azoTAB, difference spectra were calculated by subtracting the original
protein spectrum obtained under visible light from the spectrum under UV light at a
given surfactant concentration. Positive values in the difference spectra, thus, indicate
that the absorbance of a particular peak increases upon UV illumination, while
negative values occur when the absorbance is greater under visible-light exposure. As
seen in Figure 3.6, at low surfactant concentrations (pure PM and PM with 2.0 mM
azoTAB) the difference between the spectra measured under the two light conditions
is within the experimental noise. At increased azoTAB concentrations, however,
generally four areas in the amide I region appear to be affected by the light conditions.
A positive peak occurs at 1666 cm
-1
for 5.0 mM, 8.0 mM and 10.0 mM azoTAB,
indicating that more α
II
-helical segments are formed under UV illumination as the
degree of mixed-micelle formation is reduced and bR form trimers in the PM lipid
bilayers. This is accompanied by a second positive peak at 1686 cm
-1
assigned to
reverse-turns in bR,
29
indicating that folding of these reverse turns, which are
primarily located in extramembranous loops
80
connecting the individual helical
segments, is a necessary step in the formation of the α
II
segments. In general, the
loops connecting the individual α-helical segments
80; 82
have been found to be of

86
great importance in the folding and stabilizing of bR. Removing these loops has been
shown to increase the tendency of the protein to denature.
79; 83
Furthermore, in a study
of bR refolding from the denatured state in SDS micelles, the folding of these loops
was deemed critical for correct folding of the protein,
79
especially for the formation of
the so-called “I
2
intermediate” (i.e., a partially-folded intermediate that has native
secondary structure
67; 71
), which represents the rate limiting step in bR refolding.
Negative peaks at 1624 cm
-1
and 1629 cm
-1
, both assigned to β-strands in bR,
29

are also observed in the different spectra in Figure 3.6, particularly evident at 5.0 mM
and 8.0 mM azoTAB. Recall that extended β-strands are also observed upon thermal
denaturation of bR.
29; 76; 80; 84; 85
Thus, it appears that reverse β-turns (~1680 cm
-1
) are
converted to extended β-strands (~1624 cm
-1
) upon illumination with visible light as
the helical bundle spreads apart during the course of unfolding in azoTAB-based
micelles. A negative trend is also seen in the region of ~1645 cm
-1
, consistent with the
formation of unordered structures upon illumination with visible versus UV light.
Together, the difference spectra confirm that bR is relatively unfolded with enhanced
extended β-strands and unordered structures when solubilized in mixed micelles
predominant under visible light, while the protein exhibits a greater degree of folding
with enhanced α
II
and β-turn character as the protein partitions back into PM bilayers
upon UV illumination.

87

-0.004
-0.002
0
0.002
0.004
0.006
1600 1620 1640 1660 1680 1700 1720
1st cycle
2nd cycle
∆ Absorbance
Wavenumber (cm
-1
)
(b)
-0.004
-0.002
0
0.002
0.004
0.006
1600 1620 1640 1660 1680 1700 1720
∆ Absorbance
Wavenumber (cm
-1
)
(a)
8 mM
5 mM
10 mM
0 mM, 2 mM

Figure 3.6: (a) Difference spectra of PM in the amide I region as a function of
surfactant concentration, defined as spectra collected under UV light minus spectra
collected under visible light. (b) Reproducibility in the difference spectra at 8.0 mM
azoTAB during successive visible → UV → visible → UV light cycles.

The difference spectra further support the three-stage model of azoTAB-PM
interactions outlined above. The minimal changes seen in the difference spectra at 2.0
mM azoTAB are consistent with Stage I interactions at low surfactant concentrations,
with molecular azoTAB embedded into the lipid bilayers of PM and bR remaining in
the folded state. As the surfactant concentration is increased into Stage II interactions
at 5.0 mM, mixed-micelle formation begins with the relatively-hydrophobic trans
form of the surfactant exhibiting enhanced micellization compared to the
relatively-hydrophilic cis form. Thus, light illumination can be used to shift bR

88
partitioning between the micellar (unfolded) and lamellar (folded) states. Continuing
to increase the surfactant concentration eventually results in complete dissolution of
the PM bilayers and partitioning of bR into mixed micelles under both visible and UV
illumination, hence, the difference spectra again approach zero with both the trans
and cis forms of azoTAB exhibiting Stage III interactions with PM. Importantly, the
photocontrol of bR folding is found to be completely photoreversible (within the
resolution of the FT-IR data), as shown in the repeated visible ↔ UV light cycles in
Figure 3.6b at 8.0 mM azoTAB.

3.5 Conclusions
The ability to photoreversibly unfold the membrane protein bacteriorhodopsin
with a light-responsive surfactant has been demonstrated with steady-state and
time-resolved UV-vis spectroscopy as well as secondary-structure measurements with
FT-IR. A three-stage model of photosurfactant-lipid interactions has been established
by
31
P NMR and dynamic light scattering measurements, with the relative
hydrophobicity of the visible-light form of the surfactant inducing mixed-micelle
formation and lipid dissolution at lower photosurfactant concentrations compared to
the UV-light form. This leads to a greater degree of partitioning of the membrane
protein into the micelle (unfolded) state under visible versus UV light, allowing
photoreversible control of membrane protein folding. The kinetics of the

89
light-induced unfolding and refolding are found to be 20 s and 80 s, respectively,
similar to values obtained from previous rapid-mixing experiments in the literature.
Secondary-structure measurements reveal that protein partitioning from lipid bilayers
into micellar structures results in unfolding through α
II
to α
I
and reverse β-turn to
extended β-strand transitions. In previous studies, either irreversible unfolding or the
requirement of dialysis to slowly remove the denaturant to allow refolding limits
folding studies of membrane protein. In contrast, in the present study a novel
method to initiate rapid, photoreversible changes membrane protein folding is
achieved through the use of photoresponsive surfactants.

3.6 Acknowledgements
This material is based upon work supported by the National Science Foundation
under Grant No. 0554115. We thank Professor Dieter Oesterhelt of the Max Plank
Institute of Biochemistry in Martinsried for supplying the Halobacterium salinarium.

90
3.7 References
1. Opella, S. J., Nevzorov, A., Mesleh, M. F. & Marassi, F. M. (2002). Structure
determination of membrane proteins by NMR spectroscopy. Biochemistry and
Cell Biology 80, 597-604.

2. Drew, D., Froderberg, L., Baars, L. & De Gier, J.-W. L. (2003). Assembly and
overexpression of membrane proteins in Escherichia coli. Biochimica et
Biophysica Acta 1610, 3-10.

3. Kiefer, H. (2003). In vitro folding of alpha-helical membrane proteins.
Biochimica et Biophysica Acta 1610, 57-62.

4. Braiman, M. S., Stern, L. J., Chao, B. H. & Khorana, H. G. (1987).
Structure-function studies on bacteriorhodopsin. IV. Purification and
renaturation of bacterio-opsin polypeptide expressed in Escherichia coli.
Journal of Biological Chemistry 262, 9271-6.

5. Yerushalmi, H., Lebendiker, M. & Schuldiner, S. (1995). EmrE, an
Escherichia coli 12-kDa multidrug transporter, exchanges toxic cations and
H+ and is soluble in organic solvents. Journal of Biological Chemistry 270,
6856-63.

6. Qi, H. L., Tai, J. Y. & Blake, M. S. (1994). Expression of large amounts of
neisserial porin proteins in Escherichia coli and refolding of the proteins into
native trimers. Infection and immunity 62, 2432-9.

7. Surrey, T. & Jaehnig, F. (1995). Kinetics of folding and membrane insertion of
a b-barrel membrane protein. Journal of Biological Chemistry 270,
28199-203.

8. Dekker, N., Merck, K., Tommassen, J. & Verheij, H. M. (1995). In vitro
folding of Escherichia coli outer-membrane phospholipase A. European
Journal of Biochemistry 232, 214-19.

9. Chen, G.-Q. & Gouaux, J. E. (1996). Overexpression of bacterio-opsin in
Escherichia coli as a water-soluble fusion to maltose binding protein: efficient
regeneration of the fusion protein and selective cleavage with trypsin. Protein
Science 5, 455-67.

91
10. Buchanan, S. K. (1999). Overexpression and refolding of an 80-kDa iron
transporter from the outer membrane of Escherichia coli. Biochemical Society
Transactions 27, 903-908.

11. Huang, K.-S., Bayley, H., Liao, M.-J., London, E. & Khorana, H. G. (1981).
Refolding of an integral membrane protein. Denaturation, renaturation, and
reconstitution of intact bacteriorhodopsin and two proteolytic fragments.
Journal of Biological Chemistry 256, 3802-9.

12. Paulsen, H., Finkenzeller, B. & Kuehlein, N. (1993). Pigments induce folding
of light-harvesting chlorophyll a/b-binding protein. European Journal of
Biochemistry 215, 809-16.

13. Riley, M. L., Wallace, B. A., Flitsch, S. L. & Booth, P. J. (1997). Slow a Helix
Formation during Folding of a Membrane Protein. Biochemistry 36, 192-196.

14. Lau, F. W. & Bowie, J. U. (1997). A Method for Assessing the Stability of a
Membrane Protein. Biochemistry 36, 5884-5892.

15. Minetti, C. A. S., Tai, J. Y ., Blake, M. S., Pullen, J. K., Liang, S.-M. & Remeta,
D. P. (1997). Structural and functional characterization of a recombinant PorB
class 2 protein from Neisseria meningitidis. Conformational stability and
porin activity. Journal of Biological Chemistry 272, 10710-10720.

16. Prive, G. G. & Kaback, H. R. (1996). Engineering the lac permease for
purification and crystallization. Journal of Bioenergetics and Biomembranes
28, 29-34.

17. Ferguson, A. D., Breed, J., Diederichs, K., Welte, W. & Coulton, J. W. (1998).
An internal affinity-tag for purification and crystallization of the siderophore
receptor FhuA, integral outer membrane protein from Escherichia coli K-12.
Protein Science 7, 1636-1638.

18. Mohanty, A. K., Simmons, C. R. & Wiener, M. C. (2003). Inhibition of
tobacco etch virus protease activity by detergents. Protein Expression and
Purification 27, 109-114.

19. Loll, P. J. (2003). Membrane protein structural biology: the high throughput
challenge. Journal of Structural Biology 142, 144-153.

92
20. Booth, P. J., Templer, R. H., Meijberg, W., Allen, S. J., Curran, A. R. & Lorch,
M. (2001). In vitro studies of membrane protein folding. Critical Reviews in
Biochemistry and Molecular Biology 36, 501-603.

21. Happe, M. & Overath, P. (1976). Bacteriorhodopsin depleted of purple
membrane lipids. Biochem. Biophys. Res. Commun. 72, 1504-1511.

22. Oesterhelt, D. & Stoeckenius, W. (1974). Isolation of Cell Membrane of
Halobacterium Halobium and Its Fractionation into Red and Purple
Membrane. Methods Enzymol. 31, 667-678.

23. Hwang, S.-B. & Stoeckenius, W. (1977). Purple Membrane Vesicles:
Morphology and Proton Translocation. J. Membrane Biol. 33, 325-350.

24. Luecke, H., Schobert, B., Richter, H.-T., Cartailler, J.-P. & Lanyi, J. K. (1999).
Structure of Bacteriorhodopsin at 1.55 A Resolution. J, Mol. Biol. 291,
899-911.

25. Kuhlbrandt, W. (2000). Bacteriorhodopsin-the movie. Nature 406, 569-570.

26. Clandera, J., Galisteo, M. L., Sabes, M., Mateo, P. L. & Padros, E. (1992). The
role of retinal in the thermal stability of the purple membrane. Eur. J. Biochem.
207, 581-585.

27. Krimm, S. & Dwivedi, A. M. (1982). Infrared Spectrum of the Purple
Membrane: Clue to a Proton Conduction Mechanism? Science, New Series
216, 407-408.

28. Huang, K.-S. & Khorana, G. H. (1980). Delipidation of bacteriorhodopsin and
reconstitution with exogenous phospholipid. Biochemistry 77, 323-327.

29. Cladera, J., Sabes, M. & Padros, E. (1992). Fourier Transform Infrared
Analysis of Bacteriorhodopsin Secondary Structure. Biochemistry 31,
12363-12368.

30. Parodi, L. A. & Lozier, R. H. (1982). Procedure for Testing Kinetic Models of
the Photocycle of Bacteriorhodopsin. Biophys. J . 38, 161-174.

93
31. Marti, T., Otto, H., Rosselet, S. J., Heyn, M. P. & Gobind, K. H. (1992).
Consequences of Amino Acid Insertiions and/or Deletions in Transmembrane
Helix C of Bacteriorhodopsin. Proc. Natl. Acad. Sci. U.S.A. 89, 1219-1223.

32. Braiman, M., Bousche, O. & KJ, P. (1991). Protein Dynamics in the
Bacteriorhodopsin Photocycle: Submillisecond Fourier Transform Infrared
Spectra of the L, M, and N photointermediates. Proc. Natl. Acad. Sci. U.S.A.
88, 2388-2392.

33. Booth, P. J., Riley, L. M., Flitsch, S. L., Templer, R. H., Farooq, A., Curran, R.
A., Chadborn, N. & Wright, P. (1997). Evidence That Bilayer Bending
Rigidity Affects Membrane Protein Folding. Biochemistry 36, 197-203.

34. Chen, G. Q. & Gouaux, E. (1999). Probing the Folding and Unfolding of
Wild-Type and Mutant Forms of Bacteriorhodopsin in Micellar Solutions:
Evaluation of Reversible unfolding Conditions. Biochemistry 38,
15380-15387.

35. Booth, P. J., Stern, L. J., Greenhalgh, D. A., Kim, P. S. & Khorana, G. H.
(1995). Intermediates in the folding of the membrane protein
bacteriorhodopsin. Nat. Struct. Biol. 2, 139-143.

36. Mukhopadhyay, A. K., Dracheva, S., Bose, S. & Hendler, R. W. (1996).
Control of the Integral Membrane Proton Pump, Bacteriorhodopsin, by Purple
Membrane Lipids of Halobacterium halobium. Biochemistry 35, 9245-9252.

37. Joshi, M. K., Dracheva, S., Mukhopadhyay, A. K., Bose, S. & Hendler, R. W.
(1998). Importance of Specific Native Lipids in Controlling the Photocycle of
Bacteriorhodopsin. Biochemistry 37, 14463-14470.

38. Popot, J.-L., Trewhella, J. & Engelman, D. M. (1986). Reformation of
Crystalline Purple Membrane from Purified Bacteriorhodopsin Fragments.
EMBO J. 5, 3039-3044.

39. Hamill, A., Wang, S.-C. & Lee, J., C. Ted. (2005). Probing Lysozyme
Conformation with Light Reveals a New Folding Intermediate. Biochemistry
44, 15139-15149.

40. Wang, S.-C. & Lee, J., C. Ted. (2007). Enhanced Enzymatic Activity Through
Photoreversible Conformational Changes. Biochemistry submitted.

94
41. Hamill, A. & Lee, J., C. Ted. (2007). Solution Structure of an
Amyloid-Forming Protein During Photoinitiated Hexamer-Dodecamer
Transitions Revealed through Small-Angle Neutron Scattering. Biochemistry
46, 7694.

42. Hayashita, T., Kurosawa, T., Miyata, T., Tanaka, K. & Igawa, M. (1994).
Effect of structural variation within cationic azo-surfactant upon
photoresponsive function in aqueous solution. Colloid. Polym. Sci. 272,
1611-1619.

43. Lee, C. T., Jr., Smith, K. A. & Hatton, T. A. (2004). Photoreversible Viscosity
Changes and Gelation in Mixtures of Hydrophobically Modified
Polyelectrolytes and Photosensitive Surfactants. Macromolecules 37,
5397-5405.

44. Shang, T., Smith, K. A. & Hatton, T. A. (2003). Photoresponsive Surfactants
Exhibiting Unusually Large, Reversible Surface Tension Changes under
Varying Illumination Conditions. Langmuir 26, 10764-10773.

45. Holmberg, K., Jonsson, B., Kronberg, B. & Lindman, B. (2002). Surfactants
and Polymers in Aqueous Solution (edition, n., Ed.), Wiley.

46. Booth, P. J., Farooq, A. & Flitsch, S. L. (1996). Retinal Binding during
Folding and Assembly of the Membrane Protein Bacteriothodopsin.
Biochemistry 35, 5902-5909.

47. Gordeliy, V. I., Schlesinger, R., Efremov, R., Buldt, G. & Heberle, J. (2003).
Crystallization in lipidic cubic phases: a case study with bacteriorhodopsin.
Methods Mol Biol 228, 305-316.

48. Wang, S.-C. & Lee, C. T., Jr. (2006). Protein Secondary Structure Controlled
with Light and Photoresponsive Surfactants. J. Phys. Chem. B 110,
16117-16123.

49. Almgren, M. (2000). Mixed micelles and other structures in the solubilization
of bilayer lipid membranes by surfactants. Biochim. Biophys. Acta 1508,
146-163.

95
50. Lichtenberg, D., Opatowski, E. & Kozlov, M. M. (2000). Phase boundaries in
mixtures of membrane-forming amphiphiles and micelle-forming amphiphiles.
Biochim. Biophys. Acta 1508, 1-19.

51. Sportelli, L. & Bartucci, R. (1985). Electrostatic interaction on purple
membrane: a spin label study on pH and ionic-strength effects. Nuovo
Cimento Soc. Ital. Fis., D 6, 609-617.

52. He, J.-A., Samuelson, L., Li, L., Kumar, J. & Tripathy, S. K. (1998). Oriented
Bacteriorhodopsin/Polycation Multilayers by Electrostatic Layer-by-Layer
Assembly. Langmuir 14, 1674-1679.

53. Michel, H., Oesterhelt, D. & Henderson, R. (1980). Orthorhombic
two-dimensional crystal form of purple membrane. Proc. Natl. Acad. Sci.
U.S.A. 77, 338-342.

54. Cabarcos, E. L., Bermudez, S. F. & Balta, C. (1981). The paracrystalline
nature of the purple membrane of halobacterium halobium II. Packing of the
membrane. Colloid. Polym. Sci. 259, 641-644.

55. Kubota, K., Tominaga, Y., Fujime, S., Jun, O. & Ikegami, A. (1985). Dynamic
Light Scattering Study of Suspensions of Purple Membrane. Biophys. Chem.
23, 15-29.

56. Claire, K. & Pecora, R. (1997). Translational and Rotational Dynamics of
Collagen in Dilute Solution. J. Phys. Chem. B 101, 746-753.

57. Nieh, M.-P., Glinka, C. J. & Krueger, S. (2001). SANS Study of the Structural
Phases of Magnetically Alignable Lanthanide-Doped Phospholipid Mixtures.
Langmuir 17, 2629-2638.

58. Cullis, P. R. & Kruijff, B. D. (1979). Lipid Polymorphism and the Functional
Roles of Lipids in Biological Membranes. Biochim. Biophys. Acta 559,
399-420.

59. Valpuesta, J. M., Arrondo, J.-L. R., Barbero, M. C., Pons, M. & Goni, F. M.
(1986). Membrane-Surfactant Interaction. J. Biol. Chem. 261, 6578-6584.

96
60. Beyer, K. (1997). Packing and Bilayer-Micelle Transitions in Mixed
Surfactant-Lipid Systems as Studied by Solid State NMR. Progr Colloid Poly
Sci 103, 78-86.

61. London, E. & Feigenson, G. W. (1979). Phosphorus NMR Analysis of
Phospholipids in Detergents. J. Lipid Res. 20, 408-412.

62. Renthal, R. & Cha, C.-H. (1984). Charge Asymmetry of The Purple
Membrane Measured by Uranyl Quenching of Dansyl Fluorescence. Biophys.
J . 45, 1001-1006.

63. Stoeckenius, W., Lozier, R. H. & Bogomolni, R. A. (1979). Bacteriorhodopsin
and the Purple Membrane of Halobacteria. Biochim. Biophys. Acta 505,
215-278.

64. Kouyama, T. & Nasuda-Kouyama, A. (1989). Turnover Rate of the Proton
Pumping Cycle of Bacteriorhodopsin: pH and Light-Intensity Dependences.
Biochemistry 28, 5968-5970.

65. Robertson, B. & Lukashev, E. P. (1995). Rapid pH Change due to
Bacteriorhodopsin Measured with a Tin-Oxide Electrode. Biophys. J . 68,
1507-1517.

66. London, E. & Khorana, H. G. (1982). Denaturation and Renaturation of
Bacteriorhodopsin in Detergents and Lipid-Detergent Mixtures. J. Biol. Chem.
257, 7003-7011.

67. Riley, M. L., Wallace, B. A., Flitsch, S. L. & Booth, P. J. (1997). Slow Alpha
Helix Formation during Folding of a Membrane Protein. Biochemistry 36,
192-196.

68. Booth, P. J., Templer, R. H., Meijberg, W., Allen, S. J., Curran, A. R. & Lorch,
M. (2001). In Vitro Studies of Membrane Protein Folding. Crit. Rev. Biochem.
Mol. Biol. 36, 501-603.

69. Le Ny, A.-L. M. & Lee, C. T., Jr. (2006). Photoreversible DNA Condensation
Using Light-Responsive Surfactants. J. Amer. Chem. Soc. 128, 6400-6408.

97
70. Kouyama, T., Bogomolni, R. A. & Stoeckenius, W. (1985). Photoconversion
from the light-adapted to the dark-adapted state of bacteriorhodopsin. Biophys.
J. 48, 201-8.

71. Booth, P. J. & Farooq, A. (1997). Intermediates in the Assembly of
Bacteriorhodopsin Investigated by Time-resolved Absorption Spectroscopy.
Eur. J. Biochem. 246, 674-680.

72. Allen, S. J., Curran, A. R., Templer, R. H., Meijberg, W. & Booth, P. J. (2004).
Folding Kinetics of an Alpha Helical Membrane Protein in Phospholipid
Bilayer Vesicles. J. Mol. Biol. 342, 1279-1291.

73. Torres, J., Sepulcre, F. & Padros, E. (1995). Conformational Changes in
Bacteriorhodopsin Associated with Protein-Protein Interactions: A Functional
Alpha
I
-Alpha
II
Helix Switch? Biochemistry 34, 16320-16326.

74. Grigorieff, N., Ceska, T. A., Downing, K. H., Baldwin, J. M. & Henderson, R.
(1996). Electron-crystallographic Refinement of the Structure of
Bacteriorhodopsin. J. Mol. Biol. 259, 393-421.

75. Mitsuoka, K., Kazuyoshi Murata, T. H., Miyazawa, A., Kidera, A., Kimura, Y .
& Fujiyoshi, Y. (1999). The Structure of Bacteriorhodopsin at 3.0 A
Resolution Based on Electron Crystallography: Implication of the Charge
Distribution. J. Mol. Biol. 286, 861-882.

76. Wang, J. & El-Sayed, M. A. (1999). Temperature Jump-Induced Secondary
Structural Change of the Membrane Protein Bacteriorhodopsin in the
Premelting Temperature Region: A Nanosecond Time-Resolved Fourier
Transform Infrared Study. Biophys. J . 76, 2777-2783.

77. Sonoyama, M., Hasegawa, T., Nakano, T. & Mitaku, S. (2004). Isomerization
of retinal chromophore and conformational changes in membrane protein
bacteriorhodopsin by solubilization with a mild non-ionic detergent,
n-octyl-beta-glucoside: an FT-Raman and FT-IR spectroscopic study. Vib.
Spectrosc 35, 115-119.

78. Heyes, C. D. & El-Sayed, M. A. (2002). The Role of the Native lipids and
Lattice Structure in Bacteriorhodopsin Protein Conformation and Stability as
Studied by Temperature-dependent Fourier Transform-Infrared Spectroscopy.
J. Biol. Chem. 277, 29437-29443.

98
79. Kim, J.-M., Booth, P. J., Allen, S. J. & Khorana, H. G. (2001). Structure and
Function in Bacteriorhodopsin: The Role of the Interhelical Loops in the
Folding and Stability of Bacteriorhodopsin. J. Mol. Biol. 308, 409-422.

80. Lazarova, T. & Padros, E. (1996). Helical and Reverse Turn Changes in the
BR to N Transition of Bacteriorhodopsin. Biochemistry 35, 8354-8358.

81. Popot, J.-L. & Engelman, D. M. (2000). Helical Membrane Protein Folding,
Stability, and Evolution. Annu. Rev. Biochem 69, 881-922.

82. Paul, C. & Rosenbusch, J. P. (1985). Folding Patterns of Porin and
Bacteriorhodopsin. EMBO J. 4, 1593-1597.

83. Allen, S. J., Kim, J.-M., Khorana, H. G., Lu, H. & Booth, P. J. (2001).
Structure and Function in Bacteriorhodopsin: The Effect of the Interhelical
Loops on the Protein Folding Kinetics. J. Mol. Biol. 308, 423-435.

84. Wang, J., Heyes, C. D. & El-Sayed, M. A. (2002). Refolding of Thermally
Denatured Bacteriorhodopsin in Purple Membrane. J. Phys. Chem. B 106,
723-729.

85. Wang, J. & EL-Sayed, M. A. (2000). The Effect of Protein Conformation
Change from Alpha
II
to Alpha
I
on the Bacteriorhodopsin Photocycle. Biophys.
J . 78, 2031-2036.

99
Chapter 4: Light Effect on Self-Assembly of Aqueous
Mixture of Sodium Dodecyl benzenesulfonate (SDBS) and
4-ethyl-4’(trimethylaminobutoxy) azobenzene Bromide
(azoTAB) by Using DLS and SANS
Jing Zhang and C. Ted Lee Jr, Manuscript

4.1 Abstract
Aqueous “catanionic” mixtures of a cationic photoresponsive
azobenzenetrimethylammonium bromide (azoTAB) surfactant and a traditional
anionic surfactant such as sodium dodecylbenzenesulfonate (SDBS) have been
examined through combined fluorescence, UV-vis, dynamic light scattering,
small-angle neutron scattering and polarized optical microscopy measurements. Due
to the strong electrostatic and hydrophobic interactions between the
oppositely-charged surfactants, catanionic vesicles form spontaneously without the
need of an external energy source. Furthermore, a photoisomerization within the
azoTAB molecule that occurs in response to 434-nm visible (trans form) or 350-nm
UV (cis form) light, combined with the relative hydrophobicity of the trans isomer,
results in the formation of novel light-responsive vesicles that can be disrupted and
reformed with simple light illumination. Critical aggregation concentrations (CACs),
namely the surfactant concentrations where vesicle first appear, are determined using

100
the hydrophobic fluorescence probe Nile red to report on bilayer formation. The
complete ternary phase diagram was constructed using DLS and SANS measurements
as well as and polarized optical microscopy, while transitions between micelles,
vesicles and lamellar structures were observed with both visible and UV light
illumination. In most case, vesicles were observed under visible light while smaller
size vesicles were observed with UV illumination because of the different
conformations of the photoresponsive surfactants. With UV illumination, surfactant
was cis conformation which was more bent and hydrophilic while with visible
illumination, trans conformation was more planar and hydrophobic. The
photo-induced micelle ↔ vesicles and vesicles ↔ lamellar transitions could be of
interested for membrane protein folding studies as well as gene delivery applications.

4.2 Introduction
The concept of a catanionic mixture could be explained as the mixture of the
cationic and anionic surfactants. Aqueous “catanionic” systems have received
considerable attention over the last several years,
1; 2; 3; 4; 5
having been studied by
techniques such as dynamic light scattering (DLS)
4
and small-angle neutron
scattering (SANS).
6; 7
Unlike pure surfactant solutions that generally form micelles
with critical micelle concentrations (CMCs) typically in the millimolar range,
catanionic mixtures can form a rich variety of microstructures, including micelles,

101
vesicles, and lamellar phases at critical aggregation concentrations (CACs) in the
micromolar regime , a result of the strong electrostatic and hydrophobic interactions
between the oppositely-charged surfactants. Furthermore, the type of microstructure
formed is generally dependent on the surfactant properties, the cation-anion ratio,
temperature, and other environmental factors.
8
Catanionic vesicles are of particular
interest due to potential applications as mimics for biological membranes, drug
delivery, and microreactors.
9
This utility is further augmented by the fact that, unlike
traditional lipid-based vesicles, catanionic vesicles form spontaneously without the
need of mechanical shear since they reside at the true free energy minimum (hence,
often referred to as “equilibrium vesicles”).
1; 2; 3; 5

In last ten years, a new catanionic surfactant mixtures had been studied which
consisted photoresponsive surfactants, such as
4-Butylazobenzene-4’-(oxyethyl)trimethylammonium bromide (AZTMA)
10
and
bis(trimethylammoniumhexyloxy)azobenzene dibromide (BTHA)
11
. In a case of the
azobenzene-based surfactant, surfactant forms two isomers under the different light
conditions. Surfactant was at the trans form in the solution in the dark or under the
room light, and formed cis form when surfactant was illuminated with the UV light.
For trans conformation, the maximum absorption was at 350 nm with the dipole
moment across the N=N bond of ~ 0.5D under the visible light, but when surfactant

102
was illuminated under the UV light, the maximum absorption was changed to 434 nm
with the dipole moment to ~ 3.1D, as shown in Figure 4.1.
12; 13; 14; 15; 16

Figure 4.1: AzoTAB chemical structure and isomerization

In the catanionic surfactant system with photoresponsive surfactant, similar to the
pure azoTAB, surfactant mixture could form different structures under the
illumination of the lights (visible light and UV light). Taking advantage of the
different hydrophobicity of the azoTAB, when interacted with SDBS, different
aggregations could be formed with light illumination. For example, for the mixture of
AZTMA and SDBS , when total surfactant concentration at 0.05 wt% and
AZTMA/SDBS=6/4, under freeze replica TEM micrographs, vesicles were observed
under visible light, while a large elongated molecular aggregates were investigated
with UV exposure.
10
Same phenomena happened in BTHA and SDS mixture.
11
The
great advantage of the catanionic surfactant mixture containing photoresponsive
µ=0.5D µ=0.5D µ=0.5D
N O(CH
2
)
2
N+(CH
3
)
3
Br-
N
CH
3
CH
2
CH
3
CH
2
N
N O(CH
2
)
2
N+(CH
3
)
3
Br-
UV light
Visible light
µ = 0.5 D
µ = 3.1 D

103
surfactant was that the vesicle formation control was reversible by light, which is the
vesicle could be formed and disrupted reversibly with the different light illumination.
Similar to other catanionic systems, the physical properties of the system could be
studied by DLS,
4; 17
SANS,
6; 7
fluorescence spectroscopy,
18; 19
electron microscopy
ect.
20

To study the transitions between different aggregates with light illumination, such
as micelle ↔ vesicles and vesicles ↔ lamellar structure and free surfactant molecule
↔ vesicles of great important in protein reconstitution study and gene transfection.
For example, it is very different to control the folding and unfolding of
bacteriorodopsin (the only protein found in purple membrane) in general because of
the properties of the protein and purple membrane sheets. In native state,
bacteriorodopsin is embedded into the purple membrane sheets, protein was readily
to aggregate because of the large percentage of the protein and paracrystalline
structure.
21
With the photoresponsive catanionic surfactant mixture, it is possible to
reversibly control purple membrane folding by light if the thickness of the bilayer is
similar to the natural purple membrane bilayers. Specifically, due to different
microstructures in visible and UV lights, it is possible to fulfill protein refolding in
one light and protein unfolding with another light illumination depending on mixture
formation. Another application is to increase the gene transfection by light
illumination.
22
In our previous study, catanionic surfactant mixture containing

104
azobenzen-based surfactant and SDBS is used to increase gene transfection.
22
By
using the azobenzen-based surfactant and SDBS catanionic surfactant mixtures, the
gene transfection is increased by increasing the rate to approach cell membrane and
the rate of endosome-lysis.
22

In the present study, the catanionic surfactant system is made of the
azobenzenetrimethylammonium bromide (azoTAB) and sodium
dodecylbenzenesulfonate (SDBS), which was different to other catanionic surfactant
system because of the isomerization of the azoTAB surfactant. Because of the two
isomers of azoTAB and different hydrophobicity of azoTAB with light illumination,
by taking advantage of this unique propertie of azoTAB, the vesicle formation and
disruption can be initiated with simple light illumination. Under visible light the
planar, relatively-hydrophobic trans configuration of the surfactant will more readily
form ordered, bilayer structures compared to the bent, relatively-hydrophilic cis
isomer under UV exposure. DLS and SANS are used to investigate the various
microstructures formed in the aqueous solutions. From DLS and SANS, it is possible
to obtain the quantitative information of the dimensions of the surfactant aggregations
and the size distributions of the microstructures. Fluorescence spectroscopy was used
to determine critical aggregation concentration (CAC) with Nile red. Polarized optical
microscopy was applied to determine the microstructures of catanionic surfactant
systems.

105
4.3 Materials and Methods
4.3.1 Materials
The surfactant 4-Ethyl-4’(trimethylaminobutoxy) azobenzene bromide (azoTAB)
was made as described
23; 24
. Generally, the azobenzene surfactant was prepared by
azocoupling of alkylaniline with phenol, followed by alkylation and quaternalization
with dibromoalkane and trimethylamine. In order to get the high quality product,
recystallization was applied to the final step at which azoTAB was synthesized. By
measurement of
1
H-NMR in D
2
O, the recystallization might be applied more than
once depending on the purity of the azoTAB. Besides of the
1
H-NMR in D
2
O to
determine the purity, the conductivity was measured to remove all the leftover salts.
All chemicals were purchased from Sigma-Aldrich and used as received unless
otherwise mentioned.

4.3.2 Critical aggregation concentration (CAC) measurement
Fluorescence measurements using Nile red as the probe were performed on a
Quanta-Master spectrofluorometer model QM-4 (Photon Technology International) at
25℃. The results were obtained with an excitation wavelength of 560 nm and an
emission wavelength of 650 nm, with excitation and emission slit width of 4 nm. The
spectrofluorometer was loaded with 2 mL of Nile red solution (0.1 µM) with
separately adding 10 mM azoTAB and SDBS to reach the given azoTAB

106
concentration (stating from 0 µM, increments 4 µM) at the fixed azoTAB and SDBS
ratios. To avoid the photo bleach of the Nile red, only azoTAB and SDBS were
exposed under an 84 W long wave UV lamp-365 nm (Spectroline, model no. XX-15A)
to convert to cis form and added to the solution with 0.1 µM nile red in dark. After
measurements, the samples were measured by UV-Vis spectroscopy to confirm
azoTAB was kept under cis state.

4.3.3 UV-Vis spectroscopy
Stock solutions of azoTAB and SDBS surfactant at the given concentrations were
prepared separately and mixed as the destination ratios. After 1 min Votex, samples
were measured and recorded by UV-vis spectroscopy in dark and room light. Then the
same samples were exposed to an 84 W long wave UV lamp-365 nm (Spectroline,
Model no. XX-15A) for at least 2 hrs to convert the surfactant transition from trans
state to cis state. After illumination, samples were measured and recorded by UV-vis
spectroscopy again. The concentration of the azoTAB in each sample is 0.1 mM, and
SDBS concentration was changed according to the given ratios to azoTAB.

4.3.4 Dynamics detection of the formation and disruption of the vesicle
AzoTAB and SDBS mixture at the appropriate ratio was pre-prepared in DI water
at the given concentration. 2 mL prepared sample was loaded and (0.1 µM

107
concentration of Nile red) was added. The samples with gentle stirring were
performed on a Quanta-Master spectrofluorometer model QM-4 (Photon Technology
International) at 25 ℃. The results were obtained with an excitation wavelength of
560 nm and an emission wavelength of 650 nm, with excitation and emission slit
width of 4 nm with time step 1 S. Samples were operated with the illumination of a
200 W mercury arc lamp (Oriel, model no. 6283) equipped with a 400-nm long pass
filter (Oriel, model no. 59472) or 320-nm UV filter (Oriel model no. 59980) and the
heat absorbing filter (Oriel, model no. 59060) and a fiber-bundle focusing assembly
(Oriel, model no. 77557) at 25 ℃ to achieve the illumination with visible light and
UV light. Photoreversibility was assured through multiple visible-UV light cycles.
With the light illumination during the experiment, the nile red had little photobleach
but it was negligible if compared to the light effects on the azoTAB surfactants.

4.3.5 Dynamics light scattering
Dynamics light scattering measurements were operated at 25 ℃ on a Brookhaven
Model BI-200SM instrument (Brookhaven Instrument Corp.) equipped with a 35 mW
(Melles Griot, model no. 05-LHP-928) HeNe (632.8 nm) laser and an avalanche
phorodiode detector (BI-APD).
AzoTAB and SDBS were separately prepared at the given concentrations and
mixed at the appropriate ratios through 200 nm syringe filters (Anatop) separately.

108
After at least 1 min votex and 1 hr set down, samples were measured at an angle of 90
degree. The conversion of the surfactant to cis isomer was achieved by UV
illumination with an 84 W long wave UV lamp-365 nm (Spectroline, Model no.
XX-15A). The data were analyzed with the NNLS software package using
BI-9000AT digital correlator (Brookhaven Instrument Corp.).

4.3.6 Small-angle neutron scattering (SANS)
AzoTAB and SDBS were separately prepared at the given concentrations and
mixed at the appropriate ratios. Small-angle neutron scattering experiments were
performed on the 30 m NG7 SANS instruments at NIST.
25
A neutron wavelength of
λ= 6 Å and a detector offset of 25 cm with two sample-detector distances of 1.33 and
7.0 m were utilized to achieve a Q range of 0.0071-0.45 Å for 0.1wt% total surfactant
concentration and 0.0048-0.4 Å for other surfactant concentrations. The net intensities
were corrected for the background and empty cell (pure D
2
O), accounting for the
detector efficiency using the scattering from an isotropic scatterer (Plexiglas), and
converted to an absolute differential cross section per unit sample volume (in units of
cm
-1
) using an attenuated empty beam. The data were then corrected for incoherent
scattering by subtracting a constant background. The identical samples were
converted to cis isomer which were achieved under the illumination of an 84 W long
wave UV lamp-365 nm (Spectroline, Model no. XX-15A) for at least 2 hrs and UV

109
lamp was used during the data collection process. The data were reduced, analyzed
and modeled by Igor Pro (WaveMetics) program.
26

4.3.7 SANS analysis
In Igor program, for vesicle modeling, poly core shell model in which vesicles
have polydisperse radius and constant shell thickness is applied.
11; 27; 28; 29; 30
In this
case, the form factor for polydispersed spherical particles with a core-shell structure,
SANS data can be modeled with 3 parameters: average vesicle size, shell thickness,
and polydispersity of the radius while other parameters have been fixed depending on
the properties of the two surfactant components.
11; 28
The scattering intensity, I(q), is
related to the differential scattering cross section, described as below,

()
() () ( )
dq
IqRq dq
d
=
Ω
∑
∫
(1)

2
0
()
() ( ) c c
dq
n G r P qr dr
d
∞
=
Ω
∑
∫
c (2)

1
1
( ) ( ) exp( ( 1))
(1)
Z Z
c
c
cc
rZ r
Gr Z
Z rr
+
c + −
=
Γ+
+ (3)

2
2
1
1 c Z r
σ
=
+
(4)

3
4
() ( ){[sin ( ) sin ] [( )cos( ) cos ] cvs c c c c c P qr q r t qr q r t q r t qr qr
q
}c
π
ρρ = − +− − + +− (5)

110
In these functions, R(q) is the resolution function, a form factor, P(q) and a structure
factor, S(q) are considered and in this model, the solution is considered diluted
enough to set S(q)=1; n is the number density of vesicles, r
c
is the radius of the core,
G(r
c
) is the distribution of the core radii, and P
2
(qr
c
) is the form factor for a single
vesicle; Γ is the gamma fuction, c r is the mean core radius, and Z is related to the
variance of the core radii ( σ
2
) and the form factor of a single faction is described as
function (5). With these considerations, the modeling date is in good agreement with
the experiment date.
For micelles structure modeling, micelles structure is to calculate the form factor
for a monodisperse ellipsoid with uniform scattering length density coupled with
structure factor.
11; 28; 31; 32
The intensity model for monodisperse ellipsoid molecules
with interacting with other molecules is described as below,

2
2
1
2
0
(, )
() | ( , )| (1 ( () 1))
() | ( , )|
d
Fq
Iq n F q d S q
dq Fq
µ
µµ
µ
<>
== + −
Ω<
∑
∫
>
(6)

where

1 3( )
(, ) ( ) ms
ju
Fq v
u
µρ ρ =− (7)
and

22 2 2 0.5
[(1 uqa b )] µ µ = +− (8)
In these functions, a is the semimajor axis of the micelle, b is the semiminor axis, v is
the volume of the micelles, ρ
m
and ρ
s
are the coherent scattering length densities of
the micelle and the solvent, respectively and j
1
(u) is the first order spherical Bessel

111
function. For micelles modeling, if b > a, the micelles are oblate ellipsoid, otherwise,
the micelles are prolate ellipsoids. In our study, the vesicle and micelles mixtures are
modeled by the summation of the vesicle model and micelle model, starting at 1:1
ratio with variables of the volume factions of two microstructures.

4.3.8 Polarized optical microscopy
AzoTAB and SDBS stock solution at different concentrations were separately
prepared and mixed at the designed ratios. After 1 min votex, the samples were
observed with an Olympus IX71 inverted microscope equipped with a 40 × objective
lens (SLCplanFl), and were recorded with a CCD digital camera (Hamamatsu, model
no. C4742-95). At each surfactant concentration, the same solution was used to obtain
images under both visible and UV light with the samples exposed to UV light for at
least 2 hrs to convert the surfactant to the cis form and shined with the illumination of
a 200 W mercury arc lamp and a fiber-bundle focusing assembly to keep the azoTAB
at the Cis state.

4.4 Results and Discussion
4.4.1 Critical aggregation concentration (CAC) measurement
In previous study, the vesicles formation of the mixture of cationic and anionic
surfactants is spontaneous and stable, which meant they were thermodynamically

112
stable.
33

34
In a catanionic surfactant mixture, different microstructures were formed
based on total surfactant concentrations and the ratios between the two components,
35
vesicle is one of the most important. The formation of the vesicle is of particular
interest of the protein reconstitution
9
and gene delivery.
22
In our study, in order to
study the onset point of vesicle formation, critical aggregation concentrations, the
concentration vesicles formed, were measured.
The formation of the vesicles in catanionic surfactant is based on numerous
factors, such as the steric interaction of the oppositely charged head group, the
hydrophobicity interaction of the hydrocarbon chains, the ratios of the two
components and the total surfactant concentrations. In order to study the formation of
the vesicle, fluorescence probe Nile red is used.
22
Nile red is a nonionic, hydrophobic
probe molecule with a fluorescence emission that can be strongly influenced by the
local polarity of the solubilization environment.
19
For example, in water Nile red
exhibits a weak emission peak at 650 nm, while in increasingly hydrophobic
environments, this maximum exhibits a blue shift along with a corresponding increase
in fluorescence intensity.
36
Thus, Nile red provides a convenient reporter on the onset
of surfactant self assembly.
37; 38

As seen in Figure 4.2, Nile red fluorescence can be used to detect vesicle
formation at the critical aggregation concentration (CAC) of catanionic surfactant
mixtures. At low concentrations of the azoTAB and SDBS surfactants, the emission

113
of Nile red does not change relative to that of pure water. However, at the CAC Nile
red begins to partition into the nonpolar bilayer region of the vesicles, causing the
fluorescence to dramatically increase along with a blue shift in the emission peak to
640 nm with the visible illumination and to 643 nm with UV exposure. This indicates
that Nile red is experiencing a relatively-hydrophobic microenvironment,
36
a result of
surfactant assembly into vesicles. The critical aggregation concentrations are then
estimated as 8 µM (visible light) and 36 µM (UV light). Note that these catanionic
systems self-associate in the micromolar range, well below critical micelle
concentrations of the pure surfactants in the millimolar regime.

0
5 10
4
1 10
5
1.5 10
5
2 10
5
640 680 720 760
Intensity
Wavelength (nm)
(a) visible
0, 4, 8 µM
12 µM
16 µM
20 µM
24 µM
28, 32 µM
0
1 10
4
2 10
4
3 10
4
4 10
4
5 10
4
6 10
4
640 680 720 760
Intensity
Wavelength (nm)
(b) UV
0, 24, 32 µM
8, 16 µM
40, 48 µM
56 µM
64 µM
72 µM

Figure 4.2: Fluorescence emission spectra of Nile red in 60/40 (molar)
azoTAB/SDBS mixtures under (a) visible and (b) UV exposure. [Nile red] = 0.1 µM.
The concentration shown is azoTAB concentration, T = 25 ° C.

114

0
10
20
30
40
50
60
70
20:80 40:60 70:30 90:10
Trans
Cis
azoTAB Concentrantion ( µm)
Ratio (azoTAB/SDBS)

Figure 4.3: CAC of the azoTAB/SDBS catanionic mixtures at given ratios under
visible light and UV illumination. [Nile red]: 0.1 µm, T = 25 ° C.

CACs determined for a range of azoTAB/SDBS ratios are presented in Figure 4.3
under both visible and UV illumination. The onset of vesicle formation is found to
increase from 2 µM to 30 µM under visible illumination and from 6 µM to 70 µM
under UV exposure with increasing azoTAB/SDBS ratio. Vesicle formation is
expected to be governed by a combination of hydrophobic interactions amongst the
surfactant hydrocarbon tails and ionic and steric interactions of the headgroups.
18

From the critical micelle concentrations (CMC) of the pure surfactants, which
provide a relative measure of hydrophobicity, SDBS (CMC = 1-4 mM)
39
is generally
more hydrophobic than azoTAB in either the trans (CMC = 5 mM) or cis (CMC = 10
mM) conformation.
23
Thus, moving towards azoTAB-rich mixtures and converting

115
azoTAB to the cis form with UV illumination would both be expected to decrease the
hydrophobic driving force of vesicle formation, as described in Figure 4.3.
To know the onset points of the vesicle formation under both visible and UV
illumination is of great importance for gene delivery.
22
For example, in our previous
paper, the different onset vesicle formation is used to increase gene transfection. In
the study, azoTAB/SDBS=70/30 and 50 µM azoTAB are used to study gene delivery.
Two issues are considered to increase the gene delivery by using azoTAB and SDBS
catanionic surfactant system, one is to increase the rate of approaching the cell
membrane and the endosome-lysis in gene delivery process, another one is to
consider the toxicity. With azoTAB surfactant, when surfactant concentration is
higher than 500 µM, only 20-30% DNA is alive, while with 50 µM, more than 90%
DNA is alive. With catanioic surfactant mixture, gene transfection can increase from
less than 10% to more than 30%.
22

4.4.2 Dynamics detection of the formation and disruption of the vesicle
The dynamics of photo-initiated vesicle rupture and re-formation are examined
through changes in Nile red fluorescence as well as light-scattering intensity, as
shown in the Figure 4.4. An azoTAB concentration intermediate the CAC values
measured under visible and UV light (see Figure 4.3) was chosen such that the

116
vesicles formed under visible light would be completely destroyed upon UV
exposure.

0
1 10
4
2 10
4
3 10
4
4 10
4
5 10
4
0 100 200 300 400 500 600 700
Intensity
Tim e (s)
V isible light
UV light
Figure 4.4: Dynamics of vesicle formation and disruption determination with
time-based fluorescence measurements at an azoTAB/SDBS ratio of 60/40 and an
azoTAB concentration of 30 µM and nile red at 0.1 µM.

From these fluorescence measurements, light-induced vesicle formation occurs
within ~80 s upon visible light exposure, while vesicle disruption occurs in ~30 s
following UV exposure. Note that at these low azoTAB concentrations, isomerization
from the trans to the cis form occurs within 20 seconds of exposure to the mercury
arc lamp (according to UV-Vis measurement),
14
considerably faster than the dynamics
above. This indicates that the rates of vesicle transitions are controlled by the
underlying dynamics of catanionic vesicle formation and disruption. The formation
and disruption of the vesicles could be reversibly controlled by light illumination. The

117
mechanism of formation and disruption of vesicles could be described as under
visible light the planar, relatively-hydrophobic trans configuration of the surfactant
would more readily form ordered, bilayer structures compared to the bent,
relatively-hydrophilic cis isomer under UV exposure. As a result cationic vesicle will
form spontaneously under visible light and be disrupted with UV illumination, as
shown in Figure 4.5.

visible
UV
N
+
CH
2
CH
2
CH
2
CH
2
O
N
N
CH
3
CH
3
C H
3
C
H
2
CH
3
SO
3
-
Na
+
CH
3
(CH
2
)
11
N
+
CH
2
CH
2
CH
2
CH
2
O
N
N
CH
2
CH
3
CH
3
CH
3
C H
3
SO
3
-
Na
+
CH
3
(CH
2
)
11
visible
UV
N
+
CH
2
CH
2
CH
2
CH
2
O
N
N
CH
3
CH
3
C H
3
C
H
2
CH
3
SO
3
-
Na
+
CH
3
(CH
2
)
11
N
+
CH
2
CH
2
CH
2
CH
2
O
N
N
CH
3
CH
3
C H
3
C
H
2
CH
3
SO
3
-
Na
+
CH
3
(CH
2
)
11
N
+
CH
2
CH
2
CH
2
CH
2
O
N
N
CH
2
CH
3
CH
3
CH
3
C H
3
SO
3
-
Na
+
CH
3
(CH
2
)
11
N
+
CH
2
CH
2
CH
2
CH
2
O
N
N
CH
2
CH
3
CH
3
CH
3
C H
3
SO
3
-
Na
+
CH
3
(CH
2
)
11

Figure 4.5: Illustration of the mechanisms responsible for the reversible
formation and disruption photoresponsive catanionic vesicles.

Considering the concentration chosen between CACs of visible light and UV
light, the formation and disruption of the versicle can be reversibly controlled with

118
light illumination. In the stop flow study of O’Connor and Hatton, the growth of the
vesicles composed of SOS and DTAB were observed,
35
while in our study, the vesicle
keeps the same size once it is formed which is proven by dynamics light scattering.

4.4.3 UV-Vis spectroscopy
The photoresponsive azoTAB surfactant in Figure 4.1 shows the reversible
conversion under illumination of visible and UV lights, associated with the maximum
absorbance at 350 nm under visible exposure and 434 nm with UV illumination of
pure azoTAB. For a typical isomerization cycle of azoTAB, the absorption spectrum
contain three transitions, 248 nm (a π- π* transition), 350 nm (a π- π* transition) and
440 nm (a weak n- π* transition).
23; 40
Using light at 365 nm from a mercury lamp, the
azobenzene is isomerized from trans isomer to the cis isomer leading to an increase in
absorbance at 440 nm and a decrease at 350 nm. In addition, alternate exposure to
visible and UV light causes photoreversible changes in the
hydrophobicity/hydrophilicity of the surfactant species that can be used to control
various surfactant properties.
41
With visible illumination, trans conformation of
azoTAB is more planar and hydrophobic while with UV illumiaion, cis conformation
is more bent and hydrophilic. In our study, with the addition of the SDBS, depending
on the steric interaction of the oppositely charged head group and hydrophobicity
interaction of the hydrocarbon tails, the maximum peak will shift with the ratio

119
changes of azoTAB and SDBS, which indicates the environment changes around
azoTAB molecules.
42

The data of UV-Vis spectra for 0.1 mM azoTAB with SDBS at the given ratios
are shown in Figure 4.6 with light illumination. Starting from dark state, the data are
collected by the serials of dark →visible light →UV light →visible light →dark. In dark
state, the maximum absorbance is around 340 nm when catanionic mixture is at
azoTAB-rich side, while the maximum absorbance is around 350 in SDBS-rich side.
The same phenomenon is observed with the visible illumination. When solutions are
exposed to UV illumination, the typical absorbance is at 440 nm and 435 nm at
SDBS-rich and azoTAB-rich sides, respectively.
Figure 4.6a showed UV-Vis spectra at azoTAB/SDBS ratios of 30/70 and 70/30
in dark, visible and UV states. In dark, the absorbance of the 350 nm peak is higher
compared to corresponding sample under visible light. This can be presented as in
dark, azoTAB is in trans conformation, while in the visible light, the samples are at
the equilibrium of the trans and cis conformations (mostly trans form).
23
With UV
light illumination, surfactants reach equilibrium where cis form takes great
percentage, associated with the increase of the 434 nm peak. The shift of the
maximum peak to lower wavelength in SDBS-rich side in dark and visible lights
indicated the absorbance band to higher energy according to the interaction of the
molecules, accounted for dimeric chromophore.
40
. Another phenomenon will be

120
noticed is that the difference absorbance of the samples in dark and visible lights with
70/30 or 30/70 ratios indicated with SDBS-rich size, the mixture is more readily to
stay at the trans conform, proven by the spectrum under UV light illumination
(SDBS-rich size, lower 434 nm absorbance) which is consistent with the study on
BTHA and SDS mixture.
42
This can be explained by the different hydrophobicity of
azoTAB isomers and the chemical architecture of two isomers of azoTAB isomers.
Trans conformation is more hydrophobic (CMC = 5 mM) and readily to interaction
with more hydrophobic SDBS (CMC = 1-4 mM) molecules to keep at trans status
compared to more hydrophilic cis conformation (CMC = 10 mM).
22
Considering the
chemical architecture of azoTAB isomers, trans azoTAB extends fully to interact with
SDBS molecules to form bilayers which prevents the extended trans form to
converting to bent cis form.
42

In Figure 4.6b, 4.6c and 4.6d, the absorbance of azoTAB depending on SDBS
concentration according to the given concentration were shown in dark, visible and
UV illumination. In dark state, approaching to 50/50 ratio, the maximum absorbance
of trans azoTAB showed a decrease with the increase of the SDBS. The same
phenomena were observed with visible illumination in the azoTAB-rich side, while in
the opposite way, the maximum absorbance of trans azoTAB increased in the
presence of SDBS, which indicated high affinity of trans azoTAB for the anionic
surfactant and of the formation of mixed aggregates
42
and with the addition of SDBS,

121
it is more readily to form trans conform which in proven with UV light illumination.
In Figure 4.6d, with UV illumination, the maximum cis peak shifted to lower
wavelength and decreased with the increase of SDBS amount. The blue shift of the
maximum peak probably indicated the torsion of the azobenzene ring due to the
parallel interaction of the chromophores.
40
In the previous work of Czikkely and etc,
there are two forms of the system containing azobenzene component, H-aggregation
and J-aggregation.
40; 43; 44
From their theory, parallel interaction modes of the
chromophores, called H-aggregation, induces the blue shift of the maximum
absorbance, while a head to tail interaction, called J-aggregate, induces the red shift
of the maximum absorbance. The decrease of the maximum peak absorbance with the
increase of the SDBS amount could be indicated as the rearrangement of the
self-assembled structures with azoTAB and SDBS. If chemical structures of SDBS
and two isomers of azoTAB were considered, under visible light, the planar,
relatively-hydrophobic trans configuration of the surfactant would more readily
interact with the SDBS compared to that under the UV light, that meant the
arrangements prevented the isomerization of the trans surfactant to cis form which
induced more trans form with addition SDBS. Another reason could be different
extension coefficient of the cataionic surfactant with different azoTAB/SDBS ratios.

122

0
0.5
1
1.5
2
2.5
300 400 500 600 700
30_70 dark
70_30 dark
30_70 visible
70_30 visible
30_70 UV
70_30 UV
Absorbance
Wavelenght (nm)
0
0.5
1
1.5
2
2.5
300 400 500 600 700
100_0
10_90
20_80
30_70
40_60
60_40
70_30
80_20
90_10
Absorbance
Wavelength (nm)

(a) (b)
0
0.5
1
1.5
2
300 400 500 600 700
100_0
10_90
20_80
30_70
40_60
60_40
70_30
80_20
90_10
Absorbance
Wavelength (nm)
0
0.5
1
1.5
300 400 500 600 700
100_0
10_90
20_80
30_70
40_60
60_40
70_30
80_20
90_10
Absorbance
Wavelength (nm)

(c) (d)

Figure 4.6: UV-Vis absorbance sptectra of azoTAB/SDBS (a) 30/70 and 70/30 in
dark, visible illumination, UV illumination; (b)(c)(d) azoTAB absorbance as a
function of SDBS concentration in dark, visible illumination and UV illumination
separately. [azoTAB]: 0.1 mM.

123
4.4.4 Dynamic light scattering
We have measured the mixture of azoTAB and SDBS at different concentrations
(such as, 0.1%, 0.25%, 0.5% etc) and different ratios (such as 10/90, 20/80, 30/70,
40/60, 60/40, 70/30, 80/20, 90/10 etc) by dynamic light scattering, small angle
neutron scattering and polarized optical microscopy. A ternary diagrams as Figure 4.7
showed the boundary regimes of different microstructures founding in the mixtures of
azoTAB and SDBS under both visible and UV illumination. In this ternary diagrams
V indicated vesicle, M, micelles and L, lamellar structure. To describe the ternary
diagrams, dynamical light scattering, small angle neutron scattering and polarized
optical microscopy are described below separately.

124

Figure 4.7: Ternary diagrams showing the regimes of different regimes of
different microstructure found in mixtures of AzoTAB and SDBS under both visible
and UV light. V: Vesicles, M: Micelles, L: Lamellar structure.

Dynamic light scattering (DLS) has been used in several studies of catanionic
surfactant vesicles.
4; 17; 45
Through measurements of diffusion coefficient (D), the
effective hydrodynamic radius can be calculated from the Stokes-Einstein equation,
namely, R
H
= k
B
T/6 πηD,
13; 46
where k
B
is Boltzmann’s constant, T is the temperature,
and η is the viscosity of the solvent. Figure 4.8a contains the hydrodynamic diameters
measured for azoTAB-SDBS vesicles as a function of azoTAB/SDBS ratios with
fixed total surfactant concentration 0.1 wt%. Figure 4.8b presents the hydrodynamic

125
diameters for large range (0.0125 wt% to 0.5 wt%) with the given surfactant ration
30/70.
From Figure 4.8a and 4.8b, the hydrodynamic diameter is seen to increase upon
approaching the equimolar azoTAB:SDBS ratio of 1:1, consistent with the cetyl
trimethylammonium tosylate(CTAT)-SDBS catanionic system studied by Kaler et
al.
3
This is a result of an increase in the net surface charge as the system moves away
from the 1:1 cation:anion ratio, which causes the counter ions to condense on the
charged surfaces, hence, decreasing the entropy of the counter ions. The effect can be
alleviated with an increase in the curvature of the surfactant bilayer layer (i.e., smaller
vesicles), which would dramatically increase the volume occupied by the external
counter ions.
7
The measured vesicles sizes are also generally found to be larger under
visible as opposed to UV illumination except at ratios of 60/40, 70/30 and 80/20,
which are considered to form lamellar structure (the catanionic mixture sizes at these
ratios are from 800 nm to 1300 nm and with UV illumination, cloudy aggregates are
observed in the aqueous solution.). In most cases, this is likely a result of the
surfactant architecture, with the planar trans isomer favoring more planar interfaces
compared to the bent cis conformation, combined with the known correlation between
enhanced asymmetry between the lengths of the cation and anion surfactants leading
to increased vesicle size.
3

126
Finally, as the overall surfactant concentration increases, the effective vesicle
size also increases, although note that at the largest surfactant concentration studied
the measured sizes would be expected to be influenced by inter-vesicle interactions
and, thus, must be considered as only effective quantities (e.g., at 0.5 wt % surfactant,
the vesicle volume fraction is ~ 0.06, well beyond the value of ca. 1% generally
considered as the limit beyond which interaction effects start to influence light
scattering data), shown in Figure 4.8b. As the study of Silva etc,
47
vesicles are formed
of two oppositely changed surfactant ions, it is reasonable to assume that azoTAB ion
is less soluble compared to shorter chain SDBS ions,
10
which leaves the vesicles are
slightly positively charged. When the total surfactant concentration is decreased, the
fraction of the dissociated ions is increased and the charges on the vesicle surface
increased as well, which will condense the counter ions on the surface inducing the
smaller vesicle formation (larger curvature).
47

127

0
200
400
600
800

(a) (b)

Figure 4.8: (a) Hydrodynamic diameters (R
H
) of azoTAB/SDBS catanionic vesicles
under visible and UV illumination as a function of surfactant ratio at a total surfactant
concentration at 0.1 wt%. (b) as a function of the total surfactant concentration at a
given azoTAB/SDBS molar ratio of 30/70 T = 25 ° C.

A summary of DLS parameters was shown in Table 4.1 under both visible and
UV illumination. The data generally followed the trends that approaching 50/50, size
increased and with the increase of overall all surfactant concentration, size increased
as well. In Table 4.1, DLS parameters are shown with light illumination,
azoTAB/SDBS ratios and total surfactant concentrations. In the table, V indicates
vesicle, M represents micelles and L is lamellar structure. For surfactants, in previous
study, critical packing parameters are introduced to describe the microstructures in
the aqueous surfactant solution. It is calculated as
max
/l a ν , where ν is the volume of
the hydrophobic potion of the surfactant, l
max
, length of the hydrophobic parts and a,
1000
1200
1400
3:97 10:9030:7060:4080:20 95:5 9
Visible light
UV light
R
H
(nm)
Ratio (azoTAB/SDBS)
9:1
0
50
100
150
200
250
0.02% 0.10% 0.25%
Visible light
UV light
R
H
(nm)
wt%

128
the area of the head group.
10; 48
When critical packing parameters < 1/3, spherical
micelles are formed , 1/3 < critical packing parameters < 1/2, cylindrical micelles are
formed and vesicles or planar bilayers are formed when critical packing parameters >
1/2.
10; 48
Because of different hydrophobicity of surfactant conformations and
surfactant architecture, three parameters will change by light illumination, which
induce different microstructures in aqueous solution.
From Table 4.1, three transitions are investigated as vesicles ↔ lamellar
structures, vesicles ↔ micelles and micelles ↔ vesicles. Because of different
hydrophobicity of surfactant conformations, three parameters ( ν, l
max
, a) will change
by light illumination, which induce different microstructures in aqueous solution. For
example, transition between vesicles ↔ lamellar structures, with light switch from
visible light to UV light, the length of the hydrophobic part decreases probably
inducing the critical packing parameter larger to convert vesicles to lamellar
structures.
10
This phenomenon is observed when total surfactant concentration is
0.025 wt%, in SDBS-rich side with visible light illumination switching to UV
illumination, while in azoTAB-rich size, vesicle structures are observed under both
visible and UV illumination. Lamellar structures are indicated as with UV
illumination, cloudy aggregates are observed in aqueous solution and during DLS
experiment, count rate increased 4 times as those with visible illumination, which
indicates the large aggregates formation. The same phenomena are investigated when

129
total surfactant concentrations are at 0.1 wt%, 0.175 wt%, 0.25 wt% and 0.5 wt%, but
with narrower ratios. The observation states that there is a lamellar structure loop (as
shown in Figure 4.7 and Table 4.1) in SDBS-rich side with UV illumination while
they keep vesicle structures with visible light exposure. This phenomenon is not
observed in other previous catanionic surfactant study, such as CTAB/SOS,
34

BTHA/SDS,
11
CTAT/SDBS
30
mixtures etc by SANS, but investigated by freeze
replica TEM with AZTMA/SDBS mixed solutions.
10
Considering three parameters ( ν,
l
max
, a) by the surfactant architecture here, with cis isomer formation, the hydrocarbon
length of the mixture decreases which pushes the critical packing parameters
increasing to produce lamellar structure.
10

With the total surfactant concentration is 0.025 wt%, no transitions between
micelles ↔ vesicles or vesicles ↔ micelles are found which light illumination on
both sides, while with the total surfactant concentration increases, and the transition is
investigated. For example, at 0.1 wt% total surfactant concentration and
azoTAB/SDBS = 1/99, vesicles are observed with visible illumination while with UV
exposure, micelles are observed, which is indicated by the single small size peak
about 4.6 nm (shown in Table 4.1) investigated by DLS measurement. With increase
of total surfactant concentration, the phenomena approach to higher azoTAB/SDBS
ratio, for 0.25 wt% (5/95), 0.5% (7/93). The transition between vesicles ↔ micelles is
consistent with surfactant mixture of stilbenzene-containing Gemini photosurfactant

130

131
(E-SGP) and DTAB.
49
Opposite to SDBS-rich side, in azoTAB-rich side, micelle
structures observed with visible illumination and vesicle structures observed with UV
light exposure and the onset point is azoTAB/SDBS = 93/7 when total surfactant
concentration is 0.175 wt%. The transition between micelles ↔ vesicles is not
observed in previous study, such as AZTMA/SDBS,
10
E-SGP/DTAB
49
and
BTHA/SDS mixtures.
11; 29
For these transitions approaching pure surfactant directions
are of great interests and never be observed in previous study. The reason for these
phenomena is probably the competition between structure architecture of trans and
cis isomers of azoTAB surfactant and the symmetric hydrocarbon tail effects. For
trans isomer, it is more planar and would like to interact with SDBS to form planar
bilayers as forming vesicles, but for cis isomer, the alkyl chain of azoTAB is more
similar to SDBS, vesicle formation is favored because of the favorable free energy
associated with the hydrocarbon/water interface.
6; 50
For example, when total
surfactant concentration is 0.1% and azoTAB/SDBS = 1/99 vesicles are formed
because of the planar trans form is readily to form vesicles with SDBS while with
UV illumination, the effect of the surfactant architecture takes the great importance.
However, in the opposite direction, when total surfactant concentration is 0.175 wt%
and azoTAB/SDBS = 93/7, the effect of symmetric hydrocarbon chains takes of
importance inducing the micelles formation with visible illumination while vesicle
formations with UV illumination.

132
Table 4.1: DLS parameters (S indicated Structure; V: Vesicle; L: Lamellar structures; M, Micelles) the unit of size: nm
azoTAB/SDBS 0.025 wt% 0.1 wt% 0.175 wt% 0.25 wt% 0.5 wt%
visible UV visible UV visible UV visible UV visible UV
S Size S Size S Size S Size S Size S Size S Size S Size S Size S Size
1/99 V 126.3 V 89.2 V 140.07 M 4.67
3/97 V 146.01 V 109.13
5/95 V 153.26 V 86.51 V 236.13 M 3.37
7/93 V 238.36 M 3.4
10/90 V 128.55 V 83.83 V 168.66 V 124.96 V 270.16 V 183.45 V 145.47 V 118.82 V 214.32 V 201.19
20/80 V 130.82 V 104.11 V 186.51 V 133.65 V 171.36 V 148.89 V 133.58 V 114.31 V 226.19 V 208.69
30/70 V 143.33 V 135.24 V 193.61 V 148.13 V 204.8 V 179.73 V 158.81 V 156.31 V 236.88 V 180.87
40/60 V 171.37 V 142.97 V 182.09 V 158.34 V 211.35 V 201.95 V 138.82 V 140 V 195.28 V 191.46
60/40 V 232.35 L 339.43 V 224.02 L 1279 V 211.16 L 1141.49 V 151.82 L 257.52 V 187.8 L 876.35
70/30 V 192.24 L 362.83 V 191.2 L 850.07 V 221.56 L 456.42 V 151.32 V 146.89 V 172.49 L 139.08
80/20 V 193.45 L 883.10 V 200.97 L 817.84 V 196.74 V 192.04 V 197.96 V 167.61 V 142.06 L 117.43
90/10 V 179.86 L 1224.22 V 160.45 L 173.32 V 144.01 V 148.44 V 147.52 V 117.03 V 110.91 L 81.98
93/7 V 236.72 V 173.72 M 3.15 V 147.81 M 4.59 V 130.18 M 3.15 V 147.81
95/3 V 184.15 V 170
97/3 M 3.94 V 90.37
99/1 V 177.5 V 195.5 V 158.9 V 119.5
4.4.5 Small-angle neutron scattering (SANS)
SANS are used to investigate various microstructures formed in the aqueous
azoTAB and SDBS systems. Samples with given molar ratios azoTAB/SDBS = 10/90,
20/80, 30/70, 40/60, 60/40, 70/30, 80/20 and 90/10 are measured by SANS when total
surfactant concentration is 0.1 wt % Also, some samples are measured when
azoTAB/SDBS ratios are at 93/7 (0.25 wt % total surfactant concentration) and 7/93,
60/40 and 93/7 (0.5 wt% total surfactant concentration) with both visible and UV
illumination. A summary of different microstructure of different surfactant
concentrations and molar ratios under both visible and UV illumination is shown in
ternary diagram in Figure 4.7. Some examples of SANS data describing the
microstructures are shown in Figure 4.9 and the microstructures are modeled with
Igor program (models description given in material and methods section) are given in
Figure 4.10. The figures only show the different scattering with visible and UV light
illumination; the other data parameters will be described in Table 4.2.

133

134

0.0001
0.001
0.01
0.1
1
10
0.01 0.1
60/40 0.1% trans
60/40 0.1% cis
70/30 0.1% trans
70/30 0.1% cis
80/20 0.1% trans
80/20 0.1% cis
Intensity (cm
-1
)
Q (A
-1
)
In Figure 4.10, SANS data and corresponding best-fit curves of data are
presented by using different models depending on the microstructures. In Igor
program, for vesicle fitting, poly core shell model is applied ， indicated in Figure 4.8.
11;
30; 51
In this case, in polydispersed spherical particles form factor, SANS data are
modeled with 3 parameters: average vesicle size, shell thickness and polydispersity
while other parameters have been fixed due to the properties of the two surfactant
components. In Figure 4.8d, 4.8e and 4.8f, the micelles structure is to calculate the
form factor for a monodisperse ellipsoid with uniform scattering length density
coupled with structure factor. The parameters defining the fits are given in Table 4.2.
Figure 4.9: SANS data of scattering intensity as a function of scattering vector Q
for samples with surfactant ratios (a) 60/40, 70/30, 80/20 and an overall surfactant
concentration 0.1w%; (b) 93/7, 7/93, 60/40 and over all surfactant concentration 0.25
wt% and 0.5wt% as described in figure.

(a) (b)
0.0001
0.001
0.01
0.1
1
10
0.01 0.1
93/7 0.25% trans
93/7 0.25% cis
93/7 0.5% trans
93/7 0.5% cis
7/93 0.5% trans
7/93 0.5% cis
Intensity (cm
-1
)
Q (A
-1
)
Table 4.2: SANS data at all molar ratios and overall surfactant concentrations, 0.1, 0.25, 0.5 wt%. In the table, Ra: rotational
axis, Rb: radius, C: charge, R: average core radius, P: core poly dispersity, ST: shell thickness, P: parameters, V: vesicle, M:
micelles
azoTAB/SDBS 0.1 wt% 0.25 wt% 0.5 wt%
visible UV visible UV visible UV
Structure P Structure P Structure P Structure P Structure P Structure P
7/93 No measurement M Ra = 28.37 Å M Ra = 26.62 Å
Rb = 16.58 Å Rb = 16.04 Å
C = 6.79 C = 7.08
V R = 426.73 Å
P = 0.586
ST = 10.58 Å
10/90 V R = 510.53 Å V R =197.41 Å
P = 0.277 P = 0.459
ST = 24.10 Å ST = 24.10 Å
Not measured Not measured
20/80 V R = 544.8 Å V R = 553.25 Å
P = 0.223 P = 0.230
ST = 28.99 Å ST = 26.46 Å
Not measured Not measured
30/70 V R = 492.85 Å V R = 509.79 Å
P = 0.259 P = 0.261
ST = 30.61 Å ST = 28.23 Å
Not measured Not measured
40/60 V R = 519.93 Å V R = 683.90 Å
P = 0.238 P = 0.386
ST = 30.08 Å ST = 28.47 Å
Not measured Not measured

135

136
Table 4.2 continued: SANS data at all molar ratios and overall surfactant concentrations, 0.1, 0.25, 0.5 wt%. In the table, Ra:
rotational axis, Rb: radius, C: charge, R: average core radius, P: core poly dispersity, ST: shell thickness, P: parameters, V: vesicle,
M: micelles
azoTAB/SDBS 0.1 wt% 0.25 wt% 0.5 wt%
visible UV visible UV visible UV
StructureP StructureP StructureP StructureP StructureP StructureP

60/40

V R = 760.20 Å
P = 0.180 No fitting
ST = 32.22 Å

Not measured

Not measured
70/30 V R = 433.26 Å
P = 0.360 No fitting
ST = 32.84 Å
Not measured Not measured
80/20 V R = 485.99 Å
P = 0.292 No fitting
ST = 33.72 Å
Not measured Not measured
90/10 V R = 536.55 Å V R = 148.51 Å
P = 0.263 P = 0.691
ST = 32.22 Å ST = 28.59 Å
Not measured Not measured
93/7 Not measured M Ra = 54.05 Å V R = 187.07 Å
Rb = 22.17 Å P = 0.56
C = 0.05 ST = 29.62 Å

M Ra = 43.77 Å V R = 232.69 Å
Rb = 23.00 Å P = 0.42
C = 5.41 ST = 20.32 Å

0.00 1
0.01
0.1
1
10
0.001 0.01 0.1 1 1 0
60/40 0.1w t% trans
60/40 0.1w t% cis
60/40 0.1w t% trans fitting
Intensity (cm
-1
)
Q (A
-1
)
0.00 1
0.01
0.1
1
10
0.001 0.01 0.1 1 1 0
70/30 0.1w t% trans
70/30 0.1w t% cis
70/30 0.1w t% trans fitting
Intensity (cm
-1
)
Q (A
-1
)
(a) (b)

0.00 1
0.01
0.1
1
10
0.001 0.01 0.1 1 1 0
80/20 0.1w t% trans
80/20 0.1w t% cis
80/20 0.1w t% trans fitting
Intensity (cm
-1
)
Q (A
-1
)
0 .001
0.01
0.1
1
10
100
0.0 01 0 .01 0.1 1 1 0
93/7 0.25w t% trans
93/7 0.25w t% cis
93/7 0.25 w t% trans fitting
93/7 0.25w t% cis fitting
Intensity (cm
-1
)
Q (A
-1
)
(c) (d)
Figure 4.10: Igor fitting data with both visible and UV light illumination for
total surfactant concentration (a) 0.1 wt% azoTAB/SDBS=60/40, (b) 0.1 wt%
azoTAB/SDBS=70/30, (c) 0.1 wt% azoTAB/SDBS=80/20, (d) 0.25 wt%
azoTAB/SDBS=93/7.

137

(e) (f)

Figure 4.10 continued: Igor fitting data with both visible and UV light
illumination for total surfactant concentration (e) 0.5 wt% azoTAB/SDBS=93/7, (f)
0.5 wt% azoTAB/SDBS=7/93.

In Figure 4.9a and Figure 4.10a, 4.10b, 4.10c, at total surfactant concentration at
0.1 wt%, vesicle structure is found in aqueous solutions at all the molar ratios with
visible light illumination, but with UV exposure, multiple micro-structures are found,
such as lamellar, vesicle and micelles. The formation of the vesicles is determined by
slope of the curves as shown in Figure 4.9. According to Guinier approximation,
13

the slop is -2 which is the characteristics scattering of the flat surface indicating the
vesicle formation
52
with visible illumination while under UV exposure, the
microstructures change and a peak at Q = 0.8 is observed, as shown in Figure 4.10a,
4.10b, 4.10c. In these graphs, no fitting data are shown because of the multi-micro
0 .001
0.01
0.1
1
100
0.001 0.01 0.1
0.00 1
0.01
0.1
1
10
0.001 0.0 1 0 .1 1
7/93 0.5w t% trans
7/93 0.5w t% cis
7/93 0.5w t% trans fitting
7/93 0.5w t% cis fitting
Intensity (cm
-1
)
Q (A
-1
)
10
1
93/7 0.5w t% trans
93/7 0.5w t% cis
93/7 0.5w t% trans fitting
93/7 0.5w t% cis fitting
Intensity (cm
-1
)
Q (A
-1
)

138
structures formation with UV light illumination. For example, at the ratio 70/30 the
presence of a peak at Q, 0.08 Å
-1
, is attributed to the presence of a mutilamellar
structure corresponded to a lamellar spacing 2/ d q π = of 78 Å. At this ratio, the
bilayer for unilamellar vesicles have been measured about 33 Å as given in Table 4.2
which is about two layers stack of the vesicles since there are water molecules
among the layers.
With the increase of total concentration and approaching to pure azoTAB and
SDBS, the transitions between micelles and vesicles are observed which is described
in Figure 4.9b and Figure 4.10d, 4.10e, 4.10f. In Figure 4.10e, when surfactant
concentration is 0.25 %, at 93/7 ratio (azoTAB/SDBS), vesicle is observed with the
curve slop -2 with UV illumination, but with visible illumination, zero slop at low
values of Q is stated in figure and the initial value of I
0
(~ 0.2 cm
-1
) which is related
to molecular weight
13
of the aggregates is investigated indicating the formation of
small micelles.
11
The same phenomenon is observed at total surfactant concentration
0.5 wt% and azoTAB/SDBS 93/7 ratio, as shown in Figure 4.9b and 4.10d, by
comparing the initial intensity of two concentrations, larger vesicle formed when
total surfactant concentration is 0.5 wt% consistent with DLS results. In Figure 4.10e,
when total surfactant concentration is 0.5 wt%, a correlation peak (Q ~ 0.03 Å
-1
)
appears which is associated with the distance between micelle molecules
11
from
which the micelles number densities can be calculated.
11
On the opposite side,

139
SDBS-rich side, shown in Figure 4.9b and 4.10f, with visible illumination, at low
scattering vector value, slop is -2 curve observed indicating the vesicles in aqueous
solution, also a correlation peak around 0.045 Å
-1
is investigated associated with
micelles formation indicating the vesicles and micelles mixture microstructures in
aqueous solution. With UV illumination, only correction peak around 0.045 Å
-1
is
observed indicating the vesicle ↔ micelles transition with light switching form
visible illumination to UV illumination.
In Table 4.2, parameters describe the estimations obtained from fitted SANS
data at all ratios and overall surfactant concentration. In our system, three different
microstructures have formed as micelles, vesicles and lamellar structures. For the
micelles structure we observe in 93/7 0.5 wt %, the radius of the micelles is about
2.3 nm while the rotational axis is about 42 Å both under visible illumination. For
pure azoTAB, the charge of the micelle is about 20 (unpublished data), but with the
addition of the SDBS, the charge is decrease to 5. With the addition of the SDBS to
the ratio 7/93 at the same surfactant concentrations, the radius of the micelles
decreases to 16 Å while the charge increases to 7. It seems that the decrease surface
charge coupled with a slight growth in size as observed in previous SDS and BTHA
system.
11
The same size micelles are found at 7/93 ratio, 0.5 wt% under both visible
light and UV light illumination, while under visible illumination 430 Å vesicles are
found with the shell thickness 10 Å. In overall surfactant concentration 0.1 w%,

140
vesicles are observed for all the trans samples, with size varying from 480 Å to 760
Å. With UV illumination, vesicles are investigated in most samples except 60/40,
70/30 and 80/20 with size varying from 148 to 684 Å. the shell thickness of the
bilayer is from 24 Å to 33.7 Å with visible illumination while it is from 19.5 Å to
28.6 Å with UV exposure which was in agreement the tail-tail interaction of the
surfactant tails considered as 28 Å.
11
And if compared the shell thickness of the
vesicles with both light illumination, it is lager with visible illumination, related to
the structure of the azoTAB, which has longer tail length than that with UV
exposure.
Another phenomenon needs to be noted is the aggregates sizes obtained from
DLS measurements are larger than those obtained from SANS measurements.
Several factors can be considered in this issue. First, analysis of DLS doesn’t
consider the interaction between the microstructures which can induce the lager size
determination than actual size and another reason is for DLS analysis, which
strongly prefers to reflect the larger aggregates in the solution.
11; 29; 30; 53

4.4.6 Polarized Optical Microscopy
For lamellar structures formed in aqueous solution, birefringence or double
refraction phenomenon will be observed. When a light in induced into a lamellar
structure, it is divided in two rays traveling in different directions and at different

141
velocities within the structure and polarized in planes at right angles to one another.
54

Optical polarized microscopy is used to determine such properties of a lamellar
structure, which uses a set of polarizers, called polarizer and analyzer oriented at 90 °
and placed before and after the samples respectively. Lamellar structures presenting
a high degree of ordering are anisotropic and exhibit birefringence when observed
between crossed polarizers.
55; 56

In our study, with high total surfactant concentration, such as 1 wt%, DLS can
not be applied due to the high microstructure concentration, while optical
microscopy could work. Figure 4.11 shows the polarized optical microscopy pictures
of microstructures at all ratios of the system with the overall surfactant 1 wt% with
visible light illumination. With UV illumination, no structures have been observed as
same as 10/90 and 90/10 with both visible and UV exposure due to the small
structure size.

142

Figure 4.11: Polarized optical microscopy images with visible light illumination.
Overall surfactant concentration: 1 wt%. (Microruler: 5 µm).

As shown in Figure 4.11, birefringence is observed at some ratios of the
catanionic surfactant systems. Interference figures, in the shape of Maltese crosses
are specifically observed. Maltese crosses are typical of uniaxial lamellar crystals.
55;
57
In these graphs the observation of maltese crossed indicated the formation of the
lamellar structure.
Combining the study of DLS, SANS and polarized optical microscopy, with
visible light illumination, most structure found is vesicle in both SDBS-rich and
azoTAB-rich sides except when ratios approach to pure azoTAB direction, micelles
are found in aqueous solution. With UV light illumination micelles ↔ vesicles and
vesicles ↔ lamellar structures are found depending on the ratios of the cationic and
20/80
80/20
30/70 40/6
70/30 60/40

143
anionic surfactants and total surfactant concentrations. To study the transition is of
great important of protein reconstitution and gene delivery. For example, for
membrane protein, it is possible to reversibly control membrane protein folding by
utilizing the transition between micelles ↔ vesicles and vesicles ↔ lamellar
structures.

4.5 Conclusions
Photoresponsive vesicles have been developed using catanionic mixtures
consisting of the photoresponsive azoTAB cationic surfactant combined with SDBS
anionic surfactants. Fluorescence measurements using the hydrophobic probe Nile
red are utilized to measure the critical aggregation concentration (CAC) of the
catanionic pairs, identified as the onset of vesicle formation. Dynamic light
scattering and small-angle neutron scattering measurements indicate the
microstructures in the system. Through changes in the isomeric state of azoTAB,
simple light illumination allows reversibly disruption and reformation of the vesicles
and the transition between micelles ↔ vesicles and vesicles ↔ lamellar structures. A
ternary diagram is given to describe the boundary to form microstructures in aqueous
solution under both visible light and UV light.

144
4.6 Acknowledgements
We acknowledge the support of the National Institute of Standards and
Technology, U. S. Department of Commerce, in providing the neutron research
facilities used in this work.

145
4.7 References
1. Bucak, S., Robinson, B. H. & Fontana, A. (2002). Kinetics of Induced
Vesicle Breakdown for Cationic and Catanionic Systems. Langmuir 18,
8288-8294.

2. Xia, Y., Goldmints, I., Johnson, P. W., Hatton, T. A. & Bose, A. (2002).
Temporal Evolution of Microstructures in Aqueous CTAB/SOS and
CTAB/HDBS Solutions. Langmuir 18, 3822-3828.

3. Kaler, E. W., Murthy, A. K., Rodriguez, B. E. & Zasadzinski, J. A. N. (1989).
Spontaneous Vesicle Formation in Aqueous Mixtures of Single-Tailed
Surfactants. Science 245, 1371-1374.

4. Yin, H., Zhou, Z., Huang, J., Zheng, R. & Zhang, Y. (2003).
Temperature-Induced Micelle to Vesicle Transition in the Sodium
Dodecylsulfate/Dodecyltriethylammonium Bromide System. Angew. Chem.
Int. Ed 42, 2188-2191.

5. Caillet, C., Hebrant, M. & Tondre, C. (2000). Sodium Octyl
Surfate/Cetyltrimethylammonium Bromide Catanionic Vesicles: Aggregate
Composition and Probe Encapsulation. Langmuir 16, 9099-9102.

6. Bergstrom, M. & Pedersen, J. S. (1998). Small-Angle Neutron Scattering
(SANS) Study of Aggregates Formed from Aqueous Mixtures of Sodium
Dodecyl Sulfate (SDS) and Dodecyltrimethylammonium Bromide (DTAB).
Langmuir 14, 3754-3761.

7. Bergstrom, M. & Pedersen, J. S. (1999). A Small-Angle Neutron Scattering
(SANS) Study of Talet-Shaped and Ribbonlike Micelles Formed From
Mixtures of an Anionic and a Catiionic Surfactant. Journal of Physical
Chemistry B 103, 8502-8513.

8. Yashen Xia, I. G., Paul W. Johnson, T. Alan Hatton, and Arijit Bose. (2002).
Temporal Evolution of Microstructures in Aqueous CTAB/SOS and
CTAB/HDBS Solutions. Langmuir 18, 3822-3828.

146
9. Brasher, L. L., Herrington, K. L. & Kaler, E. W. (1995). Electrostatic Effects
on the Phase Behavior of Aqueous Cetyltrimethylammonium Bromide and
Sodium Octyl Sulfate Mixtures with Added Sodium Bromide. Langmuir 11,
4267-4277.

10. Sakai, H., Matsumura, A., Yukoyama, S., Saji, T. & Abe, M. (1999).
Photochemical Swiching of Vesicle Formation Using an
Azobenzene-Modified Surfactant. Jounal of Physical Chemistry B 103,
10737-10740.

11. Hubbard, F. P. J. & Abbott, N. L. (2007). Effect of Light on Self-Assembly of
Aquous Mixtures of Sodium Dodecyl Sulfate and Cationic, Bolaform
Surfactant Containing Azobenzene. Langmuir 23, 4819-4829.

12. Wang, S.-c. & Lee, C. T. J. (2006). Protein Secondary Structure Controlled
with Light and Photoresponsive Surfactants. J. Phys. Chem. B 110,
16117-16123.

13. Hamill, A. C., Wang, S.-c. & Lee, C. T. J. (2005). Probing Lysozyme
Conformation with Light Reveals a New Folding Intermediate. Biochemistry
44, 15139-15149.

14. Le Ny, A.-L. M. & Lee, C. T. J. (2006). Photoreversible DNA Condensation
Using Light-Responsive Surfactants. J. Am. Chem. Soc. 128, 6400-6408.

15. Wang, S.-c. & Lee, C. T. J. (2007). Enhanced Enzymatic Activity through
Photoreversible Conformational Changes. Biochemistry 46, 14557-14566.

16. Hamill, A. C., Wang, S.-c. & Lee, C. T. J. (2007). Solution Structure of an
Amyloid-Forming Protein During Photoinitiated Hexamer-Dedecamer
Transitions Revealed through Small-Angle Neutron Scattering. Biochemistry
46, 7694-7705.

17. Shioi, A. & Hatton, T. A. (2002). Model for Formation and Growth of
Vesicles in Mixed Anionic/Cationic (SOS/CTAB) Surfactant Systems.
Langmuir 18, 7341-7348.

18. Yu, W.-Y., Yang, Y.-M. & Chang, C.-H. (2005). Cosolvent Effects on the
Spontaneous Formation of Vesicles from 1:1 Anionic and Cationic Surfactant
Mixtures. Langmuir 21, 6185-6193.

147
19. Sackett, D. L. & Wolff, J. (1987). Nile Red As a Polarity-Sensitive
Fluorescent Probe of Hydrophobic Protein Surfaces. Analytical Biochemistry
167, 228-234.

20. Fromherz, P. & Ruppel, D. (1985). Lipid Vesicle Formation: the Transition
from Open Disks to Closed Shells. FEBS Letters 179, 155-159.

21. Zhang, J., Wang, S.-c. & Lee, C. T. J. (2008). Photoreversible
Conformational Changes in Membrane Proteins Using Light-responsive
Surfactant. J. Phys. Chem. B Summitted.

22. Liu, Y ., Le Ny, A.-L. M., Schmidt, J., Talmon, Y ., Chmelka, B. F. & Lee, C. T.
J. (2008). Photo-Assisted Gene Delivery Using Light -Responsive Catanionic
Vesicles. J. Am. Chem. Soc. summited.

23. Lee, C. T., Jr., Smith, K. A. & Hatton, T. A. (2004). Photoreversible Viscosity
Changes and Gelation in Mixtures of Hydrophobically Modified
Polyelectrolytes and Photosensitive Surfactants. Macromolecules 37,
5397-5405.

24. Hayashita, T., Kurosawd, T., Miyata, T., Tanaka, K. & Igawa, M. (1994).
Effect of structural variation within cationic azo-surfactant upon
photpresponsive function in aqueous solution. Colloid & Polymer Science
272, 1611-1619.

25. Glinka, C. J., Barker, J. G., Hammouda, B., Krueger, S., Moyer, J. J. & Orts,
W. J. (1998). The 30 m small-angle neutron scattering instruments at the
National Institute of Standards and Technology. J. Appl. Crystallogr. 31,
430-445.

26. Kline, S. R. (2006). Reduction and Analysis of SANS and USANS Data
using Igor Pro. J. Appl. Cryst. 39, 895.

27. Hassan, P. A., Fritz, G. & Kaler, E. W. (2003). Small Angle Neutron
Scattering Study of Sodium Dodecyl Sulfate Micellar Growth Driven by
Addition of a Hydrotropic Salt. J. Colloid Interface Sci. 257, 154-162.

28. Brasher, L. L. & Kaler, E. W. (1996). A Small-Angle Neutron Scattering
(SANS) Contrast Variation Investigation of Aggregation Composition in
Catanionic Surfactant Mixtures. Langmuir 12, 6270-6276.

148

29. Hubbard, F. P. J., Santonicola, G., Kaler, E. W. & Abbott, N. L. (2005).
Small-Angle Neutron Scattering from Mixtures of Sodium Dodecyl Sulfate
and a Cationic, Bolaform Surfactant Containing Azobenzene. Langmuir 21,
6131-6136.

30. Gonzalez, Y. I., Stjerndahl, M., Dganit, D. & Kaler, E. W. (2004).
Spontaneous Vesicle Formation and Phase Behavior in Mixtures of an
Anionic Surfactant with Imidazoline Compounds. Langmuir 20, 7053-7063.

31. Hansen, J.-P. & Hayter, J. B. (1982). A Rescaled MSA Structure Factor for
Dilute Charged Colloidal Dispersion. Mol. Phys 46, 651-656.

32. Kumar, S., Naqvi, A. Z., Aswal, V. K., Goyal, P. S. & Kabir-ud-Din. (2003).
A SANS Study on Growth of Anionic Micelles with Quaternary Ammonium
Bromide. Current Science 84, 1346-1349.

33. Marques, E. F., Regev, O., Khan, A., Miguel, M. d. G. & Lindman, B. (1998).
Vesicle Formation and General Phase Behavior in the Catanionic Mixture
SDS-DDAB-Water. The Anionic-Rich Side. J. Phys. Chem. B 102,
6746-6758.

34. Tsuchiya, K., Nakanishi, H., Sakai, H. & Abe, M. (2004).
Temperature-Dependent Vesicle Formation of Aqueous Solutions of Mixed
Cationic and Anionic Surfactants. Langmuir 20, 2117-2122.

35. O'Connor, A. J. & Hatton, T. A. (1997). Dynamics of Micelle-Vesicle
Transitions in Aqueous Anionic/Cationic Surfactant Mixtures. Langmuir 13,
6931-6940.

36. Eloi Feitosa, F. R. A., Anna Niemiec, M. Elisabete C. D. Real Oliveira,
Elisabete M. S. Castanheira, and Adelina L. F. Baptista. (2006). Cationic
Liposomes in Mixed Didodecyldimethylammonium Bromide and
Dioctadecyldimethylammonium Bromide Aqueous Dispersions Studied by
Differential Scanning Calorimetry, Nile Red Fluorescence, and Turbidity.
Langmuir 22, 3579-3585.

37. Liao, X. & Higgins, D. A. (2002). Scanning Probe Microscopy Studies of
Mesostructured Nonstoichiometric Polyelectrolyte-Surfactant Complexes.
Langmuir 18, 6259-6265.

149
38. Marc C. A. Stuart, J. C. v. d. P. a. J. B. F. N. E. (2005). The Use of Nile Red
to Monitor the Aggregation Behavior in Ternary Surfactant-Water-Organic
Solvent Systems. Journal of Physical Organic Chemistry 18, 929-934.

39. Flaming, J. E., Knox, R. C., Sabatini, D. A. & Kibbey, T. C. (2003).
Surfactant Effects on Residual Water and Oil Saturations in Porous Media.

40. Kuiper, H. M. & Engberts, J. B. F. N. (2004). H-Aggregation of
Azobenzene-Substituted Amphiphiles in Vesicular Membranes. Langmuir 20,
1152-1160.

41. Eastoe, J. & Vesperinas, A. (2005). Self-assembly of light-sensitive
surfactants. Soft Matter 1, 338-347.

42. Bonini, M., Berti, D., Meglio, J. M. D., Almgren, M., Teixeria, J. & Bagliono,
P. (2005). Surfactant Aggregates Hosting a Photoresponsive Amphiphile:
Structure and Photoinduced Conformational Changes. Soft Matter 1,
444-454.

43. Czikkely, V., Forsterling, H. D. & Kuhn, H. (1970). Extended Dipole Model
for Aggregates of Dye Molecules. Chem. Phys. Lett. 6, 207-210.

44. Czikkely, V., Forsterling, H. D. & Kuhn, H. (1970). Light Absorbance and
Structure of Aggregates of Dye Molecules. Chem. Phys. Lett. 6, 11-14.

45. Claire, K. & Pecora, R. (1997). Translational and Rotational Dynamics of
Collagen in Dilure Solution. Jounal of Physical Chemistry B 1997, 746-753.

46. Hassan, P. A., Narayanan, J. & Manohar, C. (2001). Vesicles and worm-like
micelles: Structure, dynamics and transformations. Current Science 80,
980-989.

47. Silva, B. F. B., Marques, E. F. & Olsson, U. (2008). Unusual Vesicle-Micelle
Transitions in a Salt-Free Catanionic Surfactant: Temperature and
Concentration Effects. Langmuir 24, 10746-10754.

48. Holmberg, K., Jonsson, B., Kronberg, B. & Lindman, B. (2002). Surfactants
and Polymers in Aqueous Solution (edition, n., Ed.), Wiley.

150
49. Eastoe, J., Dominguez, M. S., Wyatt, P. & Orr-Ewing, A. J. (2004). UV
Causes Dramatic Changes in Aggregation with Mixtures of Photoactive and
Inert Surfactants. Langmuir 20, 6120-6126.

50. Regev, O. & Khan, A. (1996). Alkyl Chain Symmetry Effects in Mixed
Cationic-Anionic Surfactant Systems. J. Colloid Interface Sci. 182, 95-109.

51. Yue, b., huang, C.-Y ., Nieh, M.-P., Glinka, C. J. & Katsaras, J. (2004). Highly
Stable Phospholipids Unilamellar Vesicles from Spontaneous Vesiculation: A
DLS and SANS Study. J. Phys. Chem. B 109, 609-616.

52. Mendes, E. & Oda, R. (1998). A small-Angle Neutron Scattering Study of a
Sheer-Induced Vesicles to Micelle Transition in Surfactant Mixtures. J. Phys.
Chem. B 102, 338-343.

53. Liu, S., Gonzalez, Y. I. & Kaler, E. W. (2003). Structural Fixation of
Spontaneous Vesicles in Aqueous Mixtures of Polymerization Anionic and
Cationic Surfactants. Langmuir 19, 10732-10738.

54. Hartshorne, N. H. & Stuart, A. (1934). Crystals and the polarising
microscope, Edward Arnold & Co.

55. Rosevear, F. B. (1954). The Microscopy of the Liquid Crystalline Nat and
Middle Phases of Soaps and Synthetic Detergents. J. Am. Oil Chem. Soc. 31,
628-639.

56. Winsor, P. A. (1966). Binary and Multicomponet Solutions of Amphiphilic
aompounds, Solubilization and the Formation of Structure, and the
Theoretical Significance of Liquid Crystalline Solutions. Chem. Rev. 68,
1-40.

57. Bortchagovsky, E. G. (2004). Reflection Polarized Light Microscopy and Its
Application to Pyrolytic Carbon Deposit. J. Appl. Phys. 95, 5192-5199.

151
Chapter 5: Suggestions for Future Work

In the previous Chapters, a novel strategy to photo-reversibly control membrane
protein folding using light-responsive surfactants was discussed. In Chapter 2,
photoresponsive surfactants were synthesized and the interfacial properties of these
surfactants were studied. In Chapter 3, bacteriorhodopsin (bR) was extracted from the
natural lipids within purple membrane fragments into photo-responsive azoTAB
surfactant micelles. In Chapter 4, photo-responsive catanionic systems were designed
to provide a potential media to solubilize membrane proteins. Further studies should
be undertaken in order to understand how to optimize the use of light-responsive
surfactants to control membrane protein folding:
1. Phase behavior of a variety of catanionic surfactant systems, and
2. Photoreversible unfolding of membrane protein (e.g., bacteriorhodopsin)
in catanionic surfactant mixtures.
Both of these topics will be discussed in further detail below.

152
5.1 Phase behavior of photoresponsive catanionic
surfactant systems.
5.1.1 AzoTAB-4 and AzoTAB-7 with and without sodium bromide as a
function of temperature
Crystallized azoTAB-4 and azoTAB-7 have been mixed at a given ratio and total
surfactant concentration (e.g., 0.1 wt %) with gentle stirring with the temperature
controlled in a water bath. Samples containing sodium bromide were prepared by
adding sodium bromide solution into azoTAB-4 and azoTAB-7 mixtures followed by
gentle stirring.
The phase behavior of the catanionic surfactant mixtures of azoTAB-1 and SDS
at various ratios with 100 mM sodium bromide are given in Table 5.2 as a function of
temperature. For comparison, azoTAB-4/azoTAB-7 mixtures without sodium bromide
were hazy (15/85, 20/80, 80/20 or 85/15) or precipitated (25/75, 30/70, 70/30, 75/25)
at various temperature (25 - 90 ° C).
For azoTAB-4/azoTAB-7 catanionic surfactant mixtures, for most ratios except
85/15, hazy and precipitated phase behaviors were investigated in aqueous solution,
which was induced by the surfactant architecture, for both azoTAB-4 and azoTAB-7,
which contain short hydrocarbon chain and it is not favorable for vesicle formation.
In previous study by Kaler etc,
1
the vesicle formation is favorable with small head
groups and large tail groups. For azoTAB-4/azoTAB-7, because of the short tails of

153
the surfactants, vesicles are not readily to form, and the oppositely charged head
group induces the precipitation.

Table 5.1: The phase behavior of azoTAB-4/azoTAB-7 catanionic surfactant
mixtures. [total surfactant] = 0.1 wt %, [sodium bromide] = 100 mM, (H: hazy, P:
precipitated, and C: clear)
azoTAB-4/ azoTAB-7 T ( ° C)
25 30 40 50 60 70 80 90
15/85 H H H H H H H H
20/80 H H H H H H H H
25/75 P P P P P P P P
30/70 P P P P P P P P
70/30 P P P P P P P P
75/25 P P P P P P P P
80/20 H H H H H H H H
85/15 C C C C C C C C

5.1.2 AzoTAB-1 and SDS with and without sodium bromide as a function of
temperature
Crystallized azoTAB-1 and SDS were mixed at various ratios and a total
surfactant concentration of 0.1 wt% with gentle stirring. The temperature was

154
controlled in water. Samples containing sodium bromide were prepared by adding
sodium bromide solution into azoTAB-1/SDS mixtures and gently stirring. The phase
behavior of the azoTAB-1/SDS catanionic surfactant mixtures is given in Table 5.2 as
a function of temperatures. For comparison, the presence of 100 mM sodium bromide
(not shown) did not affect the observed phase behaviors.

Table 5.2: The phase behavior of azoTAB-1/SDS catanionic surfactant mixtures.
[total surfactant] = 0.1 %. (H: hazy, P: precipitated, and C: clear)
azoTAB-4/SDS T ( ° C)
25 30 40 50 60 70 80 90
15/85 H H H H H H H H
20/80 H H H H H H H H
25/75 H H H H H H H H
30/70 H H H H H H H H
60/40 P P P P P P P P
70/30 P C C C C C P P
75/25 C C C C C C C C
80/20 C C C C C C C C
85/15 C C C C C C C C

155
For azoTAB-1/SDS catanionic surfactant mixtures, in SDS rich sides, hazy or
precipitated phase behavior were investigated for all the samples, which is resulted by
vesicle formation, in previous study by Yu-chuan Liu
2
, when azoTAB-1/SDBS =
31/69 and the total surfactant concentration is 0.2 wt %, vesicles were observed by
cyro-TEM. For azoTAB-rich side, clear samples were observed, and a variety of
microstructures could form, as discussed in Chapter 4.

5.1.3 AzoTAB-1 and AzoTAB-7 with and without sodium bromide as a
function of temperature
Crystallized azoTAB-1 and azoTAB-7 were mixed at various ratios and a total
surfactant concentration of 0.1 wt% with gentle stirring. The temperature was
controlled in water. Samples containing sodium bromide were prepared by adding
sodium bromide solution into azoTAB-1/ azoTAB-7 mixtures and gently stirring. The
phase behavior of the azoTAB-1/ azoTAB-7 catanionic surfactant mixtures is given in
Table 5.3 as a function of temperatures.
For azoTAB-1/azoTAB-7 catanionic surfactant mixtures, for all samples, hazy
and precipitated phase behavior were investigated in aqueous solution, which was
induced by the similarity of the two surfactant tails, in previous studies, it had been
reported that catanionic surfactant vesicles are more stable with dissimilar

156
length of the alkyl tail chain length of the cationic and anionic surfactant
components.
3

Table 5.3: The phase behavior of azoTAB-1/azoTAB-7 catanionic surfactant
mixtures. [total surfactant] = 0.1 %. (H: hazy, P: precipitated, and C: clear)
azoTAB-1/azoTAB-7 T ( ° C)
25 30 40 50 60 70 80 90
15/85 P P P P H H H H
25/75 P P P P P H H H
30/70 P P P P P H H H
70/30 P P P P P P P P
75/25 P P P P P P P P
85/15 P P P P P P P P

5.1.4 AzoTAB-1 and AzoTAB-2 with and without sodium bromide as a
function of temperature
Crystallized azoTAB-1 and azoTAB-2 were mixed at various ratios and a total
surfactant concentration of 0.1 wt% with gentle stirring. The temperature was
controlled in water. Samples containing sodium bromide were prepared by adding
sodium bromide solution into azoTAB-1/ azoTAB-2 mixtures and gently stirring. The

157
phase behavior of the azoTAB-1/ azoTAB-2 catanionic surfactant mixtures is given in
Table 5.3 as a function of temperatures.
For azoTAB-1/azoTAB-2 catanionic surfactant mixtures, cationic and anionic
surfactants contain similar alkyl chain length, meaning of the similar hydrophobicity,
inducing that vesicle formation is favorable. With increase the temperature to 90 ° C,
precipitation was observed which probably was induced by the instability of
azoTAB-2 surfactant.

Table 5.4: The phase behavior of azoTAB-1/azoTAB-2 catanionic surfactant
mixtures. [total surfactant] = 0.1 %. (H: hazy, P: precipitated and C: clear)
azoTAB-1/ azoTAB-2 T ( ° C)
25 30 40 50 90
15/85 C C C C P
20/80 C C C C P
25/75 H H H C P
30/70 H H H H P
70/30 H H H C P
75/25 H H H C P
80/20 H H H C H
85/15 H H H C P

158
5.2 Membrane proteins solubilized in catanionic
surfactant mixtures.
5.2.1 Purple membrane mixed with azoTAB-1/SDBS catanionic surfactant
mixtures (UV-vis measurement)
Purple membrane (PM) was isolated from Halobacteria Salinarium as described
in Chapter 3, Methods. PM suspended in water was then added directly into vials to
determine the concentration of the stock solution and samples were prepared by
adding the same amount of purple membrane into azoTAB-1/SDBS = 7/93 solutions
with SDBS concentrations ranging from 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1 and 2 mM. The
samples were then illuminated for 1 hr at 25 °C under gentle stirring with a 200-W
mercury arc lamp (Oriel, model no. 6283) equipped with a 400-nm long pass filter
(Oriel, model no. 59472), heat absorbing filter (Oriel, model no. 59060), and a
fiber-bundle focusing assembly (Oriel, model no. 77557) to isolate the 436-nm
mercury line. After the UV-vis spectra were recorded, the samples were then
illuminated with the arc lamp equipped with a 320-nm UV filter (Oriel model no.
59980) to isolate the 365-nm mercury line, with surfactant conversion from the trans
to the cis isomer assured with absorption measurements. The concentration of the
protein in each sample was ~0.17 mg/mL, determined spectroscopically. Preliminary
data are described below.

159
5.2.2 Preliminary results for purple membrane solubilized with
azoTAB-1/SDBS catanionic vesicles
Based on the discussion of PM with photoresponsive surfactants in Chapter 2 and
the properties of the catanionic surfactant systems in Chapter 4, the effect of
surfactant and light on the conformation of the membrane protein bR were examined
with UV-vis spectroscopy, as seen in Figures 5.1.

0
1
2
3
4
200 400 600 800
Absorbance
Wavelength (nm )
0
0.1
0.2
0.3
0.4
0.5
400 600 8 00
Absorbance
Wavelength (nm )
0
1
2
3
4
200 400 600 800
Absorbance
Wavelength (nm )
0
1
2
3
4
200 400 600 800
Absorbance
Wavelength (nm )
0
0.1
0.2
0.3
0.4
0.5
400 600 8 00
Absorbance
Wavelength (nm )

0
1
2
3
4
2 0 0 400 6 00 800
Absorbance
Wavelength (nm )
0
0.1
0.2
0.3
0.4
0.5
400 6 00 8 00
0
1
2
3
4
2 0 0 400 6 00 800
Absorbance
Wavelength (nm )
0
1
2
3
4
2 0 0 400 6 00 800
Absorbance
Wavelength (nm )
0
0.1
0.2
0.3
0.4
0.5
400 6 00 8 00
(a) (b)

Figure 5.1: Purple membrane with azoTAB-1/SDBS = 7/93 catanionic surfactant
mixture. (a) Visible light; (b) UV light. [bR] = 0.17 mg/mL. The arrow show the
effect of increasing SDBS concentration ([SDBS] = 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1 and
2 mM). The experiments were performed by Chia-hao Chang in the Lee’s lab.

From the DLS and SANS measurements in Chapter 4, vesicles are expected to
form at these concentrations under both visible and UV illumination. The residual

160
lipid concentration is approximately 0.064 mM, while azoTAB-1 concentrations
range from 0.004, 0.008, 0.012, 0.032, 0.048, 0.064, 0.08 and 0.16 mM, respectively.
As seen in Figure 5.1, when the azoTAB-1 concentration is relatively low compared
to the lipid concentration, the protein remains in the folded state as evidence by the
retinal peak at 560 nm. With increasing azoTAB-1 concentration, however, the
protein unfolded as evidenced by a loss of the peak at 560 nm, despite that fact that
vesicles are formed at this azoTAB-1/SDBS ratio. It is likely that the observed
unfolding of bR in azoTAB-1/SDBS vesicles, despite the fact that these vesicles
contain bilayers similar to the cellular bilayer, is believed to be a result of the
relatively small thickness of the vesicle bilayers compared to the natural cellular
bilayer. As discussed in Chapter 4, the bilayer thickness of azoTAB-1/SDBS vesicles
is approximately 2.8-3.0 nm, while the bilayer thickness of the natural lipids within
purple membrane is about 4.2-4.5 nm.
4
Thus, longer surfactant tails are likely
required for catanionic surfactant mixtures to solubilized folded bR (e.g., azoTAB-10,
n-octadecyl sulfate, or n-hexadecyl sulfate).

161
5.2.3 Purple membrane solubilized with azoTAB-10/SDBS catanionic
surfactant mixtures (dynamic light scattering and UV-vis measurements)
5.2.3.1 Dynamic light scattering
Dynamic light scattering measurements were performed at 25 ° C using a
Brookhaven Model BI-200SM instrument (Brookhaven Instrument Corp.) equipped
with a 35 mW (Melles Griot, model no. 05-LHP-928) HeNe (632.8 nm) laser and an
avalanche photodiode detector (BI-APD).
AzoTAB-10 and SDBS aqueous solutions were separately prepared at the given
concentrations and mixed at the appropriate ratios separately through 450 nm syringe
filters (Anatop). After vortexing for at least 1 min followed by 1 hr to thermally
equilibrate, the scattering intensity was then measured at a scattering angle of 90
degree. The conversion of the surfactant to the cis isomer was achieved through UV
illumination with an 84 W long wave UV lamp-365 nm (Spectroline, Model no.
XX-15A). The data were analyzed with the NNLS software package using
BI-9000AT digital correlator (Brookhaven Instrument Corp.).

5.2.3.2 Preliminary dynamic light scattering results for azoTAB-10/SDBS
catanionic mixtures
Dynamic light scattering measurements of azoTAB-10/SDBS mixtures at
different total surfactant concentrations (e.g., 0.1 wt %, 0.25 wt %, and 0.5 wt %, etc.)

162
and different azoTAB-10/SDBS ratios (3/93 for 0.1 wt %, 0.25 wt%, and 0.5 wt %;
1/99 for 0.5 wt %) were collected. A summary of DLS parameters are shown in Table
5.5 under both visible and UV illumination.

Table 5.5: Dynamic light scattering data for azoTAB-10/SDBS catanionic
mixtures. (V = vesicles, M = micelles)
azoTAB-10/SDBS 0.1 wt % 0.25 wt % 0.5 wt %
Vis (nm) UV (nm) Vis (nm) UV (nm) Vis (nm) UV (nm)
3/97 V: 144.7 V: 134.7 V: 159.4 V: 146.1 V: 188.2 V: 178.7
1/99 Not measured Not measured V: 161.5 V: 164.4
M: 12.1

The data generally follow the trends that vesicle size increases upon approaching
equimolar conditions, while the apparent vesicle size also increases with increases of
the overall surfactant concentration (similar to the azoTAB-1/SDBS catanionic
surfactant mixtures in Chapter 4). For the azoTAB-1/SDBS system, a vesicles ↔
micelles transition was observed at ratio a 1/99 when the total surfactant
concentration was 0.1 wt %. In contrast, for the azoTAB-10/SDBS a similar vesicles
↔ micelles transition was observed at a total surfactant concentration of 0.5 wt %.
The reason for this phenomenon appears to be that azoTAB-10, which has a longer

163
hydrocarbon tail and is thus more hydrophobic than azoTAB-1, more readily forms
vesicles in aqueous solution.

5.2.3.3 Preliminary results for purple membrane with azoTAB-10/SDBS
The sample preparation is the same as described in section 5.2.1 except that the
protein concentration was ~0.14 mg/mL or 0.045 mg/mL. For a membrane protein
concentration of 0.14 mg/mL, the catanionic surfactant mixture conditions were
azoTAB-10/SDBS = 3/97 and 93/7, while the total surfactant concentration was 0.1
wt %, as shown in Figure 5.2. For a membrane protein concentration is ~0.045
mg/mL, the catanionic surfactant mixture conditions were azoTAB-10/SDBS = 1/99,
while the total surfactant concentration was varied from 0.05 wt %, 0.1 wt %, 0.25 wt
% and 0.5 wt %, as shown in Figure 5.3.

164

0
1
2
3
4
200 4 00 600 8 00
3/97 trans
3/97 cis
7/93 trans
7/93 cis
Absorbance
Wavelength (nm )

Figure 5.2: Purple membrane with azoTAB-10/SDBS. [Protein] = 0.14 mg/mL,
[total surfactant] = 0.1 wt %, cuvette path length = 1 cm.

-1
0
1
2
3
4
200 4 00 600 8 00
Pure PM
0.05 w t% trans
0.1 w t% trans
0.25 w t% trans
0.5 w t % trans
Absorb
-1
0
1
2
3
4
200 4 00 600 8 00
0.05 w t% cis
0.1 w t% cis
0.25 w t% cis
0.5 w t% cis
Absorbance
Wavelength (nm )
ance
Wavelength (nm )
(a) (b)

Figure 5.3: Purple membrane with azoTAB-10/SDBS with light illumination, (a)
visible light; (b) UV light. [protein] = 0.045 mg/mL, azoTAB-10/SDBS = 1/99.

165
From Figure 5.2, when the total surfactant concentration was 0.1 wt% and the
protein concentration was 0.14 mM, the residual lipid concentration was about 0.053
mM, while the azoTAB-10 concentrations were about 0.07 mM (3/97) or 0.16 mM
(97/3), respectively. Bacteriorhodopsin remains in the folded state in
azoTAB-10/SDBS vesicles under visible light illumination, while upon UV
illumination the protein is unfolded. This phenomenon appears to be induced by the
shorter cis conformation which would form smaller thickness vesicles in aqueous
(meaning the thickness is not large enough for protein folding with UV illumination).
A similar phenomenon is observed in Figure 5.3, as increases in surfactant
concentration lead to higher ratios of azoTAB-10 relative to the residual purple
membrane lipids. For a purple membrane concentration 0.045 mg/ml, the lipid
concentration is about 0.016 mM, while azoTAB concentrations are 0.011 mM, 0.022
mM, 0.055 mM and 0.11 mM. Under visible light the membrane protein is observed
to be partially-folded under these conditions (i.e., a peak at 560 nm is still evident but
smaller that the peak for pure PM), while the protein is found to be unfolded under
UV light (i.e., the 560-nm peak has disappeared). Comparing the azoTAB-10/SDBS
and azoTAB-1/SDBS catanionic surfactant systems, the longer and more hydrophobic
azoTAB-10 surfactant, and the larger bilayer thickness that is expected to result for
the azoTAB-10/SDBS vesicles, appears to promote proper membrane protein folding.

166
In order to achieve larger bilayer thicknesses, azoTAB-10/n-octadecyl sulfate and
azoTAB-10/n-hexadecyl sulfate could be studied. n-Octadecyl sulfate and
n-hexadecyl sulfate are analoges of SDS with longer hydrocarbon tails, which thus
exhibit lower solubility in aqueous solution. Crystallized azoTAB-10 and n-octadecyl
sulfate or n-hexadecyl sulfate were directly added into the vials at the given ratio and
then water is added. After 1 min vortexing, the phase behaviors were investigated by
eye. As a result, for azoTAB-10/n-octadecyl sulfate and azoTAB-10/n-hexadecyl
sulfate mixtures at a ratio of about 30/70 and a total surfactant concentration of 0.025
wt %, 0.5 wt %, or 0.1 wt %, precipitation was observed.

5.3 Conclusions
Phase behaviors of a variety of catanionic surfactant mixtures have been studied
along with the solubilization of purple membrane in azoTAB-1/SDBS and
azoTAB-10/SDBS catanionic mixtures. Because the bilayer thickness is not large
enough for proper bacteriorhodopsin folding; purple membrane was unfolded in
azoTAB-1/SDBS catanionic surfactant mixtures. For azoTAB-10/SDBS catanionic
surfactant mixtures, bacteriorhodopsin keeps a partially folded conformation under
visible illumination and an unfolded conformation upon UV exposure. These results
suggest a general method by which catanionic vesicles can be designed to mimic the
natural bilayer thickness such that solubilization in the completely-folded state

167
(within bilayers under visible light) could be followed by solubilization in the
unfolded state (within micelles under UV light) to photo-control membrane protein
folding.

168
5.4 References
1. Kaler, E. W., Murthy, A. K., Rodriguez, B. E. & Zasadzinski, J. A. N. (1989).
Spontaneous Vesicle Formation in Aqueous Mixtures of Single-Tailed
Surfactants. Science 245, 1371-1374.

2. Liu, Y ., Le Ny, A.-L. M., Schmidt, J., Talmon, Y., Chmelka, B. F. & Lee, C. T.
J. (2008). Photo-Assisted Gene Delivery Using Light -Responsive Catanionic
Vesicles. J. Am. Chem. Soc. summited.

3. Sakai, H., Matsumura, A., Yukoyama, S., Saji, T. & Abe, M. (1999).
Photochemical Swiching of Vesicle Formation Using an
Azobenzene-Modified Surfactant. Jounal of Physical Chemistry B 103,
10737-10740.

4. Cabarcos, E. L., Bermudez, S. F. & Balta, C. (1981). The paracrystalline
nature of the purple membrane of halobacterium halobium II. Packing of the
membrane. Colloid. Polym. Sci. 259, 641-644.

169
Bibliography

Allen, S. J., Curran, A. R., Templer, R. H., Meijberg, W. and Booth, P. J. Folding
Kinetics of an Alpha Helical Membrane Protein in Phospholipid Bilayer Vesicles. J.
Mol. Biol. 342, 1279-1291 (2004).

Allen, S. J., Kim, J.-M., Khorana, H. G., Lu, H. and Booth, P. J. Structure and
Function in Bacteriorhodopsin: The Effect of the Interhelical Loops on the Protein
Folding Kinetics. J. Mol. Biol. 308, 423-435 (2001).

Almgren, M. Mixed micelles and other structures in the solubilization of bilayer lipid
membranes by surfactants. Biochim. Biophys. Acta 1508, 146-163 (2000).

Barni, E., Barolo, C., Quagliotto, P., Valldeperas, J. and Viscardi, G. Novel
Azobenzene Derivatives Containing a Glucopyranoside Moiety. Part I: Synthesis,
Characterisation and Mutagenic Properties. Dyes Pigm. 46, 29-36 (2000).

Berg, J. M., Tymoczko, J. L. and Stryer, L. Biochemistry (ed. Edition, F.) (W. H.
Freeman and Company, 2002).

Bergstrom, M. and Pedersen, J. S. Small-Angle Neutron Scattering (SANS) Study of
Aggregates Formed from Aqueous Mixtures of Sodium Dodecyl Sulfate (SDS) and
Dodecyltrimethylammonium Bromide (DTAB). Langmuir 14, 3754-3761 (1998).

Bergstrom, M. and Pedersen, J. S. A Small-Angle Neutron Scattering (SANS) Study
of Talet-Shaped and Ribbonlike Micelles Formed From Mixtures of an Anionic and a
Catiionic Surfactant. J. Phys. Chem. B 103, 8502-8513 (1999).

Beyer, K. Packing and Bilayer-Micelle Transitions in Mixed Surfactant-Lipid
Systems as Studied by Solid State NMR. Progr Colloid Poly Sci 103, 78-86 (1997).

Bonini, M., et al. Surfactant Aggregates Hosting a Photoresponsive Amphiphile:
Structure and Photoinduced Conformational Changes. Soft Matter 1, 444-454 (2005).

Booth, P. J. and Farooq, A. Intermediates in the Assembly of Bacteriorhodopsin
Investigated by Time-resolved Absorption Spectroscopy. Eur. J. Biochem. 246,
674-680 (1997).

170
Booth, P. J., Farooq, A. and Flitsch, S. L. Retinal Binding during Folding and
Assembly of the Membrane Protein Bacteriothodopsin. Biochemistry 35, 5902-5909
(1996).

Booth, P. J., et al. Intermediates in the folding of the membrane protein
bacteriothodopsin. Structural biology 2, 139-143 (1995).

Booth, P. J., et al. Evidence That Bilayer Bending Rigidity Affects Membrane Protein
Folding. Biochemistry 36, 197-203 (1997).

Booth, P. J., Stern, L. J., Greenhalgh, D. A., Kim, P. S. and Khorana, G. H.
Intermediates in the folding of the membrane protein bacteriorhodopsin. Nat. Struct.
Biol. 2, 139-143 (1995).

Booth, P. J., et al. In vitro studies of membrane protein folding. Critical Reviews in
Biochemistry and Molecular Biology 36, 501-603 (2001).

Bortchagovsky, E. G. Reflection Polarized Light Microscopy and Its Application to
Pyrolytic Carbon Deposit. J. Appl. Phys. 95, 5192-5199 (2004).

Braiman, M., Bousche, O. and KJ, P. Protein Dynamics in the Bacteriorhodopsin
Photocycle: Submillisecond Fourier Transform Infrared Spectra of the L, M, and N
photointermediates. Proc. Natl. Acad. Sci. U.S.A. 88, 2388-2392 (1991).

Braiman, M. S., Stern, L. J., Chao, B. H. and Khorana, H. G. Structure-function
studies on bacteriorhodopsin. IV. Purification and renaturation of bacterio-opsin
polypeptide expressed in Escherichia coli. Journal of Biological Chemistry 262,
9271-6 (1987).

Brasher, L. L., Herrington, K. L. and Kaler, E. W. Electrostatic Effects on the Phase
Behavior of Aqueous Cetyltrimethylammonium Bromide and Sodium Octyl Sulfate
Mixtures with Added Sodium Bromide. Langmuir 11, 4267-4277 (1995).

Brasher, L. L. and Kaler, E. W. A Small-Angle Neutron Scattering (SANS) Contrast
Variation Investigation of Aggregate Composition in Catanionic Surfactant Mixtures.
Langmuir 12, 6270-6276 (1996).

Bucak, S., Robinson, B. H. and Fontana, A. Kinetics of Induced Vesicle Breakdown
for Cationic and Catanionic Systems. Langmuir 18, 8288-8294 (2002).

171
Buchanan, S. K. Overexpression and refolding of an 80-kDa iron transporter from the
outer membrane of Escherichia coli. Biochemical Society Transactions 27, 903-908
(1999).

Cabarcos, E. L., Bermudez, S. F. and Balta, C. The paracrystalline nature of the
purple membrane of halobacterium halobium II. Packing of the membrane. Colloid.
Polym. Sci. 259, 641-644 (1981).

Caillet, C., Hebrant, M. and Tondre, C. Sodium Octyl
Surfate/Cetyltrimethylammonium Bromide Catanionic Vesicles: Aggregate
Composition and Probe Encapsulation. Langmuir 16, 9099-9102 (2000).

Chen, G. Q. and Gouaux, E. Probing the Folding and Unfolding of Wild-Type and
Mutant Forms of Bacteriorhodopsin in Micellar Solutions: Evaluation of Reversible
unfolding Conditions. Biochemistry 38, 15380-15387 (1999).

Chen, G.-Q. and Gouaux, J. E. Overexpression of bacterio-opsin in Escherichia coli as
a water-soluble fusion to maltose binding protein: efficient regeneration of the fusion
protein and selective cleavage with trypsin. Protein Science 5, 455-67 (1996).

Cladera, J., Galisteo, M. L., Sabes, M., Mateo, P. L. and Padros, E. The role of retinal
in the thermal stability of the purple membrane. European Journal of Biochemistry
207, 581-585 (1992).

Cladera, J., Sabes, M. and Padros, E. Fourier Transform Infrared Analysis of
Bacteriorhodopsin Secondary Structure. Biochemistry 31, 12363-12368 (1992).

Cladera, J., Torres, J. and Padros, E. Analysis of Conformatioal Changes in
Bacteriorhodopsin Upon Retinal Removal. Biophys. J . 70, 2882-2887 (1996).

Claire, K. and Pecora, R. Translational and Rotational Dynamics of Collagen in
Dilure Solution. J. Phys. Chem. B 1997, 746-753 (1997).

Clandera, J., Galisteo, M. L., Sabes, M., Mateo, P. L. and Padros, E. The role of
retinal in the thermal stability of the purple membrane. Eur. J. Biochem. 207, 581-585
(1992).

Cullis, P. R. and Kruijff, B. D. Lipid Polymorphism and the Functional Roles of
Lipids in Biological Membranes. Biochim. Biophys. Acta 559, 399-420 (1979).

172
Czikkely, V., Forsterling, H. D. and Kuhn, H. Extended Dipole Model for Aggregates
of Dye Molecules. Chem. Phys. Lett. 6, 207-210 (1970).

Czikkely, V., Forsterling, H. D. and Kuhn, H. Light Absorbance and Structure of
Aggregates of Dye Molecules. Chem. Phys. Lett. 6, 11-14 (1970).

Dekker, N., Merck, K., Tommassen, J. and Verheij, H. M. In vitro folding of
Escherichia coli outer-membrane phospholipase A. European Journal of Biochemistry
232, 214-19 (1995).

Delong, L. M. and Russo, P. S. Thermodynamics and Dynamic Behavior of
Semiflexible Polymers in the Iostropic Phase. Macromolecules 24, 6139-6155 (1991).

Doren, H. A. v., Smits, E., Pestman, J. M., Engberts, J. B. F. N. and Kellogg, R. M.
Mesogenic Surgars. From Aldoses to Liquid Crystals and Surfactants. Chem. Soc. Rev.
29, 183-199 (2000).

Dov Lichtenberg, R. J. R. a. E. A. D. Solubilization of Phospholipids by Detergents
Structural and Kinetic Aspects. Biochimica et Biophysica Acta 737, 285-304 (1983).

Drew, D., Froderberg, L., Baars, L. and De Gier, J.-W. L. Assembly and
overexpression of membrane proteins in Escherichia coli. Biochimica et Biophysica
Acta 1610, 3-10 (2003).

Eastoe, J., Dominguez, M. S., Cumber, H., Burnett, G. and Wyatt, P. Photoresponsive
Microemulsions. Langmuir 19, 6579-6581 (2003).

Eastoe, J., Dominguez, M. S., Cumber, H. and Wyatt, P. Light-Sensitive
Microemulsion. Langmuir 20, 1120-1125 (2004).

Eastoe, J., Dominguez, M. S., Wyatt, P. and Orr-Ewing, A., J. UV Causes Dramatic
Changes in Aggregation with Mixtures of Photoactives and Inert Surfactants.
Langmuir 20, 6120-6126 (2004).

Eastoe, J. and Vesperinas, A. Self-assembly of Light-sensitive Surfactants. Soft Matter
1, 338-347 (2005).

Eastoe, J., et al. Photoinduced Vesicles. American Chemical Society 22, 851-853
(2006).

173
Eloi Feitosa, F. R. A., Anna Niemiec, M. Elisabete C. D. Real Oliveira, Elisabete M.
S. Castanheira, and Adelina L. F. Baptista. Cationic Liposomes in Mixed
Didodecyldimethylammonium Bromide and Dioctadecyldimethylammonium
Bromide Aqueous Dispersions Studied by Differential Scanning Calorimetry, Nile
Red Fluorescence, and Turbidity. Langmuir 22, 3579-3585 (2006).

Ferguson, A. D., Breed, J., Diederichs, K., Welte, W. and Coulton, J. W. An internal
affinity-tag for purification and crystallization of the siderophore receptor FhuA,
integral outer membrane protein from Escherichia coli K-12. Protein Science 7,
1636-1638 (1998).

Findlay, H. E. and Booth, P. J. The biological significance of lipid-protein interactions.
J. Phys.: Condens. Matter 18, 1281-1291 (2006).

Flaming, J. E., Knox, R. C., Sabatini, D. A. and Kibbey, T. C. Surfactant Effects on
Residual Water and Oil Saturations in Porous Media. Vandose Zone Journal 2,
168-176 (2003).

Frankel, M. and Wolovsky, R. Wavelength Dependence of Photoisomerization
Equilibria in Azocompounds. J. Chem. Phys 23, 1367-1368 (1955).

Fromherz, P. and Ruppel, D. Lipid Vesicle Formation: the Transition from Open
Disks to Closed Shells. FEBS Lett. 179, 155-159 (1985).

Glinka, C. J., et al. The 30 m small-angle neutron scattering instruments at the
National Institute of Standards and Technology. J. Appl. Crystallogr. 31, 430-445
(1998).

Gonzalez, Y. I., Stjerndahl, M., Dganit, D. and Kaler, E. W. Spontaneous Vesicle
Formation and Phase Behavior in Mixtures of an Anionic Surfactant with Imidazoline
Compounds. Langmuir 20, 7053-7063 (2004).

Gordeliy, V. I., Schlesinger, R., Efremov, R., Buldt, G. and Heberle, J. Crystallization
in lipidic cubic phases: a case study with bacteriorhodopsin. Methods Mol Biol 228,
305-316 (2003).

Gouaux, G. Q. C. a. E. Probing the Folding and Unfolding of wild-Type and Mutant
Forms of Bacteriorhodopsin in Micellar Solutions: Evaluation of Reversible
Unfolding Conditions. Biochemistry 38, 15380-15387 (1999).

174
Grabner, D., et al. Phase Behavior of Aqueous Mixtures of
2-Phenylbenzimidazole-5-sulfonic Acid and Cetyltrimethylammonium Bromide:
Hydrogels, Vesicles, Tubules, and Ribbons. J. Phys. Chem. B 112, 2901-2908 (2008).

Griffiths, J. II. Photochemistry of Azobenzene and its Derivatives. Chem. Soc. Rev. 1,
481-493 (1972).

Grigorieff, N., Ceska, T. A., Downing, K. H., Baldwin, J. M. and Henderson, R.
Electron-crystallographic Refinement of the Structure of Bacteriorhodopsin. J. Mol.
Biol. 259, 393-421 (1996).

Hamill, A. and Lee, J., C. Ted. Solution Structure of an Amyloid-Forming Protein
During Photoinitiated Hexamer-Dodecamer Transitions Revealed through
Small-Angle Neutron Scattering. Biochemistry 46, 7694 (2007).

Hamill, A., Wang, S.-C. and Lee, J., C. Ted. Probing Lysozyme Conformation with
Light Reveals a New Folding Intermediate. Biochemistry 44, 15139-15149 (2005).

Hamill, A. C., Wang, S.-c. and Lee, C. T. J. Solution Structure of an
Amyloid-Forming Protein During Photoinitiated Hexamer-Dedecamer Transitions
Revealed through Small-Angle Neutron Scattering. Biochemistry 46, 7694-7705
(2007).

Hansen, J.-P. and Hayter, J. B. A Rescaled MSA Structure Factor for Dilute Charged
Colloidal Dispersion. Mol. Phys 46, 651-656 (1982).

Happe, M. and Overath, P. Bacteriorhodopsin depleted of purple membrane lipids.
Biochem. Biophys. Res. Commun. 72, 1504-1511 (1976).

Hartshorne, N. H. and Stuart, A. Crystals and the polarising microscope (Edward
Arnold & Co., 1934).

Hassan, P. A., Fritz, G. and Kaler, E. W. Small Angle Neutron Scattering Study of
Sodium Dodecyl Sulfate Micellar Growth Driven by Addition of a Hydrotropic Salt. J.
Colloid Interface Sci. 257, 154-162 (2003).

Hassan, P. A., Narayanan, J. and Manohar, C. Vesicles and worm-like micelles:
Structure, dynamics and transformations. Current Science 80, 980-989 (2001).

175
Hayashita, T. and Bartsch, R. A. Selective Concentration of Lead(II) Chloride
Complex with Liquid Anion-Exchange Membranes. Anal. Chem. 63, 1023-1027
(1991).

Hayashita, T., Kurosawa, T., Miyata, T., Tanaka, K. and Igawa, M. Effect of structural
variation within cationic azo-surfactant upon photoresponsive function in aqueous
solution. Colloid. Polym. Sci. 272, 1611-1619 (1994).

He, J.-A., Samuelson, L., Li, L., Kumar, J. and Tripathy, S. K. Oriented
Bacteriorhodopsin/Polycation Multilayers by Electrostatic Layer-by-Layer Assembly.
Langmuir 14, 1674-1679 (1998).

Helenius, A. and Simons, K. Solubiliztion of Membranes by Detergents. Biochim.
Biophys. Acta 415, 29-79 (1975).

Henderson, R., et al. Model for the structure of bacteriorhodopsin based on
high-resolution electron cryo-microscopy. J. Mol. Biol. 213, 899-929 (1990).

Heyes, C. D. and El-Sayed, M. A. The Role of the Native lipids and Lattice Structure
in Bacteriorhodopsin Protein Conformation and Stability as Studied by
Temperature-dependent Fourier Transform-Infrared Spectroscopy. J. Biol. Chem. 277,
29437-29443 (2002).

Hiemenz, P. C. and Rajogopalan, R. Principles of Colloid and Surface Chemistry
(Marcel Dekker, Inc., New Youk., 1997).

Holmberg, K., Jonsson, B., Kronberg, B. and Lindman, B. Surfactants and Polymers
in Aqueous Solution (ed. edition, n.) (Wiley, 2002).

Huang, K.-S., Bayley, H., Liao, M.-J., London, E. and Khorana, H. G. Refolding of an
integral membrane protein. Denaturation, renaturation, and reconstitution of intact
bacteriorhodopsin and two proteolytic fragments. Journal of Biological Chemistry
256, 3802-9 (1981).

Huang, K.-S. and Khorana, G. H. Delipidation of bacteriorhodopsin and reconstitution
with exogenous phospholipid. Biochemistry 77, 323-327 (1980).

Hubbard, F. P. J. and Abbott, N. L. Effect of Light on Self-Assembly of Aqueous
Mixtures of Sodium Dodecyl Sulfate and a Cationic , Bolaform Surfactant Containing
Azobenze. Langmuir 23, 4819-4829 (2007).

176
Hubbard, F. P. J., Santonicola, G., Kaler, E. W. and Abbott, N. L. Small-Angle
Neutron Scattering from Mixtures of Sodium Dodecyl Sulfate and a Cationic,
Bolaform Surfactant Containing Azobenzene. Langmuir 21, 6131-6136 (2005).

Hwang, S.-B. and Stoeckenius, W. Purple Membrane Vesicles: Morphology and
Proton Translocation. J. Membrane Biol. 33, 325-350 (1977).

Jacrot, B. The Study of Biological Structures by Neutron Scattering from Solution.
Rep. Prog. Phys. 39, 911-953 (1976).

Joshi, M. K., Dracheva, S., Mukhopadhyay, A. K., Bose, S. and Hendler, R. W.
Importance of Specific Native Lipids in Controlling the Photocycle of
Bacteriorhodopsin. Biochemistry 37, 14463-14470 (1998).

Kahn, T. W., Sturtevant, J. M. and Engelman, D. M. Thermodynamic measurements
of the contributions of helix-connecting loops and of retinal to the stability of
bacteriorhodopsin. Biochemistry 31, 8829-8839 (1992).

Kaler, E. W., Murthy, A. K., Rodriguez, B. E. and Zasadzinski, J. A. N. Spontaneous
Vesicle Formation in Aqueous Mixtures of Single-Tailed Surfactants. Science 245,
1371-1374 (1989).

Kiefer, H. In vitro folding of alpha-helical membrane proteins. Biochimica et
Biophysica Acta 1610, 57-62 (2003).

Kim, J.-M., Booth, P. J., Allen, S. J. and Khorana, H. G. Structure and Function in
Bacteriorhodopsin: The Role of the Interhelical Loops in the Folding and Stability of
Bacteriorhodopsin. J. Mol. Biol. 308, 409-422 (2001).

Kleinschmidt, J. H. Membrane Proteins-Introduction. CMLS 60, 1527-1528 (2003).

Kline, S. R. Reduction and Analysis of SANS and USANS Data using Igor Pro. J.
Appl. Cryst. 39, 895 (2006).

Kouyama, T., Bogomolni, R. A. and Stoeckenius, W. Photoconversion from the
light-adapted to the dark-adapted state of bacteriorhodopsin. Biophys. J. 48, 201-8
(1985).

177
Kouyama, T. and Nasuda-Kouyama, A. Turnover Rate of the Proton Pumping Cycle
of Bacteriorhodopsin: pH and Light-Intensity Dependences. Biochemistry 28,
5968-5970 (1989).

Krimm, S. and Dwivedi, A. M. Infrared Spectrum of the Purple Membrane: Clue to a
Proton Conduction Mechanism? Science, New Series 216, 407-408 (1982).

Kubota, K., Tominaga, Y., Fujime, S., Jun, O. and Ikegami, A. Dynamic Light
Scattering Study of Suspensions of Purple Membrane. Biophys. Chem. 23, 15-29
(1985).

Kuhlbrandt, W. Bacteriorhodopsin-the movie. Nature 406, 569-570 (2000).

Kuiper, H. M. and Engberts, J. B. F. N. H-Aggregation of Azobenzene-Substituted
Amphiphiles in Vesicular Membranes. Langmuir 20, 1152-1160 (2004).

Kumar, S., Naqvi, A. Z., Aswal, V. K., Goyal, P. S. and Kabir-ud-Din. A SANS Study
on Growth of Anionic Micelles with Quaternary Ammonium Bromide. Current
Science 84, 1346-1349 (2003).

Kuo-Sen Huang, H. H., and H. Gobind Khorana. Delipidation of bacteriorhodopsin
and reconstitution with exogenous phospholipid. Biochemistry 77, 323-327 (1980).

Lau, F. W. and Bowie, J. U. A Method for Assessing the Stability of a Membrane
Protein. Biochemistry 36, 5884-5892 (1997).

Lazarova, T. and Padros, E. Helical and Reverse Turn Changes in the BR to N
Transition of Bacteriorhodopsin. Biochemistry 35, 8354-8358 (1996).

Le Ny, A.-L. M. and Lee, C. T. J. Photoreversible DNA Condensation Using
Light-Responsive Surfactants. J. Am. Chem. Soc. 128, 6400-6408 (2006).

Lee, C. T., Jr., Smith, K. A. and Hatton, T. A. Photoreversible Viscosity Changes and
Gelation in Mixtures of Hydrophobically Modified Polyelectrolytes and
Photosensitive Surfactants. Macromolecules 37, 5397-5405 (2004).

Lee, C. T. J., Smith, K. A. and Hatton, T. A. Photocontrol of Protein Folding: The
Interaction of Photosensitive Surfactants with Bovine Serum Albumin. Biochemistry
44, 524-536 (2005).

178
Liao, X. and Higgins, D. A. Scanning Probe Microscopy Studies of Mesostructured
Nonstoichiometric Polyelectrolyte-Surfactant Complexes. Langmuir 18, 6259-6265
(2002).

Lichtenberg, D. Characterization of the Solubilization of Lipid Bilayers by
Surfactants. Biochim. Biophys. Acta 821, 470-478 (1985).

Lichtenberg, D., Opatowski, E. and Kozlov, M. M. Phase boundaries in mixtures of
membrane-forming amphiphiles and micelle-forming amphiphiles. Biochim. Biophys.
Acta 1508, 1-19 (2000).

Lichtenberg, D, Robson, R. and Dennis, E. A. Solubilization of Phospholipids by
Detergents Structural and Kinetic Aspects. Biochimica et Biophysica Acta 737,
285-304 (1983)

Liu, H., et al. Effect of Electrostatic Interactions on the Structure and Dynamics of a
Model Polyelectrolyte. I. Diffusion. J. Chem. Phys. 113, 6001-6010 (1998).

Liu, S., Gonzalez, Y. I. and Kaler, E. W. Structural Fixation of Spontaneous Vesicles
in Aqueous Mixtures of Polymerization Anionic and Cationic Surfactants. Langmuir
19, 10732-10738 (2003).

Liu, Y., et al. Photo-Assisted Gene Delivery Using Light -Responsive Catanionic
Vesicles. J. Am. Chem. Soc. summited (2008).

Loll, P. J. Membrane protein structural biology: the high throughput challenge.
Journal of Structural Biology 142, 144-153 (2003).

London, E. and Feigenson, G. W. Phosphorus NMR Analysis of Phospholipids in
Detergents. J. Lipid Res. 20, 408-412 (1979).

London, E. and Khorana, H. G. Denaturation and Renaturation of Bacteriorhodopsin
in Detergents and Lipid-Detergent Mixtures. J. Biol. Chem. 257, 7003-7011 (1982).

Luecke, H., Schobert, B., Richter, H.-T., Cartailler, J.-P. and Lanyi, J. K. Structure of
Bacteriorhodopsin at 1.55 A Resolution. J, Mol. Biol. 291, 899-911 (1999).

Lyklema, J. Fundamentals of Interface and Colloid Science: Soft Collids (Academic
Press, 2005).

179
Maire, M. l., Champeil, P. and Moller, J. V. Interaction of membrane proteins and
lipids with solubilization detergents. Biochim. Biophys. Acta 1508, 86-111 (2000).

Marc C. A. Stuart, J. C. v. d. P. a. J. B. F. N. E. The Use of Nile Red to Monitor the
Aggregation Behavior in Ternary Surfactant-Water-Organic Solvent Systems. Journal
of Physical Organic Chemistry 18, 929-934 (2005).

Marques, E. F., Regev, O., Khan, A., Miguel, M. d. G. and Lindman, B. Vesicle
Formation and General Phase Behavior in the Catanionic Mixture SDS-DDAB-Water.
The Anionic-Rich Side. J. Phys. Chem. B 102, 6746-6758 (1998).

Marti, T., Otto, H., Rosselet, S. J., Heyn, M. P. and Gobind, K. H. Consequences of
Amino Acid Insertiions and/or Deletions in Transmembrane Helix C of
Bacteriorhodopsin. Proc. Natl. Acad. Sci. U.S.A. 89, 1219-1223 (1992).

Mendes, E. and Oda, R. A small-Angle Neutron Scattering Study of a Sheer-Induced
Vesicles to Micelle Transition in Surfactant Mixtures. J. Phys. Chem. B 102, 338-343
(1998).

Michel, H., Oesterhelt, D. and Henderson, R. Orthorhombic two-dimensional crystal
form of purple membrane. Proc. Natl. Acad. Sci. U.S.A. 77, 338-342 (1980).

Minetti, C. A. S., et al. Structural and functional characterization of a recombinant
PorB class 2 protein from Neisseria meningitidis. Conformational stability and porin
activity. Journal of Biological Chemistry 272, 10710-10720 (1997).

Mitsuoka, K., et al. The Structure of Bacteriorhodopsin at 3.0 A Resolution Based on
Electron Crystallography: Implication of the Charge Distribution. J. Mol. Biol. 286,
861-882 (1999).

Mohanty, A. K., Simmons, C. R. and Wiener, M. C. Inhibition of tobacco etch virus
protease activity by detergents. Protein Expression and Purification 27, 109-114
(2003).

Mukerjee, P. and Chan, C. C. Effects of High Salt Concentrations on the Micellization
of Octyl Glucoside: Salting-out of Monomers and Electrolyte Effects on the
Micelle-Water Interfacial Tension. Langmuir 18, 5375-5381 (2002).

Mukerjee, P. and Mysels, K. J. Critical Micelles Concentrations of Aqueous
Surfactant Systems. (National Bureau of Standards., Washington, DC., 1971).

180
Mukhopadhyay, A. K., Dracheva, S., Bose, S. and Hendler, R. W. Control of the
Integral Membrane Proton Pump, Bacteriorhodopsin, by Purple Membrane Lipids of
Halobacterium halobium. Biochemistry 35, 9245-9252 (1996).

Nieh, M.-P., Glinka, C. J. and Krueger, S. SANS Study of the Structural Phases of
Magnetically Alignable Lanthanide-Doped Phospholipid Mixtures. Langmuir 17,
2629-2638 (2001).

O'Connor, A. J. and Hatton, T. A. Dynamics of Micelle-Vesicle Transitions in
Aqueous Anionic/Cationic Surfactant Mixtures. Langmuir 13, 6931-6940 (1997).

Oesterhelt, D. and Stoeckenius, W. Functions of a New Photoreceptor Membrane.
Proc. Nat. Acad. Sci. U.S.A. 70, 2853-2857 (1973).

Oesterhelt, D. and Stoeckenius, W. Isolation of Cell Membrane of Halobacterium
Halobium and Its Fractionation into Red and Purple Membrane. Methods Enzymol. 31,
667-678 (1974).

Opella, S. J., Nevzorov, A., Mesleh, M. F. and Marassi, F. M. Structure determination
of membrane proteins by NMR spectroscopy. Biochemistry and Cell Biology 80,
597-604 (2002).

Overath, M. H. a. P. Bacteriorhodopsin depleted of purple membrane lipids.
Biochemical and Biophysical Research Communication 72, 1504-1511 (1976).

Parodi, L. A. and Lozier, R. H. Procedure for Testing Kinetic Models of the
Photocycle of Bacteriorhodopsin. Biophys. J . 38, 161-174 (1982).

Paul, C. and Rosenbusch, J. P. Folding Patterns of Porin and Bacteriorhodopsin.
EMBO J. 4, 1593-1597 (1985).

Paulsen, H., Finkenzeller, B. and Kuehlein, N. Pigments induce folding of
light-harvesting chlorophyll a/b-binding protein. European Journal of Biochemistry
215, 809-16 (1993).

Popot, J.-L. and Engelman, D. M. Helical Membrane Protein Folding, Stability, and
Evolution. Annu. Rev. Biochem 69, 881-922 (2000).

Popot, J.-L. and Engelman, D. M. Membrane Protein Folding and Oligomerization:
The Two-Stage Model. Biochemistry 29, 4031-4037 (1990).

181
Popot, J.-L., Trewhella, J. and Engelman, D. M. Reformation of Crystalline Purple
Membrane from Purified Bacteriorhodopsin Fragments. EMBO J. 5, 3039-3044
(1986).

Prive, G. G. and Kaback, H. R. Engineering the lac permease for purification and
crystallization. Journal of Bioenergetics and Biomembranes 28, 29-34 (1996).

Qi, H. L., Tai, J. Y. and Blake, M. S. Expression of large amounts of neisserial porin
proteins in Escherichia coli and refolding of the proteins into native trimers. Infection
and immunity 62, 2432-9 (1994).

Regev, O. and Khan, A. Alkyl Chain Symmetry Effects in Mixed Cationic-Anionic
Surfactant Systems. J. Colloid Interface Sci. 182, 95-109 (1996).

Renthal, R. and Cha, C.-H. Charge Asymmetry of The Purple Membrane Measured
by Uranyl Quenching of Dansyl Fluorescence. Biophys. J . 45, 1001-1006 (1984).

Rhoades, E. and Gafni, A. Micelle Formation by a Fragment of Human Islet Amyloid
Polypeptide. Biophys. J . 84, 3480-3487 (2003).

Riley, M. L., Wallace, B. A., Flitsch, S. L. and Booth, P. J. Slow Alpha Helix
Formation during Folding of a Membrane Protein. Biochemistry 36, 192-196 (1997).

Robertson, B. and Lukashev, E. P. Rapid pH Change due to Bacteriorhodopsin
Measured with a Tin-Oxide Electrode. Biophys. J . 68, 1507-1517 (1995).

Romsted, L. S. and Yoon, C.-O. Counterion Affinity Orders in Aqueous Micellar
Solutions of Sodim Decyl Phosphate and Sodium Dodecyl Sulfate Determined by
Changes in 23Na NMR Relation Rates: A Surprising Dependence on Head Group
Change. J. Am. Chem. Soc. 115, 989-994 (1993).

Rosen, M. J. Surfactant and Interfacial Phenomena (John Wiley & Sons, Inc., 2004).

Rosevear, F. B. The Microscopy of the Liquid Crystalline Nat and Middle Phases of
Soaps and Synthetic Detergents. J. Am. Oil Chem. Soc. 31, 628-639 (1954).

Sackett, D. L. and Wolff, J. Nile Red As a Polarity-Sensitive Fluorescent Probe of
Hydrophobic Protein Surfaces. Anal. Biochem. 167, 228-234 (1987).

182
Sakai, H., Matsumura, A., Yukoyama, S., Saji, T. and Abe, M. Photochemical
Swiching of Vesicle Formation Using an Azobenzene-Modified Surfactant. J. Phys.
Chem. B 103, 10737-10740 (1999).

Shang, T., Smith, K. A. and Hatton, T. A. Photoresponsive Surfactants Exhibiting
Unusually Large, Reversible Surface Tension Changes under Varying Illumination
Conditions. Langmuir 26, 10764-10773 (2003).

Shioi, A. and Hatton, T. A. Model for Formation and Growth of Vesicles in Mixed
Anionic/Cationic (SOS/CTAB) Surfactant Systems. Langmuir 18, 7341-7348 (2002).

Silva, B. F. B., Marques, E. F. and Olsson, U. Unusual Vesicle-Micelle Transitions in
a Salt-Free Catanionic Surfactant: Temperature and Concentration Effects. Langmuir
24, 10746-10754 (2008).

Skibinska, L., et al. Effect of Electrostatic Interactions on the Structure and Dynamics
of a Model Polylectrolyte. II. Intermolecular Correlations. J. Chem. Phys. 110,
1794-1800 (1999).

Sonoyama, M., Hasegawa, T., Nakano, T. and Mitaku, S. Isomerization of retinal
chromophore and conformational changes in membrane protein bacteriorhodopsin by
solubilization with a mild non-ionic detergent, n-octyl-beta-glucoside: an FT-Raman
and FT-IR spectroscopic study. Vib. Spectrosc 35, 115-119 (2004).

Soroka, K., Vithanage, R. S., Phillips, D. A., Walker, B. and Dasgupta, P. K.
Fluresence Properties of Metal Complexes of 8-Hydroxyquinoline-5-Sulfonic Acid
and Chromatographic Applications. Anal. Chem. 59, 629-636 (1987).

Sportelli, L. and Bartucci, R. Electrostatic interaction on purple membrane: a spin
label study on pH and ionic-strength effects. Nuovo Cimento Soc. Ital. Fis., D 6,
609-617 (1985).

Stoeckenius, W., Lozier, R. H. and Bogomolni, R. A. Bacteriorhodopsin and the
Purple Membrane of Halobacteria. Biochim. Biophys. Acta 505, 215-278 (1979).

Surrey, T. and Jaehnig, F. Kinetics of folding and membrane insertion of a b-barrel
membrane protein. Journal of Biological Chemistry 270, 28199-203 (1995).

183
Torres, J., Sepulcre, F. and Padros, E. Conformational Changes in Bacteriorhodopsin
Associated with Protein-Protein Interactions: A Functional alfa
I
-alfa
II
Helix Switch.
Biochemistry 34, 16320-16326 (1995).

Tsuchiya, K., Nakanishi, H., Sakai, H. and Abe, M. Temperature-Dependent Vesicle
Formation of Aqueous Solutions of Mixed Cationic and Anionic Surfactants.
Langmuir 20, 2117-2122 (2004).

Ulrich, K.-H., Maire, M. l., Noel, J.-P., Tadeusz, G.-K. and Moller, J. V. Transitional
Steps in the Solubilization of Protein-Containing Membranes and Liposomes by
Nonionic Detergent. Biochemistry 32, 1648-1656 (1993).

Valentin I. Gordeliy, R. S., Rouslan Efremov, Georg Buldt, and Joachim Heberle.
Crystallization in Lipidic Cubic Phases. Methods in Enzymology 228, 305-316
(1988).

Valpuesta, J. M., Arrondo, J.-L. R., Barbero, M. C., Pons, M. and Goni, F. M.
Membrane-Surfactant Interaction. J. Biol. Chem. 261, 6578-6584 (1986).

Wang, J. and EL-Sayed, M. A. The Effect of Protein Conformation Change from
Alpha
II
to Alpha
I
on the Bacteriorhodopsin Photocycle. Biophys. J . 78, 2031-2036
(2000).

Wang, J. and El-Sayed, M. A. Temperature Jump-Induced Secondary Structural
Change of the Membrane Protein Bacteriorhodopsin in the Premelting Temperature
Region: A Nanosecond Time-Resolved Fourier Transform Infrared Study. Biophys. J .
76, 2777-2783 (1999).

Wang, J., Heyes, C. D. and El-Sayed, M. A. Refolding of Thermally Denatured
Bacteriorhodopsin in Purple Membrane. J. Phys. Chem. B 106, 723-729 (2002).

Wang, S.-C. and Lee, C. T., Jr. Protein Secondary Structure Controlled with Light and
Photoresponsive Surfactants. J. Phys. Chem. B 110, 16117-16123 (2006).

Wang, S.-c. and Lee, C. T. J. Enhanced Enzymatic Activity through Photoreversible
Conformational Changes. Biochemistry 46, 14557-14566 (2007).

Whitten, D. G. Photochemistry and Photophysics of trans-Stilbene and Related
Alkenes in Surfactant Assemblies. Acc. Chem. Res 26, 502-509 (1993).

184
Windson, C. G. An Introduction to Small-Angle Neutron Scattering. J. Appl. Cryst. 21,
582-588 (1988).

Winson, P. A. Binary and Multicomponent Solutions of Amphiphilic Compounds.
Solubilization and the Formation, Structure, and Theoretical Significance of Liquid
Crystalline Solutions. Chem. Rev. 68, 1-40 (1976).

Winsor, P. A. Binary and Multicomponet Solutions of Amphiphilic aompounds,
Solubilization and the Formation of Structure, and the Theoretical Significance of
Liquid Crystalline Solutions. Chem. Rev. 68, 1-40 (1966).

Xia, Y ., Goldmints, I., Johnson, P. W., Hatton, T. A. and Bose, A. Temporal Evolution
of Microstructures in Aqueous CTAB/SOS and CTAB/HDBS Solutions. Langmuir 18,
3822-3828 (2002).

Yerushalmi, H., Lebendiker, M. and Schuldiner, S. EmrE, an Escherichia coli 12-kDa
multidrug transporter, exchanges toxic cations and H+ and is soluble in organic
solvents. Journal of Biological Chemistry 270, 6856-63 (1995).

Yin, H., Zhou, Z., Huang, J., Zheng, R. and Zhang, Y . Temperature-Induced
Micelle to Vesicle Transition in the Sodium
Dodecylsulfate/Dodecyltriethylammonium Bromide System. Angew. Chem. Int. Ed
42, 2188-2191 (2003).

Yu, W.-Y., Yang, Y.-M. and Chang, C.-H. Cosolvent Effects on the Spontaneous
Formation of Vesicles from 1:1 Anionic and Cationic Surfactant Mixtures. Langmuir
21, 6185-6193 (2005).

Yue, b., huang, C.-Y., Nieh, M.-P., Glinka, C. J. and Katsaras, J. Highly Stable
Phospholipids Unilamellar Vesicles from Spontaneous Vesiculation: A DLS and
SANS Study. J. Phys. Chem. B 109, 609-616 (2004).

Zhang, J., Wang, S.-c. and Lee, C. T. J. Photoreversible Conformational Changes in
Membrane Proteins Using Light-responsive Surfactant. J. Phys. Chem. B Summitted
(2008).

185

Abstract (if available)

Abstract Membrane proteins are of significant importance, performing a variety of biological functions including pumps, channels, and receptors. Thus, membrane proteins represent attractive candidates as drug targets. Bacteriorhodopsin (bR), the most widely studied membrane protein, consists of seven transmembrane helical segments and functions which can work as a proton pump in Halobacterium Salinarium. In the present study, the reversible control of bR conformation with simple light illumination is examined, providing a protocol to probe membrane protein folding (a challenge even to this day due to the large, aggregation-prone hydrophobic regions of membrane proteins compared to soluble proteins). Two general methodologies are utilized to control membrane protein folding, including (1) saturation of the natural lipids with a photoresponsive surfactant resulting in partitioning of the protein into detergent-lipid mixed micelles in the unfolded state, and (2) the development of artificial bilayers through self-assembly of the photosurfactant into light-responsive vesicles to solubilize membrane proteins. The azobenzene-based photosurfactant undergoes a reversible photoisomerization upon illumination either visible (trans) or UV (cis) light. The trans isomer is relatively hydrophobic and, thus, readily forms detergent-lipid mixed micelles relative to the cis form, while the planar trans conformation also enhances the formation of artificial lamellar structures in vesicle bilayers relative to the bent cis form. Together, these strategies provide a convenient means to control membrane protein folding with light illumination.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Zhang, Jing (author)

Core Title Controlling membrane protein folding with light illumination and catanionic surfactant systems

School Andrew and Erna Viterbi School of Engineering

Degree Doctor of Philosophy

Degree Program Chemical Engineering

Publication Date 12/08/2008

Defense Date 10/28/2008

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag catanionic surfactant systems,light responsive surfactant,membrane protein,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Lee, C. Ted, Jr. (committee chair), Langen, Ralf (committee member), Shing, Katherine S. (committee member)

Creator Email sanshow800@gmail.com,zhang4@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1891

Unique identifier UC1470842

Identifier etd-Zhang-2544 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-143167 (legacy record id),usctheses-m1891 (legacy record id)

Legacy Identifier etd-Zhang-2544.pdf

Dmrecord 143167

Document Type Dissertation

Rights Zhang, Jing

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu