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Controlling membrane protein folding using photoresponsive surfactant

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CONTROLLING MEMBRANE PROTEIN FOLDING USING
PHOTORESPONSIVE SURFACTANT

by

Chia Hao Chang

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

August 2012

Copyright 2012 Chia Hao Chang
ii

Acknowledgements

I would like to express my deepest appreciation to those people who give me
guidance, collaboration and support throughout the research.
I would like to thank my advisor Dr. Ted Lee, who gives me opportunity to work
in his lab and pursue the exciting project.
I would like to thank my committee members, Dr. Katherine Shing and Dr. Ralf
Langen, who take time to read my thesis and give me helpful suggestions.
I would like to thank for my colleagues, Yu-chuan Liu, Kiki (Jing) Zhang and
Khiza Mazwi, who give me great help in experiments and discuss the projects with me. I
would like to thank for Karen, Tina and Shokry, who give me help whenever I need it.
The most importantly, I would like to thank my parents and my brother, who give
me support and love. Without their encouragement, I would not finish the dissertation.

iii

Table of Contents

Acknowledgements ii
List of Tables v
List of Figures vi
Abstract ix
Chapter 1: Introduction 1
1.1 Catanionic Surfactant System 1
1.2 Photoresponsive Surfactants 3
1.3 Membrane Proteins 4
1.4 Bacteriorhodopsin 4
1.5 References 7

Chapter 2: The Structure Change of Light Effect on Sodium Dodecyl
Benzenesulfonate (SDBS) and 4-ethyl-4’(trimethylaminobutoxy)
Azobenzene Bromide (azoTAB) Catanionic System 9
2.1 Abstract 9
2.2 Introduction 10
2.3 Experimental Section 14
2.4 Results and Discussion 18
2.5 Conclusions 44
2.6 References 48

Chapter 3: Conformational Change of Membrane Protein by Using Photo-
sensitive Catanionic Surfactant System 51
3.1 Abstract 51
3.2 Introduction 51
3.3 Material and Methods 54
3.4 Results and Discussion 57
3.5 Conclusions 69
3.6 References 70

Chapter 4: Conformation Change of Bacteriorhodopsin through Photoresponsive
Surfactant 72
4.1 Abstract 72
iv

4.2 Introduction 73
4.3 Material and Methods 77
4.4 Results and Discussion 81
4.5 Conclusions 91
4.6 References 92

Chapter 5 Conclusion and Future Work 95
5.1 Conclusion 95
5.2 References 97

Bibliography 98

v

List of Tables

Table 2.1: DLS-determined size of photoresponsive catanionic vesicles as a
function of the azoTAB/SDBS molar ratio and overall surfactant
concentration (wt %) under visible and UV illumination. Average
error ~5%. Micellar and lamellar regions are denoted in bold and
italics, respectively. 45

Table 2.2: 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, Pa: parameters, V: vesicle, M: micelles. 46

Table 3.1: Dynamic light scattering measurement of effective diameter for
azoTAB/SOS system between visible light and UV light.
(molar ratio is 3:97, standard deviations given in parentheses) 59

Table 4.1: Fits of the SANS Data of refold solutions at different azoTAB
concentration by using Ellipsoid form factor model. Radius of
gyration values obtained from PDDF and Guinier analyses. 87

vi

List of Figures

Figure 1.1: Ternary phase diagram for CTAB/SOS (cetyltrimethylammonium
bromide/ sodium octyl sulfate) water system. 2

Figure 1.2: trans and cis azoTAB chemical structures. 3

Figure 2.1: Fluorescence emission spectra of Nile red in 60/40 (molar)
azoTAB/SDBS mixtures under (a) visible and (b) UV exposure.
[Nile red] = 0.1 M. The concentration shown is azoTAB
concentration, T = 25 C. 20

Figure 2.2: CAC of the azoTAB/SDBS catanionic mixtures at given ratios
under visible light and UV illumination. [Nile red]: 0.1 m,
T = 25 C. 21

Figure 2.3: 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 M and nile red at
0.1 μM. 23

Figure 2.4: Zeta potential of azoTAB/SDBS in visible and UV illumination. 25

Figure 2.5: 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. 28

Figure 2.6: UV-Vis absorbance sptectra of azoTAB/SDBS and deconvolution
peak (a) 20/80 in dark, (b) 20/80 in visible illumination, (c) 20/80
in UV illumination; total surfactant concentration is 0.1 wt%. 30

Figure 2.7: (a) Hydrodynamic diameters (RH) 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. 33

vii

Figure 2.8: 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. 37

Figure 2.9: 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. 40

Figure 2.10: Polarized optical microscopy images with visible light
illumination. Overall surfactant concentration: 1 wt%.
(Scale: 5 m). 44

Figure 3.1: Scattered light intensities for aqueous mixtures of azoTAB/SOS.
(molar ratio 3:97) 60

Figure 3.2: Part (a) and (b) represent diameter distribution of the total
surfactant concentration at 0.24 wt% between visible light and
UV light in azoTAB/SOS mixture. Part (c) and (d) are the
diameter distribution at 0.56 wt%. The diameter distributions are
measured with DLS. The solution was aged for 7 days. 60

Figure 3.3: Folding percentage of bR as a function of surfactant
concentration. A, B, C and D are the folding fraction of the visible
↔ UV light cycles in different surfactant concentration. BR
concentration is 0.8 mg/mL in each sample. The inset shows the
folding percentage difference between visible light and UV light
at 0.56 wt%, 0.64 wt% and 0.72 wt%. 62

Figure 3.4: In-situ measurement of PM-azoTAB/SOS system. The total
surfactant concentrations are 0.24 wt% and 0.56 wt%. The
absorbance at 350 nm and 560 nm is a function of time. 64

Figure 3.5: Selected individual UV-vis spectra from in-situ measurement at
0.56 wt% (A: pure purple membrane. B: t=0, start to shine UV
light. C: t=4.5 min, UV light. D: t=11.5 min, start to shine visible
light. E: t=19.5 min, visible light.) 65

viii

Figure 3.6: Diameter distributions of 0.24 wt% and 0.56 wt%
azoTAB/SOS/PM aqueous mixtures. [bR]=0.8mg/mL. Part (a) is
the diameter distribution of 0.24 wt% solution exposed to visible
light for 20 min. Part (b) is the diameter distribution of the
solution exposed to UV light for the other 20 min. The same
solution to take the other 20 min illumination of visible light is
part (c). Part (d), (e) and (f) represent the diameter distributions
in the 20 min illumination of visible, UV and visible light in the
0.56 wt% solution. 66

Figure 4.1: Relative folding percentage of bR refolds from different
concentration of DMPC/CHAPS and DOPC/CHAPS systems
as a function of azoTAB concentration determined from the
protein absorbance at 560 nm. DMPC/CHAPS, A: 1% / 1% at
trans; B: 0.75%/ 0.75% at trans; C: 0.5%/0.5% at trans;
D: 1% / 1% at cis; E: 0.75%/ 0.75% at cis; F: 0.5%/0.5% at cis.
DOPC/CHAPS, G: 1%/ 1% at trans; H: 1%/ 1% at cis. 82

Figure 4.2: UV-vis spectra of bR fold at different azoTAB concentraion. 83

Figure 4.3: SANS data and uniform ellipsoid fits for bR fold in
DMPC/CHAPS as a function of azoTAB concentration and bR
fold in DOPC/CHAPS solution. The scattering data have been
scaled for better distinguish the curves. The inset shows the
original intensity. 85

Figure 4.4: PDDFs of bR in DMPC/CHAPS and DOPC/CHAPS solutions
as a function of the azoTAB concentration under both visible
(trans) and UV (cis) light illumination. 88

ix

Abstract

Membrane proteins perform a number of roles in biological function. Membrane
lipids can self assembly into numerous different phases in aqueous solution, including
micelles, vesicles and lamellar phases. However, the phase properties of biological
membranes are far more complex. Many membrane proteins require specific lipids to be
present in the membrane to be fully active. Therefore, artificial membrane-like
environments for protein folding are studied. Many approaches are developed to solve the
membrane protein folding problems and based on manipulating the bilayer structures.
Bacteriorhodopsin (bR) is 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 provides a method to control membrane
protein folding. The azobenzene-based photosurfactant undergoes a reversible
photoisomerization upon illumination either visible (trans) or UV (cis) light. The trans
isomer is relatively hydrophobic and planar than cis isomer. The phase behavior and
bilayer structures are changed by the effect of photoresponsive surfactant. These
strategies provide a convenient means to control membrane protein folding with light
illumination.

1

Chapter 1: Introduction
1.1 Catanionic Surfactant System
Catanionic surfactant system consists of mixtures of cationic and anionic
surfactant in water. The mixtures can produce a large variety of differently morphological
aggregate microstructures, such as spherical and rod-like micelles, vesicles and lamellar
structures.
1-5

Many examples of behaviors are shown in the triangular phase diagrams for
catanionic mixtures, for example, DTAB/SDS (dodecyltrimethylammonium bromide/
sodium dodecyl sulfate)
6
, CTAB/SOS (cetyltrimethylammonium bromide/ sodium octyl
sulfate)
6-8
and CTAT/SDBS ( cetyltrimethylammonium tosylate/ sodium
dodecylbenzenesulfonates).
5

Figure 1.1 represents the ternary phase diagram of CTAB/SOS water system.
One-phase regions are unshaded, such as vesicles (V), rodlike micelles (R), micelles (M).
Two-phase regions are shaded, such as rodlike micelles and vesicles (R +V), vesicles and
lamellar phases (V + L). The phase diagram was observed that CTAB-rich vesicle lobe is
small and narrow in extent, but SOS-rich vesicle lobe is considerably larger. The micelle
to vesicle or vesicle to micelle phase transition is also considerable interest. SOS-rich
samples exhibit different phases from lower surfactant concentration to higher surfactant
concentration. There is limited micelle growth with added CTAB or increased dilution.
Some of the structures based on a bilayer are the most important in nature. Vesicles are
self-closed bilayer aggregates in a nearly spherical shape. There are growing interests in
2

the preparation of bilayer vesicles to encapsulate biological substances, such as proteins,
enzymes or DNA. Pharmaceutical or chemical applications were developed such as
biological membrane models, gene therapy and drug encapsulation.
9-11
The formation of
catanionic vesicles has detailed investigations in the thermodynamics. A small variation,
including pH, ionic strength, temperature, etc. may induce a reorganization of the
surfactant molecules into phase separation or structural transition.
12

Figure 1.1: Ternary phase diagram for CTAB/SOS (cetyltrimethylammonium bromide/
sodium octyl sulfate) water system.
8

3

1.2 Photoresponsive Surfactants
The photoresponsive cationic surfactant 4-ethyl-4’ (trimethylaminobutoxy)
azobenzene bromide (azoTAB) was synthesized according to the published procedures.
13,
14
In summary, the azobenzene surfactant is prepared by azocoupling of alkylaniline with
phenol, followed by alkylation and quarternization. The final product was purified by
recrystallization from ethanol.

trans cis

Figure 1.2: trans and cis azoTAB chemical structures.

AzoTAB structure was shown in Figure 1.2. Azobenzene compounds are known
as to form trans and cis isomers induced by photo illumination. The presence of the
double bond and the sp
2
hybridization of the nitrogen impose the molecular to be planar
at trans form. The cis isomer of azobenzene exists at a distorted conformation due to
steric interactions between the ortho hydrogen of the benzene rings.
15
The azoTAB
surfactant exists as the trans isomers (~100% trans) in dark environment and decreases
the trans composition to ~90% in visible light. AzoTAB was converted to the cis form
when illuminated with UV light (~80% cis). UV-Vis spectroscopy can determine the
photoisomeric state. The trans isomer exhibits a maximum absorbance at 350 nm and the
cis isomer at 434 nm. The trans isomer is significantly more hydrophobic than the cis
form. Therefore, the photo illumination can induce change of many physical properties,
4

such as surface tension, electrical conductivity and critical micelle concentration
(CMC).
13

1.3 Membrane Proteins
Membrane proteins are responsible for most of the dynamic processes carried out
by membranes. Membrane lipids form a permeability barrier and establish compartments,
whereas specific proteins mediate nearly all membrane functions.
16
Membrane proteins
constitute 20-30% of all the proteins in a given cell.
17
The primary difficulty encountered
in the study of membrane proteins is that the obtainment of protein. Membrane proteins
usually have low concentration in biological membranes and get a low yield of a single
protein species. The other difficulty is the membrane proteins are naturally attached in the
lipid bilayer. It is very difficult to maintain its function in the non-native environment.
Membrane proteins are not generally soluble in aqueous solution. The special
environment needed to work in vitro to satisfy the hydrophobicity of membrane
proteins.
18, 19
Therefore, solubilization and reconstitution of membrane proteins often
require a detergent or a lipid system.
1.4 Bacteriorhodopsin
Bacteriorhodopsin (bR) is a good case to study because it is the only protein
present in the purple membrane of Halobacteria salinaria.
18
Purple membrane is laid in
patches of about 0.5 µm in diameter and contains 75 wt% bacteriorhodopsin and 25 wt%
lipids.
20, 21
90% are polar and 10% are nonpolar of these lipids.
22
Bacteriorhodopsin
consists of a single polypeptide chain of 248 amino acid residues and a retinal is
covalently attached to Lys-216 via a Schiff-base linkage.
23
Nearly 70% of amino acid
5

residues in bacteriorhodopsin are hydrophobic. Bacteriorhodopsin is a small integral
membrane protein (Mr ~ 26kD). Bacteriorhodopsin traverses the membrane seven times
in the form of α-helices which are designated A to G from the N- to C- terminus. A
retinal chromophore is covalently linked on the helix G. Bacteriorhodopsin have the
phenomenon of light-adapted and dark-adapted forms.
24
Retinal is solely in the 13-cis
configuration in the light adaption. Bacteriorhodopsin contains retinal in the mixture of
all trans and 13-cis configuration in the dark. The optical properties of the light and dark
adaption are different. Light-adapted bacteriorhodopsin has the maximum absorption
around 570 nm, but dark-adapted bacteriorhodopsin shows slightly blue shift relatively.
25

Bacteriorhodopsin has been recognizes as the prime protein for studying the
interaction between proteins and surfactants. Bacteriorhodopsin can be refolded in a
variety of detergent and lipid-based systems.
18, 26, 27
A general refolding strategy of
bacteriorhodopsin is solubilization of the apoprotein bacterioopsin in SDS (sodium
dodecyl sulfate) to remove lipids and retinals from purple membrane. Addition of mixed
phospholipid/detergent to the denatured state of bacterioopsin (bO) can restore full native
secondary structure, such as DMPC/CHAPSO,
28
DMPC/CHAPS,
26
systems. Furthermore,
all trans retinal added to the mixture can regenerate a native-like chromophore with a
maximum absorption at 560 nm.
29

The importance of study the interaction of membrane proteins and surfactants
cannot be underestimated, because of its direct effects on the stability and functionality of
these proteins outside their native biological membranes. To study how surfactants bind
to solubilized membrane proteins can provide a path toward understanding the
mechanism of the surfactant molecules interface with the hydrophobic domain of proteins.
6

An ideal situation for membrane protein to work in vitro is to resemble a similar natural
lipid bilayer environment. A perfect surfactant has the ability to preserve protein activity
and conformation in solution.

7

1.5 References
1. Hao, J. C.; Hoffmann, H. Current Opinion in Colloid & Interface Science 2004, 9,
279- 293.

2. Svenson, S. Current Opinion in Colloid & Interface Science 2004, 9, 201-212.

3. Salkar, R. A.; Mukesh, D.; Samant, S. D.; Manohar, C. Langmuir 1998, 14, 3778-
3782.

4. Marques, E. F. Langmuir 2000, 16, 4798-4807.

5. Kaler, E. W.; Herrington, K. L.; Murthy, A. K.; Zasadzinski, J. A. N. Journal of
Physical Chemistry 1992, 96, 6698-6707.

7. Shioi, A.; Hatton, T. A. Langmuir 2002, 18, 7341-7348.

8. Yatcilla, M. T.; Herrington, K. L.; Brasher, L. L.; Kaler, E. W.; Chiruvolu, S.;
Zasadzinski, J. A. Journal of Physical Chemistry 1996, 100, 5874-5879.

9. Bramer, T.; Dew, N.; Edsman, K. Journal of Pharmaceutical Sciences 2006, 95,
769-780.

10. Caillet, C.; Hebrant, M.; Tondre, C. Langmuir 2000, 16, 9099-9102.

11. Fischer, A.; Hebrant, M.; Tondre, C. Journal of Colloid and Interface Science
2002, 248, 163-168.

12. Zhu, Z. Y.; Xu, H. X.; Liu, H. W.; Gonzalez, Y. I.; Kaler, E. W.; Liu, S. Y.
Journal of Physical Chemistry B 2006, 110, 16309-16317.

13. Hayashita, T.; Kurosawa, T.; Miyata, T.; Tanaka, K.; Igawa, M. Colloid &
Polymer Science 1994, 272, 1611-1619.

14. Lee, C. T. Macromolecules 2004, 37, 5397-5405.

15. El Halabieh, R. H.; Mermut, O.; Barrett, C. J. Pure Applied Chemistry 2004, 76,
1445-1465.

16. Berg, J. M.; Tymoczko, J. L.; Stryer, L., Biochemistry. W. H. Freeman and
Company: 2002.

17. Kleinschmidt, J. H. Cellular and Molecular Life Sciences 2003, 60, 1527-1528.

18. Seddon, A. M.; Curnow, P.; Booth, P. J. Biochimica et Biophysica Acta-
Biomembranes 2004, 1666, 105-117.
8

19. Drew, D.; Froderberg, L.; Baars, L.; de Gier, J. W. Biochim Biophys Acta 2003,
1610, 3-10.

20. Blaurock, A. E. Methods in Enzymology 1982, 88, 124-132.

21. Happe, M.; Overath, P. Biochemical and Biophysical Research Communications
1976, 72, 1504-1511.

22. Kates, M.; Kushwaha, S. C.; Sprott, G. D. Methods in Enzymology 1982, 88, 98-
111.

23. Gerber, G. E.; Khoranat, H. G. Methods in Enzymology 1982, 88, 56-74.

24. Oesterhelt, D.; Meentzen, M.; Schuhmann, L. Eur J Biochem. 1973, 40, 453-463.

25. Braiman, M. S.; Stern, L. J.; Chao, B. H.; Khorana, H. G. J. Biol. Chem. 1987,
262, 9271-9276.

26. Booth, P. J.; Flitsch, S. L.; Stern, L. J.; Greenhalgh, D. A.; Kim, P. S.; Khorana, H.
G. Nat. Struct. Biol. 1995, 2, 139-143.

27. Rigaud, J. L.; Paternostre, M. T.; Bluzat, A. Biochemistry 1988, 27, 2677-2688.

28. Chen, G. Q.; Gouaux, E. Biochemistry 1999, 38, 15380-15387.

29. Huang, K. S.; Bayley, H.; Liao, M. J.; London, E.; Khorana, H. G. J. Biol. Chem.
1981, 256, 3802-3809.

9

Chapter 2: The Structure Change of Light Effect on Sodium Dodecyl
Benzenesulfonate (SDBS) and 4-ethyl-4’(trimethylaminobutoxy)
Azobenzene Bromide (azoTAB) Catanionic System
Chia Hao Chang, Jing Zhang and C. Ted Lee, Jr.
*

2.1 Abstract
We have studied the structure change of surfactant mixtures of two oppositely
charged surfactants by switching the light wavelength in expose. In mixture of positively
charged azobenzene-based photoresponsive surfactant (azobenzenetrimethylammonium
bromide, azoTAB) and a negatively charged alkyl-based surfactants (sodium
dodecylbenzenesulfonate, SDBS), the aqueous catanionic surfactants have been
examined with fluorescence and UV-vis spectroscopy, dynamic light scattering, zeta
potential measurements, small-angle neutron scattering and optical microscopy. AzoTAB
undergoes reversible photoisomerization to the relatively-hydrophobic trans isomer or
the relatively-hydrophilic cis isomer upon exposure to 434-nm visible or 350-nm UV
light, respectively. This results in the formation of light-responsive vesicles that can be
disrupted and spontaneously reformed with simple light illumination. Critical aggregation
concentrations (CACs), namely the surfactant concentrations where vesicle first appear,
are determined using the hydrophobic fluorescence probe Nile red to report on bilayer
formation. The measurement of zeta potential shows that light switch from visible light to
UV light, the surface charge of azoTAB/SDBS catanionic surfactants may have dramatic
change from positive to negative charge at some ratios. The structure of solution was
constructed by using DLS and SANS measurements as well as and polarized optical
10

microscopy Photo-initiated transitions between micelles, vesicles, and lamellar structures
were observed with both visible and UV light illumination, depending on the solution
conditions. In most cases, vesicles were observed under visible light, while micelles or
relatively small vesicles were observed upon UV illumination.

2.2 Introduction
Aqueous “catanionic” systems consisting of mixtures of cationic and anionic
surfactants have received considerable attention over the last several years,
1-5
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 at
surfactant concentrations exceeding critical micelle concentrations (CMCs) typically in
the millimolar range, catanionic mixtures can form a rich variety of microstructures,
including micelles, 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, cation-to-anion
ratio, temperature, and other environmental factors.
2
Catanionic vesicles are of particular
interest due to potential applications as mimics for biological membranes, drug delivery,
and microreactors.
8
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”).
1-3, 5

11

In last ten years, catanionic surfactant mixtures consisting of photoresponsive
surfactant such as 4-butylazobenzene-4’-(oxyethyl)trimethylammonium bromide
(AZTMA)
9
and bis(trimethylammoniumhexyloxy)azobenzene dibromide (BTHA)
10
have
been studied. Azobenzene-based surfactants exists primarily in the trans form under the
room light and the cis form under 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 was illuminated under the UV light, the maximum
absorption was changed to 434 nm with the dipole moment to ~ 3.1 D, as shown in
Scheme 2.1.
11-15

Scheme 2.1: 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
 = 3.1 D  = 0.5 D  = 3.1 D  = 0.5 D  = 3.1 D  = 0.5 D
 = 3.1 D  = 0.5 D  = 3.1 D  = 0.5 D  = 3.1 D  = 0.5 D
UV light
Visible light
 = 3.1 D
 = 0.5 D
12

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.
9
Same phenomena happened
in BTHA and SDS mixture.
10
The great advantage of the catanionic surfactant mixture
containing photoresponsive 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, 16
SANS,
6, 7
fluorescence spectroscopy,
17, 18

electron microscopy etc.
19

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.
20
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
13

depending on mixture formation. Another application is to increase the gene transfection
by light illumination.
21
In our previous study, catanionic surfactant mixture containing
azobenzen-based surfactant and SDBS is used to increase gene transfection.
21
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.
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 property 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.

14

2.3 Experimental Section
Materials. The surfactant 4-ethyl-4’(trimethylaminobutoxy) azobenzene bromide
(azoTAB) was made as previously described.
22, 23
Generally, the azobenzene surfactant
was prepared by azocoupling of ethylaniline with phenol, followed by alkylation and
quaternalization with dibromobutane and trimethylamine. Successive recrystallization
was carried out until the final azoTAB product was deemed pure by
1
H-NMR
measurements. Conductivity measurements also ensured the remove of residual salts. All
chemicals were purchased from Sigma-Aldrich in the highest purity and used as received
unless otherwise mentioned.
Critical aggregation concentration (CAC) measurements. Fluorescence
measurements using Nile red as the probe were performed on a Quanta-Master
spectrofluorometer model QM-4 (Photon Technology International) at 25 C using
excitation and emissions wavelengths of 560 nm and 650 nm, respectively, and slit
widths of 4 nm. The spectrofluorometer was initially loaded with 2 mL of a 0.1 μM Nile
red solution, followed by the separate addition of the necessary microliter quantities of
concentrated azoTAB and SDBS solutions to achieve the desired overall surfactant
concentration and azoTAB/SDBS ratio. To avoid photobleaching of Nile red, the
azoTAB solution was pre-exposed to an 84 W long wave UV lamp-365 nm (Spectroline,
model no. XX-15A) to convert to cis form and then added to Nile red in the dark. UV-vis
spectroscopy confirmed that azoTAB remained in the cis state following measurements,
as the half-life of thermal conversion back to the trans form is on the order of 24 hr.
Zeta potential measurements. Zeta potential measurements were performed
using a Zetasizer NANO ZS (Malvern Instruments) to determine the electrophoretic
15

mobility, µ(cm
2
V
-1
S
-1
) by several individual scans. Electrophoretic mobility is acquired
by performing an electrophoresis experiment on the solution and measuring the velocity
of the particles by Laser Doppler velocimetry. Solutions of azoTAB and SDBS were
individually prepared at the desired concentrations and then mixed in the appropriate
proportion and allowed to reach equilibrium over 24 hr in the lab room light. The total
surfactant concentration for each sample is 0.1wt%. The vesicle samples were then
loaded into 1-mL folded capillary cells containing electrodes, and zeta potential
measurements were performed at 37 °C. After measuring the dark samples, the same
samples conversion of the surfactant from trans to the cis isomer was achieved with UV
illumination from an 84 W long wave UV lamp-365 nm (Spectroline, Model no. XX-
15A) for 2 hr. The data were analyzed by dispersion technology software (Malvern
Instruments LTD.). The data is expressed as the zeta potential which can be calculated
by using the Henry equation.
(1)
,where u is the electrophoretic mobility, v is the rate of the particles, E is the electric field
of strength, ε is dielectric constant, ζ is zeta potential, η is viscosity, and the function
f
1
( κa ) depends on the shape of the particles. We consider the shape is spherical particles.
The parameter κ is a convenient way to characterize the thickness of the double layer. α
was considered as radius of spherical and nonconducting particles.
24

UV-Vis measurements. Stock solutions of azoTAB and SDBS were separately
prepared and stored in the dark for 2 days and then mixed and votexed for 1 min to result
in catanionic mixtures containing 0.1 mM azoTAB with varying and SDBS
concentration. Absorption measurements were performed on an Agilent model 8453 UV-
16

vis spectrophotometer using 0.2 cm path length cuvette. UV-vis absorption spectra where
then recorded in the dark and under room light conditions. The same samples were then
exposed to an 84 W long wave UV lamp-365 nm (Spectroline, Model no. XX-15A) for at
least 2 hr to convert the surfactant from the trans to cis state, and the absorption spectra
were again recorded. When specified, samples were centrifuged at 10000rpm for 30 min
(Microfuge® 18 centrifuge, Beckman Coulter) and 0.5 ml of the supernatant was
collected. We measure the UV-vis absorption and analyze the the deconvolution at peak
350nm, 440nm, 313nm and 241nm.
In situ detection of vesicle formation and disruption. Catanionic vesicles were
prepared in 2 mL DI water which contain azoTAB and SDBS in the appropriate ratios
and 0.1 μM concentration of Nile red. The time-based fluorescence intensities of Nile red
were measured at 25 C using a Quanta-Master model QM-4 spectrofluorometer (Photon
Technology International) using excitation and emission wavelengths of 560 nm and 650
nm, respectively, and slit widths of 4 nm. Samples were illuminated in situ using a 200 W
mercury arc lamp (Oriel, model no. 6283) equipped with either a 400-nm long pass filter
(Oriel, model no. 59472) or a 320-nm UV filter (Oriel model no. 59980) combined with
an IR filter (Oriel, model no. 59060) and a fiber-bundle focusing assembly (Oriel, model
no. 77557). A modest degree of photobleaching of Nile red was detected over time, but
well below the changes resulting from surfactant photoisomerization.
Dynamic light scattering. Dynamics light scattering measurements were
performed at a scattering angle of 90  and temperature of 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, an avalanche photodiode
17

detector (BI-APD), and BI-9000AT digital correlator). Separate azoTAB and SDBS
solutions were independently passed through 200-nm syringe filters (Anatop) into the
sample vials. Samples were vortexed for 1 min and then allowed to reach equilibrium
over the course of 1 hr. Conversion of the surfactant to cis isomer was achieved by
illuminating with an 84 W long wave UV lamp-365 nm (Spectroline, Model no. XX-
15A). The data were analyzed with the NNLS software package.
Small-angle neutron scattering (SANS). Separate azoTAB and SDBS solutions
were mixed at the appropriate ratios to give the desired concentrations. Small-angle
neutron scattering experiments were performed on the 30 m NG7 SANS instruments at
NIST.
25
A neutron wavelength of  = 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
-1
(0.1 wt% total surfactant concentration) or 0.0048  0.4 Å
-1
(all other
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. Following data
collection for trans azoTAB, without changing samples the SANS cuvettes were then
illuminated with an 84 W long wave UV lamp-365 nm (Spectroline, Model no. XX-15A)
for at least 2 hr to convert azoTAB to the cis isomer. The same lamp was also used for in
situ UV illumination during collection of the cis-azoTAB data. The SANS data were
reduced, analyzed and modeled using the Igor Pro (WaveMetics) program supplied by
NIST.
26

18

The SANS data of the catanionic vesicles were fit using a polydisperse core-
shell
10, 27-30
form factor supplied by NIST, with fitted parameters being the average core
radius (r
c
), the constant shell thickness (t), and polydispersity of the core radius, while all
other parameters could be fixed based on the properties of the system.
28
The solution was
considered dilute enough to set the structure factor equal to unity. The SANS data for
micelles were fit using a form factor
10, 28, 31, 32
which contains the effects on particle size,
shape and scattering power for monodisperse bi-axial ellipsoids of uniform scattering
length density coupled with structure factor both supplied by NIST. SANS data for
systems containing mixtures of vesicles and micelles were fit by the summing the two
above models scaled by the respective fitted volume factions of the two microstructures.
Polarized optical microscopy. Separate azoTAB and SDBS solutions were
mixed at the appropriate ratios to give the desired concentrations. After vortexing for 1
min, the samples were observed with an Olympus IX71 inverted microscope equipped
with a 40  objective lens (SLCplanFl). Images were recorded with a CCD digital camera
(Hamamatsu, model no. C4742-95). At each surfactant concentration, the same sample
was used to obtain images under both visible and UV light, with the samples exposed
through the fiber-bundle focusing assembly to the 365-nm line from the 200 W mercury
arc lamp isolated through the combination of the 320-nm band-pass and IR filters (Oriel,
model 59060) for 2 hrs to convert the surfactant to the cis form.

2.4 Results and Discussion
Critical aggregation concentration. In previous study, the vesicles formation of
the mixture of cationic and anionic surfactants is spontaneous and stable, which meant
19

they were thermodynamically 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
8
and gene delivery.
21

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.
21
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.
18
For example, in water Nile red exhibits a weak emission peak at 655 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

Figure 2.1 shows the fluorescence emission spectra of Nile red in 60/40 (molar)
azoTAB/SDBS mixtures under visible light and UV light. 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 of Nile red does not change relative to that of pure water (~656
nm). 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
20

the emission peak from 654nm to 644 nm with the visible illumination and from 654nm
to 650 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 M (visible light) and 36 M
(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.

Figure 2.1: Fluorescence emission spectra of Nile red in 60/40 (molar) azoTAB/SDBS
mixtures under (a) visible and (b) UV exposure. [Nile red] = 0.1 M. The concentration
shown is azoTAB concentration, T = 25  C.

CACs determined for a range of azoTAB/SDBS ratios are presented in Figure 2.2
under both visible and UV illumination. The onset of vesicle formation is found to
increase from 1 μM to 40 μM under visible illumination and from 8 μM to 80 μM under
UV exposure with increasing azoTAB/SDBS ratio. Vesicle formation is expected to be
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 M
12 M
16 M
20 M
24 M
28, 32 M
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 M
8, 16 M
40, 48 M
56 M
64 M
72 M
21

governed by a combination of hydrophobic interactions amongst the surfactant
hydrocarbon tails and ionic and steric interactions of the headgroups.
17
From the critical
micelle concentrations (CMC) of the pure surfactants, which provide a relative measure
of hydrophobicity, SDBS (CMC = 1-4 mM) is generally more hydrophobic than azoTAB
in either the trans (CMC = 5 mM) or cis (CMC = 10 mM) conformation.
22
Thus, moving
towards azoTAB-rich mixtures and converting 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 2.2.
0
20
40
60
80
trans
cis
Ratio (azoTAB/SDBS)
10/90 20/80 30/70 40/60 60/40 70/30 80/20 90/10
azoTAB Concentrantion ( m)

Figure 2.2: CAC of the azoTAB/SDBS catanionic mixtures at given ratios under visible
light and UV illumination. [Nile red]: 0.1 μm, T = 25  C.

To know the onset points of the vesicle formation under both visible and UV
illumination is of great importance for gene delivery. For example, in our previous paper,
the different onset vesicle formation is used to increase gene transfection. In the study,
22

azoTAB/SDBS=70/30 and 50 M 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 M, only 20-30%
DNA is alive, while with 50 M, more than 90% DNA is alive. With catanioic surfactant
mixture, gene transfection can increase from less than 10% to more than 30%.
21

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 2.3. An azoTAB concentration intermediate the CAC values measured under
visible and UV light (see Figure 2.2) was chosen such that the vesicles formed under
visible light would be completely destroyed upon UV exposure.
The catanionic azoTAB-SDBS solution (6:4 molar ratio, 30 μM azoTAB, 20 μM
SDBS, and 0.1 μM Nile Red, 25 ℃) was measured by these fluorescence spectrometer,
light-induced vesicle formation occurs within ~65 s ( 99% increase in fluorescence) upon
visible light exposure, while vesicle disruption occurs in ~30 s ( 99% decrease in
fluorescence) following UV exposure. The formation and disruption of the vesicles could
be reversibly controlled by light illumination. The 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
23

exposure. As a result cationic vesicle will form spontaneously under visible light and be
disrupted with UV illumination.
Considering the concentration chosen between CACs of visible light and UV
light, the formation and disruption of the versicle can be reversibly controlled with 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.

Figure 2.3: 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 M and nile red at 0.1 μM.

Zeta Potential Measurement. Zeta potential can be determined by measuring the
electrophoretic mobility in an electric field. The electrophoretic mobility is measuring the
velocity of the particles by using Laser Doppler velocimetry (LDV). Positive particles
will migrate toward the negative electrode, and negative particles migrate toward positive
electrode with a velocity proportional to the magnitude of the zeta potential. Zeta
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
Time (s)
Visible light
UV light
24

potential can be calculated from electrokinetic measurements by Henry equation.
24
The
magnitude of the zeta potential allows us to evaluate the charge density and the stability
of the dispersion. If the particles have low zeta potential value in solution, which means
low net charge on the surface, then there is no force to prevent particles aggregate and to
maintain particles in stable suspension. However, if all the particles in suspension have a
large positive or negative charge (>30mV and <-30mV), they will tend to repel each
other and imply a high stability of the dispersion.
39

Figure 2.4 shows the results of zeta potential measurements in different ratio of
azoTAB/SDBS under lab room light and illumination of UV lights. We have measured
the mixture of azoTAB and SDBS at different ratio and the total surfactant concentration
is 0.1wt%. As the diagram indicates, the zeta potential values are characterized by a
decrease from positive charge to negative charge, by increasing the ratio of anionic SDBS
in the solution. We observe that point of zero charge at lab room light environment
appears at the ratio of 54/46. On the contrary, the point of zero charge at UV illumination
has revealed the ratio of 68/32. The ratio of azoTAB/SDBS at SDBS-rich side (such as
10/90, 20/80, 30/70, 40/60) shows no big difference of zeta potential value between lab
room light and UV illumination. According to the data of DLS (dynamical light
scattering, Table 2.1), the solutions at these ratios have similar particle size under visible
and UV illumination which are all vesicles. However, azoTAB-rich side (such as 60/40,
70/30, 80/20, 90/10) shows very different zeta potential value under visible and UV
illumination. UV-exposed samples have smaller zeta potential value than samples stored
in the Lab room light. DLS shows the structure will change by light illumination. The
azoTAB-rich side solution forms vesicles under visible light illumination and the particle
25

size will become bigger with light switch from visible light to UV light. Inverse
relationship between surface potential and vesicle size has already been presented.
40, 41

The zeta potential value decreases with increasing vesicle size. Carmonaribeiro
41
states
that an increase in particle radius should lead to a decrease in Stern potential.
Furthermore, Figure 2.4 reflects that zeta potential values at the ratio 60/40 between lab
room light environment and UV illumination have the biggest difference, the value drops
from 53 mv to -20 mV. The vesicle charge can be switched from positive to negative
with simple light illumination. DLS also shows that the particle size between these two
situations is quite different from 220 nm to 1300 nm. However, the particle size grows
slightly at the ratio 90/10, it reduces the gap of the zeta potential value from 88 mv to 52
mv between lab room light environment and UV illumination.
-100
-50
0
50
100
Trans
cis
ZP (mV)
Ratio (azoTAB/SDBS)
20/80 40/60 60/40 80/20

Figure 2.4: Zeta potential of azoTAB/SDBS in visible and UV illumination.

26

UV-Vis spectroscopy. The photoresponsive azoTAB surfactant in Scheme 2.1
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 - * transition), 350 nm (a - *
transition) and 440 nm (a weak n- * transition).
22, 42
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.
43
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 changes of azoTAB and
SDBS, which indicates the environment changes around azoTAB molecules.
44

The data of UV-Vis spectra for 0.1 mM azoTAB with SDBS at the given ratios
are shown in Figure 2.5 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
27

illumination, the typical absorbance is at 440 nm and 435 nm at SDBS-rich and azoTAB-
rich sides, respectively.
Figure 2.5 (a) 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 trans and cis conformations.
22
(about 85% trans at 30/70 and 75% trans at
70/30, deconvolution and compare Peak 350nm) With UV light illumination, surfactants
reach equilibrium where cis form takes great percentage , associated with the increase of
the 440 nm peak. Another phenomenon will be 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 440 nm absorbance) which is
consistent with the study on BTHA and SDS mixture.
44

In Figure 2.5 (b), Figure 2.5 (c) and Figure 2.5 (d), 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
44
and with the addition of SDBS, it is
more readily to form trans conform which in proven with UV light illumination. In
28

Figure 2.5(d), with UV illumination, the maximum cis peak shifted to lower wavelength
and decreased with the increase of SDBS amount.

(a) (b)

(c) (d)
Figure 2.5: 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

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)
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)
29

Figure 2.6 showed UV-Vis spectra at azoTAB/SDBS ratios of 20/80 and total
surfactant concentration is 0.1wt%. The UV-Vis spectrum was deconvoluted into 4 peaks
which are around 350 nm, 440 nm, 313 nm and 241 nm. Figure 2.6 (a), Figure 2.6 (b) and
Figure 2.6 (c) showed the deconvolution of UV-Vis spectrum which were under dark,
visible and UV illumination. In Figure 2.6 (a), the dark-adapted samples were stocked in
dark for 2 days. According to the literature
44
, it is reasonable to assume that over 95% of
the absorption at 350 nm is due to the trans from. We can assume that the cis isomers are
in negligible amounts in dark-adapted samples. Therefore, we assume that dark-adapted
samples contains 100% of trans from. On this basis, we calculate the trans% by
(2)
Abs
350
comes from the peak intensity of deconvolution. In accordance with Figure 2.6 (b)
and Figure 2.6 (c), we acquire the trans form is about 90% at visible illumination and
20% at UV illumination. We centrifuge the visible-illuminated and UV-illuminated
samples, and it was observed some precipitation. Supernatant was measured by DLS and
the particles size is smaller than 30 nm. Therefore, we assume that the precipitation is
composed of bilayer-structures. The supernatant was also measured by the UV-Vis
spectrum.

30

0
0.2
0.4
0.6
0.8
1
1.2
200 300 400 500 600
20/80 dark
Peak 350
Peak 440
Peak 313
Peak 241
Absorbance
Wavelength (nm)
(a)
0
0.2
0.4
0.6
0.8
1
1.2
200 300 400 500 600
20/80 visible
Peak 350
Peak 440
Peak 313
Peak 241
Absorbance
Wavelength (nm)
(b)

0
0.2
0.4
0.6
0.8
1
1.2
200 300 400 500 600
20/80 UV
Peak 350
Peak 440
Peak 313
Peak 241
Absorbance
Wavelength (nm)
(C)

Figure 2.6: UV-Vis absorbance sptectra of azoTAB/SDBS and deconvolution peak (a)
20/80 in dark, (b) 20/80 in visible illumination, (c) 20/80 in UV illumination; total
surfactant concentration is 0.1 wt%.

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.
31

Dynamic light scattering (DLS) has been used in several studies of catanionic
surfactant vesicles.
4, 16, 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/6πηD,
12, 46
where k
B
is Boltzmann’s constant, T is the temperature, and
η is the viscosity of the solvent. Figure 2.7 (a) 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 2.7 (b) presents the hydrodynamic
diameters for large range (0.0125 wt% to 0.5 wt%) with the given surfactant ration 30/70.
From Figure 2.7 (a) and Figure 2.7 (b), 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
32

conformation, combined with the known correlation between enhanced asymmetry
between the lengths of the cation and anion surfactants leading to increased vesicle size.
3

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 2.7 (b). 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,
9
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

33

0
200
400
600
800
1000
1200
1400
3:97 10:90 30:70 60:40 80:20 95:5 99:1
Visible light
UV light
R
H
(nm)
Ratio (azoTAB/SDBS)
(a)
0
50
100
150
200
250
0.02% 0.10% 0.25%
Visible light
UV light
R
H
(nm)
wt%
(b)

Figure 2.7: (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 2.1 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 2.1, DLS parameters are shown with light illumination, azoTAB/SDBS
34

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
/la  , where  is the volume of the hydrophobic potion of
the surfactant, l
max
, length of the hydrophobic parts and a, the area of the head group.
9, 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. 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 2.1, three transitions are investigated as vesicles  lamellar
structures, vesicles  micelles and micelles  vesicles. Because of different
hydrophobicity of surfactant conformations, three parameters ( , 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.
9
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
35

phenomena are investigated when total surfactant concentrations are at 0.1 wt%, 0.175
wt%, 0.25 wt% and 0.5 wt%. The observation states that there is a lamellar structure loop
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,
10
CTAT/SDBS
30
mixtures etc by SANS, but
investigated by freeze replica TEM with AZTMA/SDBS mixed solutions.
9
Considering
three parameters (, 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. 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 2.1) 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 (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,
9
E-SGP/DTAB
49
and BTHA/SDS
36

mixtures.
10, 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.
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 =7/93, 60/40, 70/30, 80/20 and 93/7 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. Some examples of SANS data describing the microstructures are shown in
Figure 2.8 and the microstructures are modeled with Igor program (models description
given in material and methods section) are given in Figure 2.9. The figures only show the
37

different scattering with visible and UV light illumination; the other data parameters will
be described in Table 2.2.
In Figure 2.9, 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 2.9.
10, 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 2.9 (d), Figure 2.9
(e) and Figure 2.9 (f), 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 2.2.

(a) (b)
Figure 2.8: 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.

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
)
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
)
38

In Figure 2.8 (a) and Figure 2.9 (a), Figure 2.9 (b), Figure 2.9 (c), 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 2.9. According to Guinier
approximation,
12
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 2.9 (a),
Figure 2.9 (b), Figure 2.9 (c). In these graphs, no fitting data are shown because of the
multi-micro 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/ dq   of 78 Å . At this ratio, the bilayer
for unilamellar vesicles have been measured about 33 Å as given in Table 2.2 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 2.8 (b) and Figure 2.9 (d), Figure 2.9 (e), Figure 2.9 (f). In Figure 2.9 (d), 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
12
of the aggregates is investigated indicating the formation of small
micelles.
10
The same phenomenon is observed at total surfactant concentration 0.5 wt%
and azoTAB/SDBS 93/7 ratio, as shown in Figure 2.8 (b) and Figure 2.9 (e), by
39

comparing the initial intensity of two concentrations, larger vesicle formed when total
surfactant concentration is 0.5 wt% consistent with DLS results. In Figure 2.9 (e), 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 from which the micelles
number densities can be calculated.
10
On the opposite side, SDBS-rich side, shown in
Figure 2.8 (b) and Figure 2.9 (f), 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.

40

0.001
0.01
0.1
1
10
0.001 0.01 0.1 1 10
60/40 0.1wt% trans
60/40 0.1wt% cis
60/40 0.1wt% trans fitting
Intensity (cm
-1
)
Q (A
-1
)
(a)

0.001
0.01
0.1
1
10
0.001 0.01 0.1 1 10
70/30 0.1wt% trans
70/30 0.1wt% cis
70/30 0.1wt% trans fitting
Intensity (cm
-1
)
Q (A
-1
)
(b)

0.001
0.01
0.1
1
10
0.001 0.01 0.1 1 10
80/20 0.1wt% trans
80/20 0.1wt% cis
80/20 0.1wt% trans fitting
Intensity (cm
-1
)
Q (A
-1
)
(c)

0.001
0.01
0.1
1
10
100
0.001 0.01 0.1 1 10
93/7 0.25wt% trans
93/7 0.25wt% cis
93/7 0.25 wt% trans fitting
93/7 0.25wt% cis fitting
Intensity (cm
-1
)
Q (A
-1
)
(d)

Figure 2.9: 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.

41

0.001
0.01
0.1
1
10
100
0.001 0.01 0.1 1
93/7 0.5wt% trans
93/7 0.5wt% cis
93/7 0.5wt% trans fitting
93/7 0.5wt% cis fitting
Intensity (cm
-1
)
Q (A
-1
)
(e)

0.001
0.01
0.1
1
10
0.001 0.01 0.1 1
7/93 0.5wt% trans
7/93 0.5wt% cis
7/93 0.5wt% trans fitting
7/93 0.5wt% cis fitting
Intensity (cm
-1
)
Q (A
-1
)
(f)

Figure 2.9: 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 Table 2.2, 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 43 Å both under visible illumination. For pure azoTAB,
the charge of the micelle is about 20, 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.
10
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 426 Å vesicles are found with the shell thickness 10 Å . In overall surfactant
concentration 0.1 w%, vesicles are observed for all the trans samples, with size varying
from 430 Å to 760 Å . With UV illumination, vesicles are investigated in most samples
42

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 Å with visible illumination while it is from 24 Å to 28 Å
with UV exposure which was in agreement the tail-tail interaction of the surfactant tails
considered as 28 Å . 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 larger size determination
than actual size and another reason is for DLS analysis, which strongly prefers to reflect
the larger aggregates in the solution.
10, 29, 30, 53

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

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
43

work. Figure 2.10 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.
As shown in Figure 2.10, 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.
54, 55
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 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.
44

Figure 2.10: Polarized optical microscopy images with visible light illumination. Overall
surfactant concentration: 1 wt%. (Scale: 5 m).

2.5 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. Zeta potential measurement shows that the
vesicle charge can be switched from positive to negative with simple light illumination.
UV-vis spectroscopy is used to measure the change of conformations. 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.

20/80
80/20
30/70
70/30 60/40
40/60

Table 2.1: DLS-determined size of photoresponsive catanionic vesicles as a function of the azoTAB/SDBS molar ratio and overall
surfactant concentration (wt %) under visible and UV illumination. Average error ~5%. Micellar and lamellar regions are denoted in
bold and italics, respectively.

azoTAB/SDBS 0.025 wt % 0.10 wt % 0.175 wt % 0.25 wt % 0.50 wt %
vis UV vis UV vis UV vis UV vis UV
1/99 126 89.2 140 4.7 1.8 1.1 1.9 1.0 1.9 1.0
3/97 116 89.3 146 109 56.3 1.4 1.3 1.0 1.6 1.0
5/95 102 83.0 153 86.5 122 2.7 236 3.4 2.4 1.0
7/93 89.9 84.3 146 82.0 156 4.0 151 1.9 238 3.4
10/90 129 83.8 169 125 270 183 145 119 214 201
20/80 131 104 187 134 171 149 134 114 226 209
30/70 143 135 194 148 205 178 159 156 237 181
40/60 171 143 182 158 211 202 139 140 195 191
60/40 232 339 224 1280 211 1140 151 258 188 870
70/30 192 363 191 850 222 456 151 146 172 139
80/20 193 880 201 820 197 192 198 168 142 117
90/10 180 1220 160 173 144 148 148 117 111 82.0
93/7 133 311 237 174 3.2 148 4.6 130 3.2 148
95/5 191 264 184 170 175 153 3.7 84.7 7.9 15.3
97/3 89.8 493 202 120 164 132 3.9 90.4 1.4 1.2
99/1 178 196 159 120 93.6 39.3 1.2 1.0 3.0 1.0

45

Table 2.2: 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, Pa: parameters, V: vesicle, M: micelles

azoTAB/SDBS 0.1 wt% 0.25 wt% 0.5 wt%
visible UV Visible UV visible UV
Structure Pa Structure Pa Structure Pa Structure Pa Structure Pa Structure Pa
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
46

Table 2.2: 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, Pa: parameters, V: vesicle,
M: micelles
azoTAB/SDBS 0.1 wt% 0.25 wt% 0.5 wt%
visible UV visible visible
Structure Pa Structure Pa Structure Pa Structure Pa

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

47
48

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24, 1973-1978.

40. Nascimento, D. B.; Rapuano, R.; Lessa, M. M.; Carmona-Ribeiro, A. M. Langmuir 1998,
14, 7387-7391.

41. Carmonaribeiro, A. M.; Midmore, B. R. Journal of Physical Chemistry 1992, 96, 3542-
3547.

42. Kuiper, J. M.; Engberts, J. Langmuir 2004, 20, 1152-1160.

43. Eastoe, J.; Vesperinas, A. Soft Matter 2005, 1, 338-347.

44. Bonini, M.; Berti, D.; Di Meglio, J. M.; Almgren, M.; Teixeira, J.; Baglioni, P. Soft
Matter 2005, 1, 444-454.

45. Claire, K.; Pecora, R. Journal of Physical Chemistry B 1997, 101, 746-753.

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47. Silva, B. F. B.; Marques, E. F.; Olsson, U. Langmuir 2008, 24, 10746-10754.

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Aqueous Solution (Second Edition). John Wiley & Sons, Ltd: 2003.

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2004, 20, 6120-6126.

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Chemistry B 2005, 109, 609-616.

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102, 338-343.

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55. Bortchagovsky, E. G. Journal of Applied Physics 2004, 95, 5192-5199.

51

Chapter 3: Conformational Change of Membrane Protein by Using
Photo-sensitive Catanionic Surfactant System
3.1 Abstract
Surfactants are important in the isolation and purification of the protein which are
also used in the solubilization and reconstitution of protein. In this study, we want to use
photo sensitive azoTAB and SOS (sodium octyl sulfate) catanionic surfactant system.
The surfactant system undergoes a photoisomerization between 434 nm visible light and
350 nm UV light can result phase transition in specific concentrations. Phase transtion
can be possible to reversibly control purple membrane folding by light if the thickness of
the bilayer is similar to the natural purple membrane bilayers. In mixtures of purple
membrane and surfactants was used to control the folding and unfolding of
bacteriorodopsin. Many different strategies for membrane protein reconstitution are to
remove surfactant. We use phase transition to decrease the formation of micelles for
membrane solubilisation. The study provides a rapid and photo responsive method to
change protein conformation.

3.2 Introduction
Membrane proteins perform very important role in numerous biological processes,
including transport of substances across membranes, signal transduction, a variety of
metabolic pathways and support considerable enzymatic activity. Predicting around 30%
of the cellular proteins is integral to the membrane.
1
A detailed knowledge of the
structure and function of membrane proteins is essential to understand drug delivery
52

mechanisms. A primary challenge in the research on membrane protein is water soluble
and to maintain the function active.
2
Membrane proteins are not generally soluble in
aqueous solution. It has high hydrophobicity that require specific environment to work in
vitro. Membrane proteins are naturally embedded in a lipid bilayer. It is important for
membrane proteins to form a good seal with the lipid bilayer to maintain a stable
structure. Membrane proteins require to incorporate into a native-like membrane or
surfactant bilayer that mimic the original environment in biological membrane.
Otherwise, membrane proteins will be in unfolding state which leads to irreversible
aggregation. However, it is not easy to conduct a native environment for membrane
protein and this frequently restricts applications after protein extracted from its native
membrane. Solubilization in surfactants or reconstitution in a non-native environment is
frequently loss of activity.
3

Despite the challenge will be encountered when work outside the original lipid
environment, membrane proteins remain play an important area to study owing to the
importance of biochemical process and pharmaceutical targets. A number of approaches
have been developed to solubilize and reconstitute membrane protein in vitro, including
ionic surfactants,
4, 5
nonionic surfactants,
6
lipid-surfactant system,
7, 8
extraction into
organic solvents,
9
dilution
10
and dialysis.
Purple membrane (PM) has been recognized the prime candidate to study
membrane-surfactant interactions. This is because that bacteriorhodopsin (bR) is the only
protein in the purple membrane isolated from Halobacterium salinarium and one of the
few membrane proteins whose native structure is analyzed near atomic resolution.
11

Purple membrane is composed of 75 wt% bR and 25 wt% lipids, which is equivalent 10
53

lipid molecules per protein.
12
These lipids are 60% phospholipids and 30% glycolipids.
13

Each bR is composed of seven helixes and covalently binds a retinal chromophore
through a Schiff-base link to a lysine residue. The secondary structure of
bacteriorhodopsin was studied by FTIR. There are 70% α-helix structure corresponding
to the transmembrane helices, 20% β-strands and 10% unordered structures.
14

Bacteriorhodopsin can be spontaneously refolded to a native state from a denature
state in vitro. Refold efficiencies can easy to be determined by the extent of recovery of
purple absorption band of the bound retinal chromophore.
15
The absorption band λ
max
is
570nm for light-adapted bR and the change of the absorption can be easily observed.
Bacteriorhodopsin can be unfolded in SDS micelles and refolded to native state when
transfer to mixed surfactant/lipid solution (SDS/DMPC/CHAPS). In the denature state,
60% of the native helical is present but the protein is not bound to its retinal chromophore.
After regeneration, the efficiency is about 80-90% in several minutes.
15-17
Other nonionic
surfactant systems like DMPC/CHAPSO and DMPC/DHPC are also used to exchange of
SDS to refold the structure.
18, 19

Surfactants are vital in the isolation and purification of the protein which are also
used in the solubilization and reconstitution of protein. Surfactants are amphiphilic that
consist of a hydrophilic head group and a hydrophobic carbon chain. Surfactants exhibit
unique properties in aqueous solutions which can spontaneously form different phases,
such as micelles, vesicles and lamellars. Membrane proteins are usually soluble in
micelles. Critical micelle concentration (CMC) becomes an important property in the
interaction of membrane protein and surfactants. CMC is defined as the minimum
concentration of individual surfactant molecules to form micelles.
54

In this study, we want to use cationic azoTAB and anionic SOS (sodium octyl
sulfate) to form catanionic surfactant system. We can control membrane protein folding
by using simple light illumination to change phase of azoTAB/SOS system. The
surfactant system undergoes a photoisomerization between 434 nm visible light and 350
nm UV light can result phase transition in specific concentrations. Photoreversible
control of phase transition between vesicles and micelles can change bacteriorhodopsin
conformation. Lipid bilayer environment can promote refolding versus unfolding in
micellar environment. Even the refold efficiency is not high (~ 10%), but greatly
simplifies the refolding process.

3. 3 Material and Methods
Preparation of Surfactant. The photoresponsive surfactant 4-ethyl-4’
(trimethylaminobutoxy) azobenzene bromide (azoTAB) is synthesized according to the
literature procedures.
20, 21
The surfactant undergoes a reversible photoisomerization by
switching the appropriate wavelength of visible light (434nm) to UV light (350nm). The
conformation of azoTAB will transfer from trans isomer to cis isomer. Trans isomer
exhibits a lower dipole moment and more hydrophobic than cis isomer. Other physical
properties also change as the photoswitch such as critical micelle concentration,
20
surface
tension and electrical conductivity. SOS (Sodium n-octyl sulfate, 99%) was purchased
from Alfa Aesar. All other chemicals were purchased from Sigma-Aldrich and used as
received unless otherwise mentioned.
55

Preparation of Purple Membrane. Purple membrane contains both lipids and
the membrane protein bacteriorhodopsin which is isolated from Halobacterium
salinarium (strain S9, a kind gift from Professor Dieter Oesterhelt at the Max Planck
Institute of Biochemistry in Martinsried). The prepare procedure was established by
Oesterhelt and Stoeckenius.
22
In brief, bacterial cells were grown in 1 L cultures
incubated at 37°C in the dark in a shaker at 100 rpm for 5 days until the absorption at 560
nm was between 1.0-1.5. The purification of PM was then isolated from the medium at
4000 rpm for 40 min in centrifuge. Further purification, impurities in PM were removed
by washing with DI water at least 15 times at 24000 rpm for 45 min (Beckman Coulter
Avanti J-25 Ι Centrifuge, Beckman rotor JA 25.5) until the water no longer exhibited a
purple color. The obtained purple membrane was stored at -20°C.
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). AzoTAB/SOS solution
was prepared in the assigned concentration in 0.1 M phosphate buffer, pH 6.0 and settled
for 8 hours. Before the measurement, the solution was exposed to visible light (440nm)
from the arc lamp with the fiber bundle focusing assembly for 20 min. The measurement
presents the trans state. Then, the solution was exposed to UV light (365nm) from the
same assembly for the other 20 min. AzoTAB/SOS solution shows cis state. The results
reveal the phase behavior of catanionic surfactant between visible and UV light.
Furthermore, purple membrane was directly added into the appropriate concentration of
56

azoTAB/SOS solution (prepared in phosphate buffer, pH 6.0). The concentration of the
protein in each sample was ~0.8 mg/mL, determined spectroscopically. The samples were
then stirred in the dark for 8 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. The dynamic light
scattering data were analyzed with the nonnegative least-squares (NNLS) routine
supplied by Brookhaven. Hydrodynamic diameters were obtained from the Stokes-
Einstein equation by the experimentally determined diffusion coefficient.
UV-Vis Spectrometer measurement. Purple membrane was suspended into the
appropriate concentration of azoTAB/SOS solution (prepared in 0.1 M sodium phosphate
buffer, pH 6.0). The samples were then stirred in the dark for 8 hrs. Before the
measurement, the solution was illuminated for 20 min 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 for 20 min. The solution mixtures are alternately exposed to visible and UV
light with surfactant conversion from the trans to the cis isomer assured with absorption
measurements. The concentration of the protein in each sample was ~0.8 mg/mL,
determined spectroscopically. The dynamics of photo-induced bR conformational
changes were studied by in situ UV-vis spectroscopy. The results were obtained over a
period of 20 min with spectra collected every 30 s.
57

3.4 Results and Discussion
Phase Behavior of Catanionic surfactant. AzoTab/SOS/water surfactant system
was prepared and aged for 7 days. Samples are initially prepared, the solution often form
a wide range of composition.
23
The phase state of solution was measured by dynamic
light scattering (DLS). All the samples were measure both under visible and UV light.
The measurements analyzed with the nonnegative least-squares (NNLS) routine by a
series of correlation functions which can resolve the simultaneous relaxations of multiple
scattering species. Stokes-Einstein equation can determine hydrodynamic diameter from
the diffusion coefficients. All the samples were average 3 runs to determine the average
diameter and along with the sample scattering count rates at each condition. Table 3.1
shows the average effective diameters of the solution that molar ratio azoTAB/SOS= 3/97
and the total surfactant concentration percentage is from 0% to 0.8% under visible and
UV light. Figure 3.1 shows the relationship between the intensity of scattered light and
the total surfactant concentration in azoTAB/SOS solutions between visible light and UV
light. The intensity of scattered light steeply increased with the increasing concentration
in visible light. However, as the sample was illuminated under the UV light, the intensity
of scattered light increases with the increasing concentration. With further increase in
concentration, the intensity decreases abruptly. We can compare the data between Table
3.1 and Figure 3.1. The scattering intensity generally varies as the square of particle
volume. The sample at low concentration 0.08 wt% shows ca. 30 nm which is smaller
than other samples in visible light. The measurement of effective diameter is ca. 100 nm
between 0.16 wt% and 0.4 wt% and ca. 200 nm between 0.56 wt% and 0.8 wt%. The
visible light sample shows two different slopes in Figure 3.1. AzoTAB/SOS mixtures has
small vesicle phase at region A. In contrast to region A, region B has much higher
58

scattering intensity and larger vesicle size. The sample which concentration is lower than
0.4 wt% was exposed under UV light. The scattering intensity and particle size drop
slightly. Therefore, region C is still in vesicle phase beside the sample at low
concentration 0.08 wt%. With concentration increase beyond region C, the intensity has
dramatic drop over an order of magnitude. The intensity in this region is only slightly
larger than the scattering intensity of DI water. DLS cannot determine the size at this low
intensity at 90° of scattering angle which means the phase changes. The vesicle to micelle
transition occurs around 0.5 wt% when the visible light switches to UV light. Abrupt
decrease in the intensity of scattered light appeared at 0.56 wt%, 0.64 wt%, 0.72 wt% and
0.8 wt%.
We can operate the phase behavior by changing the concentration and switching
the wavelength of illuminated light according to the result of Table 3.1 and Figure 3.1.
Figure 3.2 shows the particle size distribution at 0.24 wt% and 0.56 wt% between visible
light and UV light illumination in azoTAB/SOS mixed solution. The particle size
distribution has two main peaks at about 1-2 nm and 80-150 nm where vesicles were
observed both under visible light and UV light irradiation at 0.24 wt%. The sample at
0.56 wt% under visible light shows two peaks at 45-55 nm and 200-260 nm which is
corresponding to large vesicles. After the sample at 0.56 wt% illuminated by UV light,
the particle distribution becomes a very simple peak at 1 nm. These results could be
considered vesicle to micelle phase transition from low surfactant concentration to high
surfactant concentration under UV light. However, the vesicle phase is not transfer to
micelle phase under visible light in the same concentration range. Many catanionic
surfactant systems have one or two vesicle lobe region in ternary phase diagram, such as
59

CTAT/SDBS
24
and CTAB/SOS
25
. As the surfactant concentration increasing along the
vesicle lob region, the phase can change from vesicle to micelle. In our case, we utilize
the photo switch to vary the phase composition instead of concentration change. The
method is more convenient and easy reversible control. We can control the phase
transition simply by switching the illuminated light at specific concentration range.
Table 3.1: Dynamic light scattering measurement of effective diameter for azoTAB/SOS
system between visible light and UV light. (molar ratio is 3:97, standard deviations given
in parentheses)

azoTAB/SOS visible light UV light
wt% Deff (nm) Deff (nm)
0.08
31.7 (2.4) 7 (2.2)
0.16
97 (1.9) 73 (6)
0.24
93(6.5) 81 (3.5)
0.32
92 (1.9) 80 (2.2)
0.4
88 (1.1) 87 (2.6)
0.48
123 (4.1) 135 (3.9)
0.56
207 (7) *
0.64
198 (9) *
0.72
197 (10) *
0.8
198 (13.8) *
60

0
5000
10000
15000
20000
25000
30000
35000
40000
0 0.2 0.4 0.6 0.8 1
visible light
UV light
Intensity (cps)
azoTAB/SOS wt%
(A) Vesicle
(B) Vesicle
(D) Micelle
(C) Vesicle

Figure 3.1. Scattered light intensities for aqueous mixtures of azoTAB/SOS. (molar ratio
3:97)

0 100 200 300 400 500
0
20
40
60
80
100

Intensity
(a) 0.24% visible
0 100 200 300 400 500
0
20
40
60
80
100

(c) 0.56% visible
0 100 200 300 400 500
0
20
40
60
80
100

Intensity
Diameter (nm)
(b) 0.24 % UV
0 1 2 3 4 5
0
20
40
60
80
100

Diameter (nm)
(d) 0.56% UV

Figure 3.2. Part (a) and (b) represent diameter distribution of the total surfactant
concentration at 0.24 wt% between visible light and UV light in azoTAB/SOS mixture.
Part (c) and (d) are the diameter distribution at 0.56 wt%. The diameter distributions are
measured with DLS. The solution was aged for 7 days.
61

Photo reversible control of bacteriorhodopsin folding in PM-azoTAB/SOS
mixtures. Purple membrane fragments are mixed with azoTAB/SOS catanionic
surfactants. Based on the phase transition of catanionic surfactants, purple membrane
bilayers were affected by the effect of surfactant and light. Figure 3.3 shows folding
percentage of bR as a function of surfactant concentration. A, B, C and D are the folding
fraction of the visible ↔ UV light cycles in different surfactant concentration. The
folding fraction decreases under both visible and UV light with increasing surfactant
concentration. When total surfactant concentration is smaller than 0.4 wt%, the folding
fractions do not change too much between visible light and UV illumination. However, a
given surfactant concentration is higher than 0.48 wt%, the folding fraction difference
can be seen. The percentage of folding is observed to be higher with azoTAB/SOS under
visible light illumination compared to UV illumination. Besides, the folding fraction was
observed to be reversible according to the illumination under light cycle A (visible light)
→ B (UV light) → C (visible light) → D (UV light). For example, at 0.56% total
surfactant concentration, ~72% of the bR molecules are folded under visible light (A).
The folding fraction drops to ~62% under the UV light (B). When the light switches to
visible light again (C), the folding fraction goes up to ~72%. The folding fraction still
drops to ~62% at UV light (D). The folding fraction was reversible by switching the light
between visible light and UV light. Phase transition happened by photo switch when the
total surfactant concentration is higher than 0.48%. Compare with Figure 3.2, we can
assume that PM-azoTAB/SOS mixture contains more bilayer sturctures under visible
light and more micelles under UV light when the concentration is higher than 0.48%. The
phase does not change between visible light and UV light when the total surfactant
62

concentration is lower than 0.4% which indicates that the folding fraction stay stable
between visible and UV light illumination at lower concentration.

Figure 3.3. Folding percentage of bR as a function of surfactant concentration. A, B, C
and D are the folding fraction of the visible ↔ UV light cycles in different surfactant
concentration. BR concentration is 0.8 mg/mL in each sample. The inset shows the
folding percentage difference between visible light and UV light at 0.56 wt%, 0.64 wt%
and 0.72 wt%.

Figure 3.4 represents light-induced changes in membrane protein folding. The
results were measured over a period of 20 min with spectra collected every 30 s. It
indicates the absorbance at 350 nm and 560 nm as a function of time at total surfactant
concentration at 0.24 wt% and 0.56 wt%. The maximum absorption is at 350 nm for trans
conformation. The sample starts from trans structure, followed by the drop of A
350
as the
0.3
0.4
0.5
0.6
0.7
0.55 0.6 0.65 0.7 0.75
Folding Fraction
surfactant concentraion (wt%)
~10%
~10%
~8%
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.2 0.4 0.6 0.8
A: visible light
B: UV light
C: visible light
D: UV light
Folding Fraction
surfactant concentraion (wt%)
63

UV illumination, and the absorbance of A
350
goes up by shining the visible light again
both happened in 0.24 wt% and 0.56 wt%. The absorbance of 560 nm decreases about
10% as the UV light illumination and increases about 7% by shining visible light again.
The absorbance change of PM peak is fairly small, but the change were reproducible
between visible and UV light cycle. Bacteriorhodopsin molecules are unfolded in the
formation of micelle. Vesicle formed as the expose of visible light, bacteriorhodopsin
was folded in the bilayer structure which causes the increase of folding fraction. Compare
to the concentration at 0.24 wt%, the absorbance of 560 nm has slightly increase (2%) as
the expose of UV light. The reason is the contribution of cis peak (A
440
) induces the
slightly increase of the absorbance at 560 nm. Phase of surfactant does not have dramatic
change between UV and visible light, which explains that folding fraction is quite
equivalent.
64

0 200 400 600 800 1000 1200
0.29
0.30
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0 200 400 600 800 1000 1200
0.20
0.21
0.22
0.23
0.24
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Visible light

Absorbance
350nm
560nm
UV light
2%
3%
0.24wt%
0.56wt%
Visible light UV light

Absorbance
time (sec)
350nm
560nm
10%
7%

Figure 3.4. In-situ measurement of PM-azoTAB/SOS system. The total surfactant
concentrations are 0.24 wt% and 0.56 wt%. The absorbance at 350 nm and 560 nm is a
function of time.

65

300 400 500 600 700 800
0.0
0.5
1.0
1.5
2.0

max
=555nm

max
=560nm

Absorbance
wavelength
A
B
C
D
E

max
=570nm
520 540 560 580 600 620
0.16
0.18
0.20
0.22
0.24
0.26
0.28

Absorbance
wavelength

Figure 3.5: Selected individual UV-vis spectra from in-situ measurement at 0.56 wt% (A:
pure purple membrane. B: t=0, start to shine UV light. C: t=4.5 min, UV light. D: t=11.5
min, start to shine visible light. E: t=19.5 min, visible light.)

In figure 3.5, spectra A represents the pure PM in aqueous solution (concentration
of the protein is ~0.8 mg/mL). Spectra B, C, D, E are selected individual UV-vis spectra
at 0.56 wt% of total surfactant concentration from figure 3.4. The λ
max
for light adapted
bR is 570 nm which is corresponding to spectra A. When PM mixes with surfactant, λ
max

shifts to 560 nm, such as spectra B. Bacteriorhodopsin has a characteristic of protein
folding which is the absorption band of the retinal chromophore at 560 nm.
Bacteriorhodopsin unfolded both causes loss in the absorbance at 560 nm and increase in
the absorbance at 380 nm (chromophore free in solution). However, 380 nm peak was
obscured by the strong 350 nm trans surfactant absorption peak. Spectra D is the
surfactant in cis conformation, the absorbance of maxima peak decreases and blue shift to
555 nm, which represents bacteriorhodopsin solubilized in micelles. According to the
66

reference, bacteriorhodopsin solubilized in micelles has maxima absorbance in the region
of 550 to 555 nm.
26
However, bacteriorhodopsin is unable to reconstitute or sustain the
native protein structure when maxima absorbance reaches to 550 nm.
27
Spectra E shows
the maxima peak is back to 560 nm and the intensity of absorbance increases. The
process is reversible by switching the light illumination.
0 100 200 300 400 500 600 700
0
20
40
60
80
100

Intensity
(a) 0.24% visible
0 100 200 300 400 500 600 700
0
20
40
60
80
100

(d) 0.56% visible
0 100 200 300 400 500 600 700
0
20
40
60
80
100

Intensity
(b) 0.24% UV
0 100 200 300 400 500 600 700
0
20
40
60
80
100

(e) 0.56% UV
0 100 200 300 400 500 600 700
0
20
40
60
80
100

Intensity
Diameter (nm)
(c) 0.24% visible
0 100 200 300 400 500 600 700
0
20
40
60
80
100

Diameter (nm)
(f) 0.56% visible

Figure 3.6: Diameter distributions of 0.24 wt% and 0.56 wt% azoTAB/SOS/PM aqueous
mixtures. [bR]=0.8mg/mL. Part (a) is the diameter distribution of 0.24 wt% solution
exposed to visible light for 20 min. Part (b) is the diameter distribution of the solution
exposed to UV light for the other 20 min. The same solution to take the other 20 min
illumination of visible light is part (c). Part (d), (e) and (f) represent the diameter
distributions in the 20 min illumination of visible, UV and visible light in the 0.56 wt%
solution.

67

Figure 3.6 is the diameter distributions of azoTAB/SOS/PM aqueous mixtures.
Part (a), (b) and (c) are the diameter distributions in the 20 min illumination of visible,
UV and visible light in 0.24 wt% surfactant concentration. Relatively, part (d), (e) and (f)
are the diameter distributions in the 20 min illumination of visible, UV and visible light
in 0.56 wt% surfactant concentration. There are two distributions in part (a). The large
particle distribution is corresponding to the size of PM disclike sheet which is about 500
nm in diameter. The other distribution around 100 nm should be the vesicles composed
by azoTAB and SOS surfactant system. Part (d) has smaller large particle distribution
because higher surfactant concentration enhances the lysis of the membrane. From part (a)
to part (c), the large particle distribution decreases as the time owing to the lysis of
membrane gradually. The vesicle phase still exists in part (a), (b) and (c). Part (e) shows
a lot of micelles in the solution which can help solubilization. Hence, the PM has smaller
size compare to other part.
The interaction of surfactant with cell membrane resembles with lipid bilayers.
When azoTAB/SOS surfactants mix with purple membrane, surfactants insert into lipid
bilayer, and then saturation of the membrane with monomeric surfactant form. By adding
more surfactants, the membrane may lyze gradually. Membrane breakdown occurs into
lipid-protein-surfactant complexes and mixed lipid-surfactant micelles. When the
solution was exposed under UV light, azoTAB/SOS surfactants form more micelle to
decompose the lipid-protein-surfactant complexes. Reversely, solution was exposed
under visible light to transfer azoTAB/SOS surfactants into vesicle bilayer structure. It
can enhance the bilayer structure of lipid-protein-surfactant complexes. The membrane
proteins are anchored to the bilayer of lipid-azoTAB-SOS.
68

We can consider that the method of bilayer lipid membrane reconstitution using
surfactant removal is the reverse of the solubilization process. There are several possible
mechanisms suggested for the constitution of membrane protein, but basically all depend
on the initial solubilisation of the membrane protein by surfactant followed by removal of
surfactant, such as dilution or dianalysis, resulting the surfactant to lipid ratio is reduced.
Here, we mix purple membrane with vesicles which composed by photo-sensitive
catanionic surfactant (azoTAB/SOS). In the beginning, there are still some monomeric
surfactant insert into membrane bilayer with the formation of lipid-protein-surfactant
complexes. Therefore, azoTAB/SOS vesicle and lipid-protein-surfactant complexes
coexist in aqueous solution. As the surfactant concentration increases, more and more
monomeric surfactants insert into membrane bilayers and disrupt the structure into
fragments.
However, the solution was exposed under UV light when the total concentration
is above 0.48%. AzoTAB/SOS vesicles will transfer into micelles phase, which means
more micelles form in the solution to help solubilization by micellar attack and protein
folding fraction drops. Owing to the same phase of azoTAB/SOS surfactants both
exposed under visible light and UV light, we don’t observe folding fraction drops
apparently when the total concentration is lower than 0.4 wt%. Then, the light switches to
visible explosion; the folding fraction climbs when the concentration above 0.48%.
Lipid-surfactant mixed micelles can form lipid-surfactant vesicles in the presence of
protein-surfactant complexes. Also, the process is reversible.
AzoTAB/SOS solution exposed under light by switching from UV to visible
light can changes the phase from micelles to vesicles. The surfactant to lipid ratio in
69

membrane may be reduced when exposed under visible light, resulting in the
conformation of lipid-protein-surfactant vesicles.

3.5 Conclusions
Bacteriorodopsin in native state is embedded into the purple membrane sheets.
Protein was readily to aggregate because of the large percentage of the protein and
paracrystalline structure
28
. 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. In this paper, we use vesicle-micelle transition in mixtures of lipids and
surfactants to control the folding and unfolding of bacteriorodopsin. Many different
strategies for membrane protein reconstitution are to remove surfactant, such as dialysis
and dilution. In contrast, the refolding efficiency is not high (~10 %) in our result. Phase
transition can only decrease part of micelle formation for membrane solubilisation. The
study provides a rapid and photo responsive method to change protein conformation.

70

3.6 References
1. Nath, A.; Atkins, W. M.; Sligar, S. G. Biochemistry 2007, 46, 2059-2069.

2. Seddon, A. M.; Curnow, P.; Booth, P. J. Biochimica Et Biophysica Acta-
Biomembranes 2004, 1666, 105-117.

3. Kleinschmidt, J. H. Cellular and Molecular Life Sciences 2003, 60, 1527-1528.

4. Dong, M. Q.; Baggetto, L. G.; Falson, P.; LeMaire, M.; Penin, F. Analytical
Biochemistry 1997, 247, 333-341.

5. Lau, F. W.; Bowie, J. U. Biochemistry 1997, 36, 5884-5892.

6. Fleming, K. G.; Ackerman, A. L.; Engelman, D. M. Journal of Molecular Biology
1997, 272, 266-275.

7. Putman, M.; van Veen, H. W.; Poolman, B.; Konings, W. N. Biochemistry 1999,
38, 1002-1008.

8. Knol, J.; Sjollema, K.; Poolman, B. Biochemistry 1998, 37, 16410-16415.

9. Yerushalmi, H.; Lebendiker, M.; Schuldiner, S. Journal of Biological Chemistry
1995, 270, 6856-6863.

10. Rigaud, J. L.; Pitard, B.; Levy, D. Biochimica Et Biophysica Acta-Bioenergetics
1995, 1231, 223-246.

11. Henderson, R.; Baldwin, J. M.; Ceska, T. A.; Zemlin, F.; Beckmann, E.; Downing,
K. H. J Mol Biol 1990, 213, 899-929.

12. Oesterhelt, D.; Stoeckenius, W. Nat New Biol 1971, 233, 149-52.

13. Kushwaha, S. C.; Kates, M.; Stoeckenius, W. Biochim Biophys Acta 1976, 426,
703-10.

14. Cladera, J.; Sabes, M.; Padros, E. Biochemistry 1992, 31, 12363-8.

15. Booth, P. J.; Flitsch, S. L.; Stern, L. J.; Greenhalgh, D. A.; Kim, P. S.; Khorana, H.
G. Nat Struct Biol 1995, 2, 139-43.

16. Huang, K. S.; Bayley, H.; Liao, M. J.; London, E.; Khorana, H. G. J Biol Chem
1981, 256, 3802-9.

17. London, E.; Khorana, H. G. J Biol Chem 1982, 257, 7003-11.

71

18. Booth, P. J.; Riley, M. L.; Flitsch, S. L.; Templer, R. H.; Farooq, A.; Curran, A.
R.; Chadborn, N.; Wright, P. Biochemistry 1997, 1997, 197-203.

19. Chen, G. Q.; Gouaux, E. Biochemistry 1999, 38, 15380-15387.

20. Hayashita, T.; Kurosawa, T.; Miyata, T.; Tanaka, K.; Igawa, M. Colloid and
Polymer Science 1994, 272, 1611-1619.

21. Lee, C. T.; Smith, K. A.; Hatton, T. A. Macromolecules 2004, 37, 5397-5405.

22. Oesterhelt, D.; Stoeckenius, W. Methods Enzymol. 1974, 31, 667-678.

23. Nakanishi, H. T., K. Ohkubo, T. Journal of Oleo Science 2005, 54, 443-451.

24. Kaler, E. W.; Herrington, K. L.; Murthy, A. K.; Zasadzinski, J. A. N. Journal of
Physical Chemistry 1992, 96, 6698-6707.

25. Yatcilla, M. T.; Herrington, K. L.; Brasher, L. L.; Kaler, E. W.; Chiruvolu, S.;
Zasadzinski, J. A. Journal of Physical Chemistry 1996, 100, 5874-5879.

26. Massotte, D.; Aghion, J. Biochem Biophys Res Commun 1991, 181, 1301-1305.

27. Tan, E. H.; Birge, R. R. Biophysical Journal 1996, 70, 2385-2395.

28. Hwang, S. B.; Stoeckenius, W. J Membr Biol 1977, 33, 325-50.

72

Chapter 4: Conformation Change of Bacteriorhodopsin through
Photoresponsive Surfactant
4.1 Abstract
The interactions between membrane proteins and solubilizing surfactants directly
effect on the stability and function. The study provides a photo responsive method to
change protein folding state in the presence of a light responsive surfactant.
Bacteriorhodopsin can be spontaneously refolded to a native state from a denature state in
vitro which was formed in a bicelle structure. Bicelles were thought to be aqueous lipid–
surfactant assemblies in which compose of bilayer fragments. Compare the bR folding
percentage in DMPC/CHAPS and DOPC/CHAPS bicelle systems by addition of
photosensitive azoTAB surfactant between trans and cis conformation. UV-vis
spectroscopy will be used to study the effect of the photosensitive surfactant and light
conditions on bR folding by study absorbance changes at 560 nm of the retinal
chromophore. Furthermore, the lipid-surfactant assembly was modeled by using small
angle neutron scattering and pair distance distribution function of the shape. The effect of
azoTAB concentration and conformation alter the stability of bR folding. AzoTAB
Switching from trans to cis probably causes a change in the curvature stress and bilayer
thickness. Membrane protein folding in bicelles is controled through the use of
photoresponsive surfactants.

73

4.2 Introduction
Membrane proteins provide a physical barrier between the cell and its
environment for living systems, serve as the mediators between cell and the surrounding,
energy conversion and cell-to-cell signaling and motility. Each living cell surrounded by
a biological membrane which was assembled by lipids and associated membrane proteins.
The lipids composed as a bilayer form. Integral membrane proteins were attached to the
membrane and embedded in the lipid bilayer, which simultaneously associate in
hydrophobic and hydrophilic regions. The primary challenge in the research is that
membrane proteins are not soluble in aqueous solution. The hydrophobic nature of
membrane proteins makes their isolation and purification extremely difficult. Membrane
proteins need to reside in a specific environment to satisfy the high hydrophobicity in
vitro work. Thus, membrane proteins require to incorporate into a native-like structure to
maintain structure integrity. Otherwise, membrane proteins will be in unfolding state
which leads to irreversible aggregation. An environment that imitates natural lipid bilayer
surrounding the membrane protein will be an ideal situation for membrane protein to
work in.
1

The interactions between membrane proteins and solubilizing surfactants directly
effect on the stability and function of these proteins outside of their native biological
membranes.
2
The ability to preserve protein activity and conformation in solution is also
important to choose optimal surfactants for membrane protein crystallization. Besides,
proteins have an affinity for specific lipids. The interaction of surfactants and general
features of cell membranes must be considered, such as hydrophobic: hydrophilic balance
74

(HLB) and critical micelle concentration (CMC) and the properties of the membrane
components.
3

A number of techniques have been developed to meet these requirements to
solubilize and reconstitute membrane proteins in vitro, including ionic surfactants,
4, 5

nonionic surfactants,
6
lipid/surfactant system,
7, 8
extraction into organic solvents,
9

dilution
10
and dialysis. Lipid/surfactant system was used in this paper. Phospholipid and
surfactant mixtures in aqueous solutions can aggregate in either micellar or lamellar
structures depending on the concentration of the two components. The mixture with
relatively high detergent concentration tends to form micellar aggregates and relatively
low detergent concentration contains lamellar aggregates. Two structures coexistence in
an intermediate range of detergent concentration.
11, 12
Many issues are concerned on the
phenomenon of solubilization and reconstitution of lipid bilayers and protein-containing
membranes, such as, the effects of proteins and different phospholipids with different
chain length and head groups act on the phase behavior of mixtures.
13

Some surfactants such as DHPC or CHAPS are mixed with short chain lipids such
as DMPC, bilayered discoidal structures known as bicelles may be formed in the correct
composition and correct temperature. For example, based on varying-chain-length PC
lipids with the bile salt detergent can reconstituted DAGK in functional form within the
bicelle. The optimal activity of DAGK in this bicellar system can be improved by
adjusting the lipid to detergent ratio.
14, 15
Bicelles have characteristic of lower surfactant
concentration and bilayer structure that make them potentially more useful as an
environment to solubilize functional membrane proteins.
16
Bilayer structure is dynamic.
Both proteins and lipids can lateral diffusion within the plane of the membrane. The
75

lipids can laterally diffuse and exchange from one bilayer surface to the other (flip–flop).
Lateral diffusion is generally faster than flip–flop although for some lipids flippase
proteins aid flip–flop. Bilayer contains both lamellar and non-lamellar forming lipids. In
general, saturated lipids are lamellar forming lipid, whereas highly unsaturated lipids
form non-lamellar phases. Non-lamellar lipids and unsaturated acyl chains tend to curve
towards water.
Bacteriorhodopsin (bR) is one of the most studied membrane proteins (molecular
mass, 26 kDa), the only protein in the purple membrane (PM) of Halobacterium
halobium.
17
BR within the PM was organized into a two-dimensional hexagonal lattice
with each unit containing a trimer of bR molecules. Purple membrane is made up of 75
wt% bR and 25 wt% lipids, which is equivalent 10 lipid molecules per bR.
18
These lipids
composed of 60% phospholipids and 30% glycolipids.
19
The protein is 248 amino acids
long and seven transmembrane helices connected by short extramembranous loops. A
retinal chromophore is covalently bound to the seven helix bundle through a Schiff-base
link to Lys-216. The secondary structure of bR studied by FTIR contains 70% α-helix
structure corresponding to the transmembrane helices, 20% β-strands and 10% unordered
structures.
BR can be spontaneously refolded to a native state from a denature state in vitro
which is one of the few membrane proteins possible to date.
20
A general strategy for bR
refolding in vitro involves solubilization of the apoprotein bacterioopsin (bO) in SDS
(sodium dodecyl sulfate) after delipidation and removal of retinal from the purple
membrane. Bacterioopsin can be refolded in mixed detergent/lipid system such as
DMPC/CHAPSO, DMPC/CHAPS, and DMPC/DHPC
21-23
.
76

Photoswitchable azobenzene-based surfactant was used to control the physical
property and structure of lipids.
24, 25
Also, simple light illumination will be used to
control membrane protein folding. Photocontrol of bR folding will be achieved through
the photoisomerization of a light-sensitive surfactant that can be switched from the trans
to the cis state from visible light to UV light. 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 and enhanced
enzymatic activity by using the unique property.
26, 27
This study will extend this
methodology to membrane proteins by utilizing the photosensitive azoTAB partitioning
into bilayer. Light-induced azoTAB affecting the structure of bilayer structure result in
the variation of protein folding. DMPC/CHAPS complex were chosen for study because
bR could be solubilized in to the mixture solution and refold at very high percentage.
Unsaturated DOPC lipid has longer carbon chain than saturated DMPC lipid. We try to
compare the bR folding percentage in DMPC/CHAPS and DOPC/CHAPS systems. The
addition of photosensitive azoTAB surfactant in DMPC/CHAPS lipid system also
changes the bR folding percentage between trans and cis conformation.
UV-vis spectroscopy will be used to study the effect of the photosensitive
surfactant and light conditions on bR folding by study absorbance changes at 560 nm of
the retinal chromophore. Furthermore, the lipid-surfactant assembly was modeled by
using small angle neutron scattering where the form factor and pair distance distribution
function of the shape can be computed. Comparing experiment data with variable of
azoTAB concentration, light and lipid length, the subtle difference of micelle structure
that dramatically changes the folding state.
77

4.3 Materials and methods
Surfactant and Lipid. The photoresponsive surfactant 4-ethyl-4’
(trimethylaminobutoxy) azobenzene bromide (azoTAB) was synthesized according to the
literature procedures.
28, 29
The surfactant undergoes a reversible photoisomerization by
switching the appropriate wavelength of visible light (434nm) to UV light (350nm). The
conformation of azoTAB will transfer from trans isomer to cis isomer. Trans isomer
exhibits a lower dipole moment and more hydrophobic than cis isomer. Other physical
properties also change as the photoswitch such as critical micelle concentration,
28
surface
tension and electrical conductivity.
1,2-Dimyristoyl-sn-glycero-3-phophocholine (DMPC) and 1,2-Dioleoyl-sn-
glycero-3-phosphocholine (DOPC) was purchased from NOF Corporation. 3-[(3-
Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), Sodium Dodecyl
Sulfate (SDS) and all-trans-retinal were purchased from Sigma-Aldrich. 2% (w/v)
DMPC stirs for 2 h at room temperature in 50 mM sodium phosphate, pH 6 buffer.
CHAPS was then added to 2% (w/v) final concentration to get mixed DMPC/CHAPS
micelles (or bicelles). The mixture sonicated in a bath sonicator for 30 min.
DOPC/CHAPS solution was made in the same procedures. The bicelle solution could be
stored at 4 °C for several days.
30

Preparation of Bacterioopsin. Purple membrane contains both lipids and the
membrane protein bacteriorhodopsin which is isolated from Halobacterium salinarium
(strain S9, a kind gift from Professor Dieter Oesterhelt at the Max Planck Institute of
Biochemistry in Martinsried). The prepare procedure was established by Oesterhelt and
78

Stoeckenius.
31
In brief, bacterial cells were grown in 1 L cultures incubated at 37°C in the
dark in a shaker at 100 rpm for 5 days until the absorption at 560 nm was between 1.0-1.5.
The purification of PM was then isolated from the medium at 4000 rpm for 40 min in
centrifuge. Further purification, impurities in PM were removed by washing with DI
water at least 15 times at 24000 rpm for 45 min (Beckman Coulter Avanti J-25 Ι
Centrifuge, Beckman rotor JA 25.5) until the water no longer exhibited a purple color.
The obtained purple membrane was stored at -20°C. Typical yields of purified purple
membrane were 20 mg/L. Delipidated bacterioopsin (bO) was prepared from purple
membrane according to the method of Braiman el al.
32
Purple membrane was mixed as
aqueous suspension with an organic solvent containing
chloroform/methanol/triethylamine (100:100:1 volume ratio) to reach a single phase.
Hydroxylamine was added to remove retinal, and phase separation was effected by
addition of a buffer containing 0.1 M phosphate, pH 6.0. Followed by centrifugation to
remove any insoluble material and get the bO pellet. The final bO pellet was stored at -20
°C. About 50% of the protein present in the purple membrane was recovered as bO.
30, 33

Refold of Bacteriorhodopsin. The procedure of bacteriorhodopsin refold from
bO was described from Huang et al.
20
8 µM bO dissolves in 0.2% SDS, 10 mM sodium
phosphate buffer. DMPC/CHAPS bicelles at 2%/2%, 1.5%/1.5%, 1%/1% (w/v) were
mixed with an equal volume of bO/SDS solution. 7 µm all-trans-retinal in ethanol stock
was added, and the final concentration of ethanol never exceeds 0.5% (v/v).
21, 30
BO/SDS
solution and DMPC/CHAPS bicelles are allowed to equilibrate for 30 min prior to add
retinal. DOPC/CHAPS was in the same procedures. Azotab surfactant was added
directly into refold solutions with the appropriate amount.
79

UV-Vis Spectrometer measurement. Refold bR was suspended into the
appropriate concentration of DMPC/CHAPS/azoTAB solution. Before the measurement,
the solution was illuminated for 20 min 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 for 20 min. The solution mixtures are alternately exposed to visible and UV light
with surfactant conversion from the trans to the cis isomer assured with absorption
measurements. The concentration of the protein in each sample was ~0.11 mg/mL,
determined spectroscopically. The ratio of the refold bR concentration to the initial bO
concentration is the folding fraction. The concentration of bO and refold bR were
determined by extinction coefficient. The extinction coefficient of bO in SDS is 66 000
cm
-1
M
-1
at 280 nm.
30
The extinction coefficient of refold bR is 51000 cm
-1
M
-1
at 560
nm.
34
Measured spectra were corrected for scattering effects using a spline routine with a
baseline choice of 700-900 nm.
Small-angle neutron scattering (SANS). Small-angle neutron scattering (SANS)
data were collected on the Bio-SANS CG-3 instrument at Oak Ridge National Laboratory
by using a neutron wavelength of λ=6 Å. Two sample detector distances (0.3 and 6.0 m)
were utilized to achieve a Q-range of 0.007–0.45 Å
−1
, 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
80

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. Following data collection for containing trans azoTAB samples,
without changing samples the SANS cuvettes were then illuminated with an 84 W long
wave UV lamp-365 nm (Spectroline, Model no. XX-15A) for at least 2 hr to convert
azoTAB to the cis isomer. All the SANS measurements, DMPC/CHAPS and
DOPC/CHAPS concentration were all at 1wt%/1wt%. Protein concentration is 0.1mg/
mL in all samples. The SANS data were reduced, analyzed and modeled using the Igor
Pro (WaveMetics) program supplied by NIST.
35

The SANS data were analyzed using three complementary techniques: Guinier
analysis to determine the radius of gyration, calculation of the pair distance distribution
functions (PDDFs), and fits of the data to an ellipsoid form factor with a charged sphere
repulsive structure factor.
36, 37
Estimated errors in radii of gyration are ±5%. The PDDFs
were calculated assuming a monodisperse system using GNOM
38
over a Q-range of ca.
0.03–0.3 Å -1 to avoid the need to account for protein intermolecular interactions. The
lowest maximum particle diameter (Dmax) was selected to give a smooth return of the
PDDF to zero at Dmax. The SANS data of the refold samples were fit using an ellipsoid
form factor supplied by NIST with fitted parameters being the rotation axis Ra and Rb,
while all other parameters could be fixed based on the properties of the system. Bicelles
were fit using a form factor which contains the effects on particle size, shape and
scattering power for monodisperse bi-axial ellipsoids of uniform scattering length density
coupled with structure factor both supplied by NIST.
39, 40
Model is smeared by the
instrumental resolution, which was calculated based on the instrument’s scattering
81

geometry. The neutron scattering length density (SLD) of the bilayer shell is assumed to
be a constant, since the SLD of D
2
O is much higher than SLD of the bilayer shell.
4.4 Results and Discussion
Effect of photosurfactant on protein folding. SDS-solubilized bacterioopsin
was mixed with DMPC/CHAPS and DOPC/CHAPS bicelles in the presence of the retinal
chromophore. Bacterioopsin can be refolded in mixed surfactant/lipid solution. The effect
of azoTAB and light on the conformation of the refold membrane protein bR was
examined by UV-vis spectroscopy.
Figure 4.1 shows the relative folding percentage of bR refolds from different
concentration of DMPC/CHAPS and DOPC/CHAPS systems as a function of azoTAB
concentration. A, B and C represent bR refold under 1%/1%, 0.75%/0.75% and
0.5%/0.5% DMPC/CHAPS solution exposed in visible light. The folding percentage
without azoTAB is nearly 85% which is in consistence with reference.
21
The small
percentage of protein which does not regenerate bR represents irreversibly aggregate in
solution. Folding percentage drops slightly when the solution contains azoTAB surfactant.
Phase behavior was affected by the addition of surfactant. D, E and F represent bR refold
exposed under UV light. D and E both have very similar folding percentage at all
azoTAB concentration. The folding fraction drops about 10% when the azoTAB switch
from trans state to cis state in 0.1 mM azoTAB. As the increase of azoTAB concentration
under UV light, the bR has much lower folding percentage under cis state compare to the
solvent at trans state.
82

It is noteworthy that folding percentage drop dramatically (~30%) between trans
and cis state when the lipid to azoTAB mole ratio close or smaller than 50. Therefore,
sample F 0.5%/0.5% DMPC/CHAPS at 0.3mM azoTAB has folding fraction at 53%.
Sample D and E need higher azoTAB concentration to reach this folding fraction under
UV light than sample F. Sample G and H are the bR folding fraction at DOPC/CHAPS
system. The folding fraction without azoTAB is only ~65%. The folding fraction still
decrease with the increase of azoTAB under UV illumination. It is clearly that
DMPC/CHAPS system provides a better bilayer environment to fold bR. UV illuminated
azoTAB also change the conformation of lipid bilayer which affect bR folding
performance.
0.4
0.5
0.6
0.7
0.8
0.9
1.0
A: 1%/1% trans
B: 0.75%/0.75% trans
C: 0.5%/0.5% trans
D: 1%/1% cis
E: 0.75%/0.75% cis
F: 0.5%/0.5% cis
Folding Fraction
azoTAB concentration (mM)
0 0.1 0.3
0.5 0.7 0.9
DMPC/CHAPS

0.9
G: 1%/1% trans
H: 1%/1% cis
azoTAB concentration (mM)
0 0.1 0.3 0.5 0.7
DOPC/CHAPS

Figure 4.1. Relative folding percentage of bR refolds from different concentration of
DMPC/CHAPS and DOPC/CHAPS systems as a function of azoTAB concentration
determined from the protein absorbance at 560 nm. DMPC/CHAPS, A: 1% / 1% at trans;
B: 0.75%/ 0.75% at trans; C: 0.5%/0.5% at trans; D: 1% / 1% at cis; E: 0.75%/ 0.75% at
cis; F: 0.5%/0.5% at cis. DOPC/CHAPS, G: 1%/ 1% at trans; H: 1%/ 1% at cis.
83

450 500 550 600 650 700
0.000
0.005
0.010
0.015
0.020
(A) DMPC/CHAPS=1%/1% trans
Absorbance
wavelength
0 mM
0.1 mM
0.3 mM
0.5 mM
0.7 mM
0.9 mM

450 500 550 600 650 700
0.000
0.005
0.010
0.015
0.020
(B) DMPC/CHAPS=1%/1% cis
Absorbance
wavelength
0 mM
0.1 mM
0.3 mM
0.5 mM
0.7 mM
0.9 mM

450 500 550 600 650 700
0.000
0.005
0.010
0.015
0.020
(C) DMPC/CHAPS=0.5%/0.5% trans
Absorbance
wavelength
0 mM
0.1 mM
0.3 mM
0.5 mM
0.7 mM
0.9 mM
450 500 550 600 650 700
0.000
0.005
0.010
0.015
0.020
(D) DMPC/CHAPS=0.5%/0.5% cis
Absorbance
wavelength
0 mM
0.1 mM
0.3 mM
0.5 mM
0.7 mM
0.9 mM
450 500 550 600 650 700
0.000
0.005
0.010
0.015
0.020
Absorbance
wavelength
0 mM
0.1 mM
0.3 mM
0.5 mM
0.7 mM
0.9 mM
(E) DOPC/CHAPS=1%/1% trans

450 500 550 600 650 700
0.000
0.005
0.010
0.015
0.020
(F) DOPC/CHAPS=1%/1% cis
Absorbance
wavelength
0 mM
0.1 mM
0.3 mM
0.5 mM
0.7 mM
0.9 mM

Figure 4.2. UV-vis spectra of bR fold at different azoTAB concentraion.

Figure 4.2 is UV-vis spectra of bR fold at different azoTAB concentration. Here
we can observe the change of absorbance at 560nm. Folding percentage was determined
by the ratio of the refold bR concentration to the initial bO concentration. BR exhibits a
convenient measure of protein folding. Retinal chromophore covalently attached to bR
shows an absorption band at 560 nm (native state), whereas chromophore free in solution
84

at 280 nm (denature state).The concentration of bO and refold bR were determined by
extinction coefficient based on the absorbance at 280 nm and 560 nm respectively.
The spectrum in Figure 4.2 (C) is shaky which means bR fold in 0.5%/0.5%
DMPC/CHAPS solution is in a very unstable environment. In fact, protein aggregates
with time and precipitates in 1 day for 0.5%/0.5% DMPC/CHAPS solution. On the
contrary, bR fold in 1%/1% DMPC/CHAPS solution provides a very stable environment
that can maintain in stable for weeks. Figure 4.2 (A) have a smooth spectrum than Figure
4.2 (C). Figure 4.2 (E) fold in DOPC/CHAPS solution is also in an unstable environment
and bR tends to aggregate in solution.
SANS experiments were also performed for bR refold in the presence of the
azoTAB surfactant, as shown in figure 4.3. The SANS experiment provides a relatively
wild Q range (0.007 −0.45 Å −1) allowed a broad range of length scales (L=2 π/Q=14 −
900 Å ) to be investigated simultaneously. It gives detailed information to study protein
structure in solution. SANS data collected from bR refold from DMPC/CHAPS and
DOPC/CHAPS with different azoTAB concentrations.
85

0.01 0.1
0.01
0.1
1
10
X25
X20
X12
X6
X4
X2
X1
Intensity (cm
-1
)
Q ( 

)
0 mM trans
0.3 mM trans
0.9 mM trans
2 mM trans
0.9 mM cis
2 mM cis
0 mM trans (DOPC)

0 2 4 6 8 10
0
2
4
6
8
10
0.01 0.1
0.1
1
A

Figure 4.3 SANS data and uniform ellipsoid fits for bR fold in DMPC/CHAPS as a
function of azoTAB concentration and bR fold in DOPC/CHAPS solution. The scattering
data have been scaled for better distinguish the curves. The inset shows the original
intensity.

The bicelle SANS curve should have the characteristic that low Q plateau region
followed by a Q
-2
and then a Q
-4
decay at hight Q region. Figure 4.3 shows a scattering
peak at Q~0.3 Ǻ
-1
when sample has less azoTAB concentration. As the azoTAB
concentration was increased, the effective charge decreased, a plateau region revealed at
low Q region as expected that cationic azTAB neutralize the net–negative bR. At high Q
regime, Porod’s law is the best described by a Q-4 decay related to the lipid bilayer
thickness. Q
-2
and Q
-4
regions are corresponding to the bicellar planar region and the lipid
bilayer respectively. The scattering profiles of protein complexes in solvent at low Q
86

were fitted using Guinier equation, I(Q)=I(0)exp(-R
2
g
Q
2
/3). Rg is the radius of gyration
of the protein complex and I(0) is the extrapolated intensity at Q=0. Two independent
slopes are detected in the Guinier plots at low-Q guinier region and intermediate-Q
guinier range. The first slope corresponds to the z-average radius of gyration of the
mixture in the region of Q
2
<0.003 Ǻ
-2
and is often a characteristic of oligomers (R
g
z
). The
second slope is Q
2
value in the region between 0.005 to 0.02 Ǻ
-2
which is a characteristic
of protein molecule (R
g
1
). It is noticed that the range of data used should abide by QR
g
<
1.3. While the Rg
1
fit span Q
2
=0.005-0.02 (QR
g
=1-3). The approximate expression exp(-
R
2
g
Q
2
/3) can be replaced by [3j
1
(QR
g
)/QR
g
]
2
as suggested by Guinier
41
. (j1(x) is
spherical Bessel function.) As seen in Table 4.1, the R
g
1
value increases from a value of
25.9Ǻ for DMPC/CHAPS without azoTAB solution, to ~27Ǻ with different
concentration of trans azoTAB –DMPC/CHAPS solution. UV illumination of the R
g
1

value decreases to 26.2 Ǻ. Calculation of the physical dimensions corresponding to these
measured R
g
1
values (true diameter=2(5/3)
0.5
R
g
1
) indicates that the diameter can be
approximated as 66.8Ǻ (DMPC/CHAPS), 69.7Ǻ (DMPC/CHAPS with trans azoTAB)
and 67.6Ǻ (DMPC/CHAPS with cis azoTAB). Guinier fit assumes that the structure is
sphere-like micelles. However, a more precise model fitted in the next paragraph reveals
an ellipsoid shape of micelles. Micelles have an ellipsoidal cross section rather than the
circular one. In the case, radius of gyration = ¼ ( a
2
+ b
2
), where a and b are semi-axes of
ellipsoid.
42
The radii of gyration were also obtained from the pair-distance distribution
functions, which utilizes the entire Q range and less influenced by interparticle
interference effects that are typically only important at low Q. The R
g
z
values obtained
from PDDFs are shown in table 4.1 which is consistent with the trend estimated from the
87

Guinier analysis. The R
g
z
obtained from PDDFs is bigger than the value from Guinier
analysis.
Table 4.1: Fits of the SANS Data of refold solutions at different azoTAB concentration
by using Ellipsoid form factor model. Radius of gyration values obtained from PDDF and
Guinier analyses.
[azoTAB] (mM) Ellipsoid model
Ra Rb Bkg(cm
-1
) X
2
/(N-1)
PDDF
D
max
R
g
z

Guinier
Rg
1
Rg
z

0 mM 19.8 36.5 0.0122 2.65 73 25.2 25.9 22.0
0.3 mM trans 20.6 39.3 0.0111 2.62 80 26.8 27.0 25.4
0.9 mM trans 20.1 40.6 0.0133 2.50 88 27.8 26.8 25.6
2 mM trans 19.8 40.8 0.0128 2.07 90 28.1 27.1 25.7
0.9 mM cis 19.1 41.4 0.0113 2.79 110 29.6 26.1 28.7
2 mM cis 19.2 41.6 0.0117 3.05 115 30.9 26.2 28.9
0 mM * 21.1 50.5 0.0143 5.60 130 39.7 27.6 37.4
*DOPC/CHAPS system. Other samples are in DMPC/CHAPS system.

To further investigate the effect of lipid, azoTAB surfactant and light illuminatin
on environment of protein folding, the PDDFs (pair-distance distribution functions)
shown in Figure 4.4 were calculated from the experimental scattering data. PDDFs
represents a measure of the probability P(r) to find two scattering centers at a distance r
apart.
43, 44
This provides detailed information regarding the structure effect on protein
folding using a model-independent procedure. As seen in the Figure 4.4, increasing
surfactant concentration results in a progressive shift in the peak position from an r-value
88

of 29 Å for DMPC/CHAPS solution to 31 Å in the presence of azoTAB. The peak
position only have modest shift between different concentration and trans/cis switch. The
biggest change is the pair-distance probabilities in the tail region. D
max
increases with
azoTAB concentration at trans from 73 Ǻ to 90 Ǻ. There is a prolonged tail when the
azoTAB switch to cis conformation. Large value of the maximum distance is 115 Ǻ at 2
mM cis azoTAB-DMPC/CHAPS. DOPC/CHAPS appears the biggest r-value in the peak
and D
max
than any other DMPC/CHAPS solution. D
max
can be considered as a diameter of
a flat disc (bicelle). The bicelles composed from DMPC/CHAPS have a smaller disc
diameter than DOPC/CHAPS micelles.

0 10 20 30 40 50 60 70 80 90 100 110 120 130
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
P(r)
r (  
0 mM
0.3 mM trans
0.9 mM trans
2 mM trans
0.9 mM cis
2 mM cis
0 mM (DOPC)

Figure 4.4 PDDFs of bR in DMPC/CHAPS and DOPC/CHAPS solutions as a function of
the azoTAB concentration under both visible (trans) and UV (cis) light illumination.

Besides, the SANS data fitted by assuming the folding adopts an ellipsoidal shape
in solution and interparticle interference effects due to screened coulomb repulsion
between charged particles. The ellipsoid is rotated about the axis of Ra to define the
89

ellipsoid. The object is an oblate ellipsoid (disk-like), if the radius Rb >Ra. The object is
a prolate ellipsoid (needle-like), when Rb < Ra. If the two radii are equal, then the
ellipsoid is a sphere. The fitting plots using an ellipsoid model are showed in figure 4.3.
The results of the fitting model were given in Table 4.1. The data indicate that the
morphology does not change dramatically. All the samples are disk-like shape.
According to the lecture
45, 46
, DMPC/CHAPS mixtures are thought to form disc-shaped
micelles (or named bicelles), with a small disc of DMPC bilayer. The CHAPS is capping
the edges of a DMPC disc. Therefore, we can estimate the semi-axes (Ra) as half of the
thickness of DMPC bilayer and major semi-axes (Rb) as the radius of the bicelle disc.
The fitted SANS data of DMPC/CHAPS with 0 mM azoTAB shows the half thickness
Ra=19.8 Ǻ and disc radius Rb=36.5. The total length of the DMPC molecules in the fully
extended conformation is 26.7Ǻ which is longer than the fit Ra at 19.8 Ǻ
(DMPC/CHAPS without azoTAB). It may be the CHAPS molecules induce some
conformational disorder in the mixed bilayer and the effective length of DMPC
hydrocarbon chain is shortened. Fit of DOPC/CHAPS with 0 mM azoTAB shows
Ra=21.5 Ǻ and Rb=50.5 Ǻ which are all larger than DMPC/CHAPS. DOPC is an
unsaturated lipid with double bonds along tails and the hydrocarbon chain is longer than
DMPC. The double bonds in the unsaturated lipids do not let the lipid tails to get as close
as the DMPC lipid. The longer chain of DOPC also composes of thicker bilayer than
DMPC does. Protein stability is related to lipid chain length.
42, 47
BR has high folding
percentage in DMPC/CHAPS solution. DMPC appears to be the optimal lipid chain
length for bR stability in bicelles. DOPC alters the bicelle radius and bilayer stress.
Therefore, DOPC/CHAPS is not a suitable environment for the stability of bR folding
90

like figure 4.1 shown. With increase in the trans azoTAB concentrations, the scattering
intensity increases for Q < 0.05 Ǻ
-1
. The sharpness of the peak reflects the interparticle
interference. A lower intensity at the low Q regime and a more prominent scattering peak
means a higher charge density. The charge density on the bicellar disks decreases as the
increase of azoTAB concentration. There are more hydrophobic trans azoTAB with net
positive charge to neutralize the protein in solution.
DMPC and DOPC are both amphipathic lipids made up of long chains of
hydrogen and carbon with a polar head group. Each DMPC tail comprises fourteen
carbons. Each DOPC tail is eighteen-carbon long and contains one double-bond in its
center. DMPC is a saturated lipid because the hydrocarbon tails only comprise single
bonds. DOPC is an unsaturated lipid which means double bonds along their tails. The
double bonds in the hydrocarbon chain create kinks which creates spaces when the lipids
pack into a bilayer. The double bonds in the unsaturated lipids do not let the lipid tails to
get as close as the saturated lipid. CHAPS is a mild detergent because of the zwitterionic
nature of its head group. Membrane proteins are often unstable in detergent micelles and
usually denature or inactivated. However, certain mixture of detergent and lipid assemble
as bilayer disc which can be used for stabilization of membrane proteins. Bicelle
morphology can both assist protein solubilization and stabilize the correct membrane
protein fold. Bilayer thickness can be varied by phospholipid length.
48
A compatible
bilayer thickness can enhance protein in correct folding state. DMPC based bicelle
mixtures will not suitable for the reconstitution of all integral membrane proteins. Some
proteins may prefer other lipids to acquire a suitable environment, such as longer acyl
chains or higher degrees of chain unsaturation. Besides, the radius of bilayer can be
91

varied by the ratio of long chain lipid to short chain lipid in solution. Some researches
utilize physical properties of bilayer to control yield of membrane protein folding. For
example, manipulation of bilayer with different curvature stress and lipid lateral pressure
can impact on folding condition. Unsaturated lipid chains contain more cis double bonds,
which kink the chains and increase monolayer curvature. Saturated lipid chains decrease
monolayer curvature. Introducing a saturated chain PC to an unsaturated PC bilayer (e.g.
DMPC added to DOPC) will reduce the stored curvature elastic stress. On the contrary,
the curvature stress of the bilayer was increased by the addition of the unsaturated
lipids.
22, 49, 50
The yield of correctly folded protein was found to depend on the optimal
curvature stress of bilayers.
4.5 Conclusions
In this study, the effect of azoTAB concentration and conformation alters the
bicelle radius, thickness, curvature stress and stability. The azoTAB shortens the bilayer
thickness and widens disc diameter by switch the illumination from visible to UV light.
Switching from trans to cis probably causes a change in the lateral pressure profile,
resulting from a change in curvature stress along the acyl chains of the lipid, and some
thinning of the membrane. The slight difference of bicelle structure changes the folding
environment of bR. Membrane protein folding in bicelles is affected through the use of
photoresponsive surfactants.

92

4.6 References
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23. Chen, G. Q.; Gouaux, E. Biochemistry 1999, 38, 15380-15387.

24. Backus, E. H. G.; Kuiper, J. M.; Engberts, J. B.; Poolman, B.; Bonn, M. The
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95

Chapter 5: Conclusion and Future Work
5.1 Conclusion
In the previous Chapters, a novel method to photocontrol membrane protein
folding using light responsive surfactants was discussed. In chapter 2, we have studied
the structure change of surfactant mixtures of two oppositely charged surfactants by
switching the light wavelength in expose. In mixture of positively charged azobenzene-
based photoresponsive surfactant (azobenzenetrimethylammonium bromide, azoTAB)
and negatively charged alkyl-based surfactants (sodium dodecylbenzenesulfonate, SDBS).
AzoTAB undergoes reversible photoisomerization to the relatively-hydrophobic trans
isomer or the relatively-hydrophilic cis isomer upon exposure to visible or UV light,
respectively. This results in the formation of light-responsive vesicles that can be
disrupted and spontaneously reformed with simple light illumination.
In Chapter 3, reversible control membrane protein folding was using photo
responsive surfactant. Catanionic surfactant system provides a phase transition to change
conformation of membrane proteins. In mixtures of purple membrane and surfactants was
used to control the folding and unfolding of bacteriorodopsin. In Chapter 4, the effect of
azoTAB concentration and conformation alters the bicelle radius, thickness, curvature
stress and stability. Switching from trans to cis probably causes a change in the lateral
pressure profile, resulting from a change in curvature stress along the acyl chains of the
lipid, and some thinning of the membrane. Membrane protein folding in bicelles was
affected through the use of photoresponsive surfactants.
96

Further studies should be undertaken in order to understand how to optimize the
use of light responsive surfactants to control protein folding. The interactions between
membrane proteins and solubilizing surfactants directly effect on the stability and
function. Our goal is to demonstrate the photoresponsive surfactant can control phase
transition and structure change.
1. Phase behavior of saturated and unsaturated phospholipids interacts with
different alkyl chain length of azoTAB at trans and cis state.
2. Rational design for protein regeneration based on the stored curvature elastic
stress and lipid length of bilayers.
Membrane proteins are often unstable in detergent micelles and usually denature
or inactivated. However, certain mixture of surfactant and lipid assembles which can be
used for stabilization of membrane proteins. Different types of membrane mimics will be
considered, such as micelles, bicelles, unilamellar lipid vesicles, and planar lipid
membranes.
1
Degree of chain unsaturation and acyl chain length of lipid, temperature,
critical micelle concentration, total surfactant concentration, surfactant to lipid ratio,
trans/cis conformation are all the factors can affect phase behavior.
2-4
A rational design
bases on these factors can be utilized to control the state of membrane protein.

97

5.2 References
1. Serebryany, E.; Zhu, G. A.; Yan, C. Y. Biochim Biophys Acta 2012, 1818, 225-
233.

2. Booth, P. J. Curr Opin Struct Biol. 2005, 15, 435-440.

3. Mitchell, D. C. Biochim Biophys Acta 2012, 1818, 951-956.

4. Judge, P. J.; Watts, A. Curr Opin in Chem. Biol. 2011, 15, 690-695.

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Abstract (if available)

Abstract Membrane proteins perform a number of roles in biological function. Membrane lipids can self assembly into numerous different phases in aqueous solution, including micelles, vesicles and lamellar phases. However, the phase properties of biological membranes are far more complex. Many membrane proteins require specific lipids to be present in the membrane to be fully active. Therefore, artificial membrane-like environments for protein folding are studied. Many approaches are developed to solve the membrane protein folding problems and based on manipulating the bilayer structures. Bacteriorhodopsin (bR) is 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 provides a method to control membrane protein folding. The azobenzene-based photosurfactant undergoes a reversible photoisomerization upon illumination either visible (trans) or UV (cis) light. The trans isomer is relatively hydrophobic and planar than cis isomer. The phase behavior and bilayer structures are changed by the effect of photoresponsive surfactant. These strategies provide a convenient means to control membrane protein folding with light illumination.

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Asset Metadata

Creator Chang, Chia Hao (author)

Core Title Controlling membrane protein folding using photoresponsive surfactant

School Andrew and Erna Viterbi School of Engineering

Degree Doctor of Philosophy

Degree Program Chemical Engineering

Publication Date 07/10/2012

Defense Date 06/11/2012

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

Tag bacteriorhodopsin,fold,membrane protein,OAI-PMH Harvest,photosurfactant

Language English

Contributor Electronically uploaded by the author (provenance)

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

Creator Email chang7@usc.edu,coucal.tw@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-54493

Unique identifier UC11289163

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Document Type Dissertation

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

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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