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Flipping the switch on protein activity activity: elastin-like polypeptides assemble into cell switches and vesicles

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Content i

FLIPPING THE SWITCH ON PROTEIN ACTIVITY: ELASTIN-LIKE
POLYPEPTIDES ASSEMBLE INTO CELL SWITCHES AND
VESICLES

by

Martha K. Pastuszka

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERISTY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirement of the degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)

May 2014

ii

Dedicated to Urszula and Jacek Pastuszka-
for their endless love, support, and encouragement

iii
ACKNOWLEDGEMENTS
This thesis would not be here if I was not for the support and mentorship of my PI, Dr.
Andrew MacKay. Thanks to his teaching, I have developed skills to think critically as a scientist.
He taught me not only the hard skills needed to answer scientific questions, like how to properly
set up an experiment, but soft skills like patience and motivation, both necessary in scientific
inquiry.
Besides my advisor, I would like to thank members of my thesis committee, Dr. Sarah
Hamm-Alvarez and Dr. Curtis Okamoto. Their expertise in cell biology and intracellular
trafficking helped guide my project into its current direction. I appreciate their valued insights
and encouragement.
Many thanks go out to my fellow MacKay lab members: Dr. Siti Janib, Dr. Suhaas Aluri,
Dr. Wan Wang, Dr. Pu Shi, Jugal Dhandhukia, Jiawei Wang, Jordan Despanie, Zhe Li, Dr.
Joshua Gustafson, Vinit Gohlap, Isaac Weinzheimer, Alexa Hudnut, Aarti Jashnani, and Sejal
Parkah. An extra special thanks goes to Siti Janib, not only did she teach me how to purify ELPs,
she was an excellent sounding board for ideas. Many thanks also goes to our collaborators from
the Hamm-Alvarez lab: Dr. Maria Edman-Woolcott, Francie Yarber, Aaron Hsueh, and Mihir
Shah.
And finally, I would not have pursured my PhD if not for the support and encouragement
from my parents, Urszula and Jacek Pastuszka. They believed in my abilities even when I didn’t
believe in them myself. Nie ma slow ile ja was kocham!

iv
TABLE OF CONTENTS
Flipping the switch in protein activity: elastin-like polypeptides assemble into cell switches
and vesicles

LIST OF FIGURES vii
LIST OF TABLES ix
LIST OF ABBREVIATIONS x

PROLOGUE 1

CHAPTER 1 Introduction

1.1 Molecular self assembly 4
1.1.1 DNA Nanostructures 5
1.1.2 Polysaccharides 9
1.1.3 Protein polymers 12

1.2 Orthogonal protein control 17
1.2.1 Transcriptional control 19
1.2.2 Translational control 20
1.2.3 Post-translational control 22

1.3 Environmentally responsive peptides 23
1.3.1 Elastin-like polypeptides 23

1.4 Clathrin-mediated endocytosis 26

CHAPTER 2 A tunable and reversible platform for the intracellular formation of
genetically engineered protein microdomains

2.1 Abstract 28

2.2 Introduction 29

2.3 Materials and methods 32
2.3.1 Plasmid constructs 32
2.3.2 Cell culture 32
2.3.3 Confocal microscopy and thermal control 33
2.3.4 Fluorescence recovery after photobleaching (FRAP) 33

2.4 Results 35
2.4.1 Tunable assembly of GEPMs 35
2.4.2 Clustering of ELP microdomains is fast and reversible 37
2.4.3 Mobility of polymers depends on transition state 43

v
2.5 Conclusions 45

CHAPTER 3 Flipping the switch on clathrin-mediated endocytosis using genetically
engineered protein microdomains

3.1 Abstract 46

3.2 Introduction 47

3.3 Materials and methods 50
3.3.1 Vector construction 50
3.3.2 Immunofluorescence 52
3.3.3 Receptor Internalization Assays 52
3.3.4 Quantification of receptor internalization in single cells 53
3.3.4 Statistics 54

3.4 Results
3.4.1 Design and characterization of a thermally responsive post-translational
switch
55
3.4.2 ELP-CLC expressed in cytosol shows colocalization with early markers
of CME
57
3.4.3 Temperature triggered pathway modulation in mammalian cells 61
3.4.4 Inhibition of GEPM medicated CME knockdown is reversible 65
3.4.5 ELP-CLC GEPMs are CME knock-down specific 67
3.4.6 Proposed mechanism of V96-CLC CME knock-down 69

3.5 Conclusions 73

CHAPTER 4 An amphipathic alpha-helical peptide from apolipoprotein A1
stabilizes protein polymer vesicles

4.1 Abstract 76

4.2 Introduction 77

4.3 Materials and methods 80
4.3.1 Construction of L4F ELP fusions 80
4.3.2 Optical density characterization of ELP phase diagrams 81
4.3.3 Temperature dependent dynamic light scattering 81
4.3.4 Transmission electron microscopy (TEM) sample preparation 81
4.3.5 α-smooth muscle actin assay 82

4.4 Results 83
4.4.1 Characterization of L4F ELP fusions 83
4.4.2 L4F mediates assembly of ELP nanoparticle 87
vi
4.4.3 L4F nanoparticles prevent hepatic stellate cell activation 91

4.5 Conclusions 93

CHAPTER 5 Conclusions and future directions

5.1 Significance 97

5.2 Conclusions 99

5.3 Proposed Improvements 102

5.4 Future Applications 104

References 105

vii
LIST OF FIGURES
Chapter 1
1.1 Cellular switches act at different points on protein expression 19
1.2 ELP cloning and characterization 25
Chapter 2
2.1 Tunable assembly of genetically-encoded polymer microdomains (GEPMs) 31
2.2 GFP-ELP expression in Hek293 cell culture 35
2.3 Transition temperature of ELP is slightly altered by the fusion to GFP 37
2.4 Clustering of ELP microdomains is fast and reversible 39
2.5 GFP-V72 microdomain formation is rapid and reversible during controlled heating
and cooling

40
2.6 GFP-2VA192 microdomain formation is rapid and reversible during controlled
heating and cooling

41
2.7 Microdomain formation and temperature fluctuations are nontoxic to cells 42
2.8 Mobility of polymers depends on transition state 44
Chapter 3
3.1 Triggered microdomain assembly in the cytosol inhibits clathrin-mediated
endocytosis

49
3.2 CLC microdomain assembly is tunable and rapid 56
3.3 ELP microdomain formation speed is retained across cell lines and polymer
constructs

56
3.4 Soluble A96-CLC does not co-localize with CLC or CHC 59
3.5 V96-CLC colocalizes with early markers of CME in CHO cells 59
3.6 V96-CLC colocalizes with early markers of CME in Hek293 cells 60
3.7 V96-CLC self-assembles into hollow spheres with CHC 60
viii
3.8 Angiotensin II receptor internalization is significantly reduced by microdomain
assembly
63
3.9 Internalization of the M1 muscarinic receptor is knocked down by V96-CLC
microdomains

64
3.10 CLC-V96 microdomain assembly blocks clathrin-dependent internalization as
effectively as chemical inhibition
64
3.11 Resolubilizing V96-CLC microdomains confers function back to CME 66
3.12 Knock-down of CME with CLC microdomains does not affect a marker of caveolin-
mediated internalization

68
3.13 Proteins not associated with CME do not associate with V96-CLC GEPMs 69
3.14 Mechanism of V96-CLC CME knock-down 70
Chapter 4
4.1 Fusion of an amphipathic peptide and an ELP designed to assemble nanostructures 84
4.2 Fusion of the L4F peptide depresses the ELP transition temperature 86
4.3 Fusion to the L4F peptide mediates nanostructure assembly 88
4.4 Nanostructures formed by L4F fusions are peptide vesicles 90
4.5 L4F-A192 nanoparticles inhibit activation of hepatic stellate cells (HSCs) 92
Chapter 5
5.1 ELP transfection efficiency is dependent on transition temperature of ELP 103

ix
LIST OF TABLES
2.1 Microdomain formation temperatures of thermally responsive polypeptides 36
3.1
Gene and protein sequences of clathrin-light chain fusions with elastin-like polypeptides

51
3.2
CLC microdomains colocalize with markers of early endocytosis 58
4.1 Recombinant protein polymers examined during this study 80

x
ABBREVIATIONS

AngII angiotensin II
AP1 adaptor-related protein complex 1
ApoA1 apolipoprotein A1
BSA bovine serum albumin
Cav1 caveolin 1
CHC clathrin-heavy chain
CHO chinese hampster ovary
CLC clathrin-light chain
CME clathrin-mediated endocytosis
CTxB cholera toxin B subunit
DAPI 4',6-diamidino-2-phenylindole
DLS dynamic light scattering
EEA-1 early endosome antigen 1
ELP elastin-like polypeptide
FACS fluorescent activated cell sorter
FRAP fluorescence recovery after photobleaching
GEPM genetically engineered protein microdomain
GFP green fluorescent protein
GPCR G-protein coupled receptor
HA human influenza hemagglutinin
Hek293 human embryonic kidney 293
HSCs hepatic stellate cells
LAMP1 lysosomal-associated membrane protein 1
LC3 microtubule-associated protein 1A/1B-light chain 3
M1R muscarinic receptor
MDC monodansylcadaverine
MW molecular weight
NAFLD non-alcoholic fatty liver disease
OD optical density
PBS phosphate buffered saline
PCR polymerase chain reaction
PEG polyethylene glycol
T1 transition 1
T2 transition 2
TEM transmission electron microscope
T
t
transition temperature

1
Prologue

This research project primarily focuses on the application of elastin-like polypeptides
(ELPs) as intracellular protein switches. ELPs are biologic, environmentally responsive polymers
comprised of repeats of the Val-Pro-Gly-Xaa-Gly motif, a sequence derived from the gene for
human tropoelastin. Modulating the molecular weight and amino acid composition allows for
precise control over the temperature that triggers polymeric self assembly, allowing for the
formulation of polymers that form structures at physiologically relevant temperatures.
Intracellular protein switches mainly focus on the genetic deletion of the protein of interest using
methods such as siRNA or tetracycline inducible promoters. Some protein switches targeting the
fully formed protein exist, however ease of implementation as well as reversibility remain a
hindrance. With this limitation in mind, a novel ELP based switch that sequesters a protein of
interest into an insoluble cytosolic microdomain would permit for the rapid and reversible
inhibition of that protein’s function, effectively acting as a functional knock-out. Studies
assaying the feasibility of this approach reveal that ELPs can knock out a protein’s activity by
sequestering it with a half life of minutes and can reinstate protein function equally quickly. The
genetic encodability of ELPs make it easy to transfer this paradigm to alternate systems,
presenting a novel, easily adaptable tool to study protein behavior in real time.

ELP’s environmental responsive switch from disorder to order has previously been used
in thermally directed chemotherapeutic applications, protein purification, as well as the
formulation of thermally responsive hydrogels for cell culture and tissue engineering. However,
this reversible self assembly had only been characterized in PBS and outside the cell, with no
2
extensive intercellular applications having previously been described. Chapter 2 characterizes,
for the first time, the biophysics of ELP assembly in mammalian cytosol. It supports the
hypothesis that cytoplasmic GFP-ELP fusion proteins will phase separate rapidly and reversibly.
ELPs comprised of varying molecular weights and compositions were assessed for their ability
to self assemble into microdomains upon thermal stimulation. A correlation between ELP self
assembly in solution versus the cytosol is found, leading to the identification of an optimal
formulation for further ELP protein switch applications.

Chapter 3 looks extensively at the conditional blockage of clathrin-mediated
endocytosis. It seeks to address the postulated hypothesis that temperature-triggered aggregation
of clathrin-light chain ELP fusion proteins will halt clathrin-mediated endocytosis. ELPs are
genetically engineered onto the N-terminus of the clathrin-light chain molecule, a key effector in
the clathrin-mediated endocytosis (CME) pathway. Prior to ELP assembly, the ELP – CLC
fusion functions normally and allows for normal receptor mediated internalization. Raising the
temperature induces ELP microdomain assembly, which brings in sequesters the fused CLC
protein. The absence of CLC to function as a scaffold protein for CME inhibits the
internalization of G-protein coupled receptors. Dropping the temperature back down causes the
ELPs to re-solublize and restores CME activity.

Fusing a polymer to a small peptide is a common technique used to increase the
molecular weight of a low molecular weight drug. Testing the hypothesis that increasing the
molecular weight of a peptide by attachment of an ELP will increase that peptide’s
hydrodynamic radius, Chapter 4 describes a surprising new formulation for the assembly of
3
polymeric vesicles using L4F-ELP fusion proteins. Below their phase transition, ELPs assume
monomeric form. Attaching L4F, an alpha helical peptide derived from the lipid-binding domain
of the ApoA1 protein, drives ELP assembly into nanoparticles below the ELP assembly
temperature, greatly increasing the hydrodynamic radius neither L4F nor the ELP would have
alone. Cell assays show that despite this vesicle formation, the L4F peptide still retains anti-
immunogenic properties, holding promise for future systemic applications of this nanoparticle.

4
Chapter 1
Introduction

1.1 Molecular Assembly
Multifunctional biomaterials offer the possibility to engineer systems with mechanical,
chemical, and physical properties that respond to environmental changes in nuanced ways
endowing material properties dramatically better than current technologies
1-3
. In the pursuit of
these biomaterials, enhanced control is needed over their assembly at the nanoscale. The rational
design of self-assembling nanoscale systems is one strategy of fabricating these materials from
the bottom-up. In the medical arena, self-assembled nanostructures are under evaluation as both
diagnostics
4
and therapeutics
5
. Beyond medical applications, nanoscale self assembly is being
used in electronics to create clusters of molecules that act as insulators, semiconductors, and
even superconductors
6
. Non-covalent forces drive the formation of ordered structures and
patterns at the nanoscale, imbuing novel functions to systems that would not occur in the
disordered state
7
. The shapes of higher order nanostructures or patterns are largely determined by
the individual parts comprising the system. Therefore, the ability to engineer precision
macromolecular parts is expected to enable the rational design of multifunctional biomaterials.
The most complex examples of self-assembly are biological. Through the evolution of
life, interconnected networks of proteins, sugars, lipids, and oligonucleotides have been
genetically ‘programmed’ to assemble the multifunctional systems essential for life. Inspired by
these natural systems, we have entered a new era of engineering, where intrinsic properties of
these natural building blocks can be reengineered for diverse applications. In addition to
harnessing the physical properties inspired by natural biomaterials, biologically derived building
5
blocks can result in more uniform synthesis, better biocompatibility
8
, and utilization of
endogenous biodegradation pathways
9
. Biologically derived systems can often be processed by
endogenous nucleases, proteases, and glycosylases, after which the degraded products are
naturally excreted or reutilized by the body
10
.
Many polymers and biological materials undergo thermally-mediated assembly
processes, which have been proposed as strategies to enhance the assembly and functionality of
biomaterials
1-2,11
. Assembly of nanostructures from synthetic polymers such as poly N-
isopropylacrylamide (PNIPAM) and poly lactic acid (PLA) have been extensively studied and
reviewed
12-13
. These ‘smart’ materials have been engineered into temperature responsive
hydrogels, micelles, and vesicles
14-16
. However, an emerging body of work now characterizes the
use of biological materials to self-assemble structures using temperature as an environmental
cue. The purpose of Section 1.1 is to review the current status of thermally responsive
biopolymers, which for the purposes of this section is divided into three broad groups: nucleic
acids, polysaccharides, and proteins. Aspects of each biopolymer can be manipulated to undergo
morphological changes with response to temperature. Here we identify examples where these
materials are exploited to assemble or significantly alter their nanostructures in response to
heating.
1.1.1 DNA Nanostructures
The high fidelity base-pairing specificity of nucleotides has been exploited by molecular
biologists for over 20 years through applications of the polymerase chain reaction (PCR)
17
. As
part of PCR, small oligonucleotides are used to recognize and pair with a very specific sequence
of melted single stranded DNA. A nucleobase on one DNA strand only binds its complementary
nucleobase on the opposite strand
18
. Purines hydrogen bond with pyrimidines and vice versa. An
6
adenine base will only form a hydrogen bond with thymine and cytosine will only bond to
guanine. While having only 2 types of bonds sounds limiting, the linear combinations of 4
nucleotides of length, n, yield 4
n
possible permutations. A family of oligonucleotides with 10
nucleobases, could thus recognize and bind to over one million unique sequences of DNA. Since
nucleotide base pairs do not bond covalently, DNA strands can be easily melted apart in a
zipper-like fashion simply by increasing the temperature. The melt temperature for these
interactions can be tuned by adjusting the guanine-cytosine content, the oligonucleotide length,
or by introducing mismatches into the recognition sequence. Thus, oligonucleotides possess
thermally responsive properties, tunability, and the ability to multiplex a large number of specific
interactions within the same system.
Despite these opportunities to engineer oligonucleotide nanostructures, their full power to
assemble diverse structures has only recently exploded as a source of tunable and biodegradable
nanostructures. In part, this has been facilitated by the recent advancement of DNA synthesis
technology, which now allows for the production of kilobase strands of nucleotides
19
. When the
design of these base pairings is aided by a computer, recent studies reveal a vast array of
nanostructures, including cubes, spheres, and even a map of the western hemisphere
20
. Typically,
these structures are formed using simple ssDNA to ssDNA oligonucleotide bonding schemes
21
,
folding long ssDNA scaffolds using shorter oligonucleotide staples
20
, and also through the
multimerization of branched DNA tiles
22-24
.
Nucleic acid base pairing and any resulting structural assembly is a thermodynamic
process, which makes them amenable to thermal stimulation. Each annealed oligonucleotide
sequence has a specific melting temperature, which can easily be monitored using intercalating
fluorescent probes
23
. The oligonucleotide melting temperature occurs where half of all the
7
nucleotide strands are paired with their complementary strand, which can adopt a double helical
that is relatively rigid in comparison with ssDNA. Sacca et al. characterized the first and second
temperature transitions of DNA strands that self-assembled into DNA tiles
23
. For example,
oligonucleotides mixed at equimolar concentrations are rapidly heated to 90
o
C. While the
samples are slowly cooled, the first transition occurs around 60
o
C. At this point, single stranded
nucleic acid sequences hybridize and form double helices. After a controlled cooling step, these
DNA double helices self organize further based on their tile structure into three-dimensional
structures determined by their sequence. Both annealing processes are fully reversible.
Large scale DNA origami structures have recently been described that are comprised of
megadalton (MD) sized DNA-origami tiles
24
. Previous work describes the formation of
polyhedra using a three arm “tripod” tile
22
. The edges of the tile are comprised of 3 double
helical structures, which are connected by single stranded hinges. These tiles form shapes such as
nanoprisms and buckeyballs. However, scaling up this scheme to megadalton sized tiles initially
failed to produce well-formed shapes. Iinuma et al
24
circumvented this size limitation by creating
tripod tiles out of 3 distinct DNA strands. DNA strands are mixed with p8064, a single stranded
circular DNA scaffold, and quickly heated to 80
o
C. The mixture anneals when the system is
rapidly cooled from 80 to 65
o
C over the course of 1 hour, followed by a slow annealing step,
where the sample is cooled from 62
o
C to 24
o
C over 42 hours. The shape the DNA origami
“tripod” tiles formed is determined by the angle between the tripod legs. For example,
tetrahedron formation requires monomeric tripod tiles with 60
o
-60
o
-60
o
angles between the
tripod legs while cubes require 90
o
-90
o
-90
o
angles between tripod legs. This scheme produces
20 MD tetrahedrons, 40 MD cubes, and 60 MD hexagonal prisms. The edges on these polyhedra
are approximately 100 nm, comparable to intracellular organelles.
8
As mentioned in the introduction, a thermally responsive system can be created by the
additional of a thermally responsive element. For example, functionalizing a molecule or particle
with a thermally responsive DNA strand will impart thermal sensitivity to the parent system. Qi
et al.
25
describe a system where DNA is used to functionalize hydrogel cubes to self assemble
into structures as large as 1 mm in length. The system is created by conjugating a short DNA
primer to a PEG-NHS molecule. This DNA-PEG-NHS solution is mixed with PEGDA and
exposed to UV light. The mixture conforms to the mold it is in while exposed to UV light and
creates hydrogel cubes with short DNA primers extending out. Using a circular DNA as a
template, the rolling circle amplification technique
26
can extend DNA primers mounted in the
hydrogel. This results in a hydrogel cube with sequences of “giant DNA” extending off the faces
of the hydrogel. Mixing giant-DNA functionalized hydrogels with hydrogels functionalized
using complementary DNA further yields even larger nanostructures. The shape of these
nanostructures is controlled by limiting the face of the hydrogel that is functionalized with giant-
DNA as well as varying complementary DNA pairs. This method has generated hydrogel
cuboids that self-assemble into 1 mm long chains as well as 2 x 2 hydrogel squares. As with all
DNA-mediated assembled structures, as sharp increase in heat is expected to disassemble the
structure; however, it remains to be seen if the assembly temperature for these nanostructures can
be tuned across a range of relevant temperature.
Much of the literature on DNA nanostructure has described complex 2-D and 3-D
designs, however all of these are rigid, fixed structures. Zhou et al. introduces a method for
creating DNA nanostructures with tunable mechanical properties
27
. Using the same annealing
process describe above, they designed a structure with ends made up of two 18 helical bundles
comprising 3 layers of 6 helices flanking an inner structure that is half double stranded DNA
9
bundles, and half single stranded DNA oligonucleotides. This inner structure, specifically the
single stranded DNA portion, allows for the facile formulation of a flexible structure. A loop of
ssDNA is connected to the ssDNA located at the center of the structure. By keeping the number
of single stranded nucleotides constant and varying the size of the loop, Zhou et al. is able to
change the angle adopted by the structure. A large ssDNA loop pulls on the system and causes
the distance between the outside 18 helical bundles to shrink due to the tension from the ssDNA
and results in a decreased angle. A smaller ssDNA extending from the center of the structure
relaxes the tension put on the system and results in a wider, more linear angle.
1.1.2 Polysaccharides
Polysaccharides are polymeric carbohydrates obtained or inspired from a broad array of
sources including animals, plants, and microorganisms
28
. The majority of polysaccharides are
anionic with the exception of cationic chitosan
29
. While their chemical structure is not directly
determined by the genetic code, they are biologically synthesized via well-coordinated
biochemical pathways resulting from conserved polysaccharide synthases. Thermal
responsiveness is mostly conferred to polysaccharides using synthetic polymers such as
PNIPAM; however, gellan and xanthan gums, produced by microorganisms, are intrinsically
thermally responsive
29
.
Chitosan, created by the partial deacetylation of chitin, is the second most abundant
natural polysaccharide after cellulose
30
. It is found in bacterial and fungal cell walls as well as in
the exoskeletons of crustaceans and insects. While chitosan does not undergo thermally inducible
conformational changes, temperature sensitivity has been conferred to it using thermally
responsive molecules. For example, complexes of chitosan with ovalbumin create stable, self-
assembled nanogels
31
. To form these gels, a mixture of chitosan and ovalbumin is allowed to
10
complex at pH 5.4, and the mixture is heated to 70
o
C, past the denaturation temperature of
ovalbumin. Once the solution reached 70
o
C, ovalbumin undergoes a conformational change
from an α-helix to a mixed β-sheet and coil structure. Additionally, it is believed that
temperatures above 70
o
C induce both intermolecular hydrophobic association and the formation
of disulfide bonds, which stabilize the chitosan-ovalbumin association. When optimized, this
strategy results in monodisperse chitosan-ovalbumin nanogels with a 50 nm radius, as confirmed
using TEM imaging. Alternatively, thermal sensitivity has been conferred to chitosan using
PNIPAM and Pluronic
32-33
.
Thermo-reversible assembly is commonly explored in hydrogel systems; furthermore,
many of these are mediated by synthetic polymers such as PNIPAM, which confer lower critical
solution temperature properties to a protein or peptide
34
. Extensively used in the food industry as
a thickening and gelating agent, κ-carrageenan has recently gained prominence as a
thermosensitive material for nanogel formulations
35
. Extracted from red seaweed, κ-carrageenan
is a high molecular weight (200 to 800 kDa) polysaccharide made up of repeating galactose and
3,6 anhydrogalactose units. Above the gelation temperature of carrageenan, the carrageenan
adopts a random coil conformation. Cooling below the gelation point causes the polysaccharide
to undergo a conformational change into a double helix, which ultimately self assembles. This
gelation has been utilized to form 100 nm nanoparticles that can be induced to release drug
above their gel to solution transition temperature
36
. κ-carrageenan has also been mixed with
methylcellulose to create a three-phase system. While κ-carrageenan solubilizes when heated,
methylcellulose undergoes gelation when heated. By varying the concentrations of κ-carrageenan
and methylcellulose from 1-2 wt%, Tomsic et al
37
formulated a system that undergoes a gel-sol-
gel transition with a solution window in the physiologic range, from 30-60
o
C.
11
In addition to carrageenan, polysaccharide-based hydrogels have recently been explored
in the tissue engineering community using gellan gum. Gellan gum is derived from the
fermentation of bacterium Sphingomonas elodea
38
. It is comprised of tetrasaccharide repeats of
glucose, D-glucuronic acid, glucose, and rhamnose. At high temperatures, gellan gum
polysaccharide forms random coils and is highly viscous in solution. Decreasing the temperature
allows the gellan to cross link through double helices, which are stabilized by the carboxylic
acids on the glucuronic acid moieties. The carboxylic acid participates in internal hydrogen
bonds that stabilize antiparallel double helices. When in an antiparallel double helical
conformation, gellan gum’s viscosity decreases dramatically, forming a gel. The mechanical
properties of the gel can be tuned by controlling the degree of acetylation. Highly acetylated
gellan gum forms soft, elastic, pliable hydrogels. However, gellan gum that is non-acetylated
forms more brittle hydrogels. Pereira et al
38
explores mixtures of gellan gum with two degrees of
acetylation, which together can form microparticles. Mixing highly acetylated gellan gum with
non-acetylated gellan gum at a ratio of 1:1 resulted in microparticles 741 um in size, larger than
when either acetylated or non-acetylated gellan gum are mixed in a 3:1 ratio. Oliveira et al
39

utilized gellan gum’s thermal responsiveness to form hydrogels in a broad range of geometrical
forms. The gels were created by heating to 90
o
C to disperse the polysaccharide. CaCl
2
was
added before slowly cooling the solution to 50
o
C, at which point the solution was cast into
cylindrical molds and allowed to cool to room temperature to form a solid gel. This hydrogel
formation is thermally reversible upon heating. Depending on the mold used, the gel formed can
maintain a variety of geometries ranging from 10 mm discs, membranes, and porous scaffolds.
Another example of a thermally responsive polysaccharide hydrogel comes from Xanthan
gum. Xanthan gum is a branched, high molecular weight exopolysaccaride produced by
12
Xanthomonas campestris
40
. It is composed of D-glycosyl, D-mannosyl, and D-glucuronyl acid in
2:2:1 molar ratio along with variable amounts of O-acetyl and pyruvyl residues
41
. A thermal
stimulus causes xanthan gum to undergo a conformational change in aqueous solution from an
ordered helix, stabilized by non-covalent bonds, to disordered coil. This order-disorder transition
is irreversible but can be modulated to occur at a range of temperatures by altering the salt
concentration and side chain substitution. Sereno et al.
42
used xantham gum to create disperse
particles through extrusion. Xanthan particles can be extruded below their order-disorder
transition temperature. Microcalorimetry indicates these extruded particles lose their helical
structure during the extrusion process and adopt more amorphous non-helical regions. This
results in intermolecular cross-linking, which creates large interconnected networks. The
extrusion process also aligns xanthan gum during particle formation. Similar to other
polysaccharides, this cross-linking property of xanthan can be reversed by reheating the system
past its order-disorder transition. Dropping the temperature back down, xanthan re-adopts its
original helical conformation and stabilizing non-covalent bonds.
1.1.3 Protein Polymers
Like polysaccharides and oligonucleotides, proteins display a range of thermally-
responsive properties that are often strongly associated with their assembly of specific secondary
structures. Most notably, the protein making machinery can be used to generate high molecular
weight (8 to 80 kDa) repetitive amino acid sequences called protein polymers. Protein polymers
can be genetically engineered into a multitude of compositions by varying the DNA sequence
that encodes for them. By selection of the repetitive motif, the protein polymer can be designed
to adopt specific secondary structures, which can be used to mediate assembly. Multiple classes
of thermally responsive protein polymers are currently utilized for material sciences applications,
13
which include leucine zippers, collagen motifs, elastin-like polypeptides, resilin biomimetics,
and protein polymers rationally designed de novo. While elastin-like polypeptides stabilize into
ordered structures with the application of heat, leucine zippers and collagen motifs destabilize
into smaller particles under increased temperature conditions
11
. Resilin mimetic polymers exhibit
both of these properties: they assemble at low and high temperatures with a window from 6 -70
o
C where they are soluble
43-44
. Another physical property of protein polymers to note is the
reversibility of this physical change. Elastin-like polypeptides, leucine-zippers, and resilin
polymers under this conformational change reversibly, while collagen motifs and undergo
conformational changes irreversibly
11,44
.
Leucine zippers are structural motifs used endogenously by cells as transcription factor
binding domains
45
. The name leucine zipper is derived from the leucine residue region of the
zipper. Leucine is found every seven residues on the polymer sequence, which allows for the
formation of an amphipathic alpha helix with the leucine residue forming a hydrophobic region
on one side
45
. This hydrophobic leucine region drives dimer formation between the two strands,
creating a coiled coil of parallel alpha helices
46
. Leucine zippers reversibly unfold at elevated
temperatures. Dimers dissociate when the polymer secondary structure changes from an alpha
helix to a random coil. One example of a leucine-zipper derived polymer motif used in the
formation of structures is a parallel coiled coil derived from the yeast transcription factor
GCN4
47
. This peptide takes two GCN4 sequences and separates them with 2 alanine inserts. The
insertion produces a phase shift in the GCN4 repeats that creates two hydrophobic ridges 200
o

apart. The addition of a polarizing solution such as salt, or an increase in temperature causes the
peptides to disassociate from large nanofibrils into smaller, more discreet nanoropes. At 4
o
C, the
molecular weight of the polymer is 1,800 kDa, but a shift in temperature up to 25
o
C causes a
14
decrease in polymer weight to 420 kDa. Atomic force microscopy imaging illustrates the
formation of nanoropes from these peptides.
One of the most abundant proteins in the body, collagen has a unique tertiary structure: a
right-handed triple helix made up of three helical peptide strands. Collagen is comprised of the
amino acid sequence Xaa-Yaa-Gly with proline being the most abundant Xaa and L-
dydroxyproline being the most abundant Yaa
48
. Multiple collagen-mimetic peptides have been
described, however one of the best characterizations of a self-assembling collagen mimetic is
done on (Pro-Lys-Gly)
4
(Pro-Hyp-Gly)
4
(Asp-Hyp-Gly)
4
, a collagen-mimetic polypeptide that
replaces arginine and glutamate residues with lysine and aspartate
49
. This peptide adopts a triple
helix secondary structure, which mediates assembly of nanofibers and a hydrogel. The assembly
of this gel is temperature sensitive; furthermore, above 40-41
o
C the collagen triple helix unfolds
into a random coil and the hydrogel disassembles
50
.
Most of the collagen mimetic peptides are able to crosslink into larger hydrogels;
however, a recent advance using collagen-mimetic peptides to assemble two-dimensional
nanoscale assemblies referred to as nanosheets
51
. Using 3 blocks of 4 amino acid triads with
differing electrostatic properties allowed the Conticello group to uniaxially orient collagen-
mimetic fibrils. By replacing the original positively charged arginine residue with the non-
cannonical amino acid aminoproline, the resulting polypeptides form a collagen triple helical
conformation with thermal stability up to 32
o
C
51
. Altering the original collagen-mimetic
formulation further, the central block of triads is lengthened from 4 repeats of (Pro-Hyp-Gly) to
7, which greatly increases the thermal stability of the nanosheets up to 60
o
C. The resulting
collagen-mimetic assembles into multi-layered nanosheets with well-defined morphology. This
15
is the first collagen formulation to form two-dimensional structures without the need for a non-
native structural interaction such as metal promoted crosslinking or β-helical peptides.
Elastin-like polypeptides, derived from the human gene for tropoelastin, are used in a
variety of applications ranging from protein purification, drug delivery, to hydrogel formation,
due in part to their temperature responsive properties
52-54
. ELP physical properties of protein
polymers made up of solely ELPs as well as previous and potential future applications are
described in Section 3.3.2. However, ELPs have been incorporated into hybrid systems along
with silk-like polypeptide (SLP) domains to confer thermal responsiveness and decrease
crystallinity of SLP domains
62
. Like ELPs, SLPs are composed of repetitive sequences of amino
acids, namely glycine and alanine. The most commonly used SLP motif is Gly-Ala-Gly-Ala-Gly-
Ser, derived from the silkworm Bombix Mori
63
. Alone, GAGAGS domains assemble into
insoluble, β-sheets, making them difficult to use in aqueous environments. Incorporating blocks
of ELP allows for increased solubility of the silk-elastin-like polypeptide (SELP).
While SELPs have been extensively used for their solution to gel transition in direct
tumor injections
64
, the Kaplan group has recently engineered SELP micelles for systemic
administration
65
. The SELP nanoparticles described by Xia et al.
66
, undergo a two step thermal
transition. Above 20
o
C, but below 40
o
C, SELP polymers with a 1:8 silk to elastin ratio and 55.7
kDa spontaneously form spherical structures with a 60 nm radius. Soluble ELP domains
expected to localize at the corona of the micelle with silk domains stabilizing the core with
intermolecular hydrogen bonding. The second transition occurs above 60
o
C when interactions
between SELP particles result in larger coacervate formations with a 241 nm radius. While this
assembly is reversible, it is interesting to note that upon cooling down to 20
o
C, SELP
morphology will vary based on silk to elastin ratio. For example, after cooling, a 1:8 ratio silk to
16
elastin polymer will return to solution as a monomer. However, a 1:4 silk to elastin ratio, upon
cooling, adopts a worm-like nanostructure composed of small spherical particles.
While elastin-like polypeptides are derived from the repetitive region of the mammalian
gene tropoelastin, the resilin-mimetic polymer rec1-resilin is derived from the repeat sequence
found in the Drosophila melanogaster CG15920 gene
44
. Resilin is highly elastic. For example,
Drosophila melanogaster only expresses resilin during its pupa stage, yet it remains functional
throughout the insect’s lifetime
44
. Rec1-resilin is composed of 18 copies of the 15-residue repeat
sequence Gly-Gly-Arg-Pro-Ser-Asp-Ser-Tyr-Gly-Ala-Pro-Gly-Gly-Gly-Asn
67
. Like an ELP, this
sequence is high in Gly and Pro residue; however, in contrast to an ELP it lacks aliphatic
residues with bulky side chains. As a stimulus responsive biopolymer, rec1-resilin is of interest
because it possesses not only lower critical solution temperature (LCST) behavior observed for
ELPs, but also an upper critical solution temperature (UCST). Below its UCST at 6
o
C , recl-
resilin is turbid in solution. Cryo-TEM images show rec1-resilin assembles a high-density
network of spherical particles approximately 5.4 nm. As the temperature is increased above the
UCST, the rec1-resilin solution becomes transparent. Cryo-TEM images show discreet
nanoparticles 9 nm in radius, and DLS confirms this by characterizing rec1-resilin as having a D
h

of 11 nm with low polydispersity in the 15 to 70
o
C temperature span. Above res1-resilin’s 70
o
C
LCST, rec1-resilin form larger discreet spherical nanoparticles between 100 and 130 nm in size.
In addition to naturally occurring or biomimetic polymers such as leucine zipper
domains, a number of stimulus responsive polypeptides have been rationally designed de novo.
One such polypeptide, 17-H-6, is an alanine-rich helical polypeptide. Amino acids like alanine,
glutamine, and glutamic acid are commonly used in de novo polypeptide designs because of their
high helical propensity
68
. The 17-H-6 alanine-rich polypeptide is comprised of (Ala-Ala-Ala-
17
Gln-Glu-Ala-Ala-Ala-Ala-Gln-Ala-Ala-Ala-Gln-Ala-Glu-Ala-Ala-Gln-Ala-Ala-Gln). At acidic
pH and low temperature, 17-H-6 takes on an alpha-helical, folded conformation, which was
confirmed using circular dichroism. This results in a globular nanostructure between 10 and 20
nm in diameter. The increase in pH to 7.4 dissociates the polypeptide. However, while
maintaining an acidic pH yet increasing the temperature to 80
o
C for 18 hr, the polypeptide
dissociated and unfolds before undergoing a conformational change to a β-sheet 2 stabilized
fibrils 5-10 nm in diameter
69
. While the conformational change induced by a low temperature
and increase in pH is reversible, the temperature mediated conformation change into nanofibrils
is not.
Another example of thermally responsive fibrillar structures is the 687 amino acid
polypeptide pioneered by the Welch group, termed YEHK. The YEHK polypeptide contains the
repeating units (Gly-Ala)
3
Gly-Tyr(Gly-Ala)
3
Gly-Glu(Gly-Ala)
3
Gly-His(Gly-Ala)
3
Gly-Lys. The
GA residues drive the antiparallel sheet formation with YEHK residues designed to induce
turns. At room temperature, the polypeptide forms face to face antiparallel β-sheets, stabilized by
internal hydrogen bonds, that result in a fibrillar structure 15 nm in width and 10 to 1000 nm in
length. At 90
o
C, the polypeptide reversibly denatures and adopts a random coil conformation
70-
71
. YEHK’s ability to reversibly unfold and refold due to temperature stimuli makes it an
excellent model to study protein folding mechanisms of globular β-sheet proteins.

1.2 Orthogonal Protein Control
There is a sustained need to target specific cellular pathways in a timely and efficient
manner, for which new tools must be developed. The ability to selectively control cellular
function is advantageous, be it for modeling a disease state or for evaluating pathway-specific
18
therapeutics. To achieve this, various cellular switches have been developed that turn isolated
molecular pathways “on” or “off” depending on the selected signal (Fig 1.1). In our view, an
ideal cell switch is: i) reversible, possessing the ability to return to its original state; ii) accurate,
targeting only the action of interest; iii) rapid, works quickly enough to minimize the
confounding actions of compensatory pathways; and iv) adaptable, the general technology
should be robust enough to address multiple cellular targets.

Cellular switches are widely used to study cell and molecular biology; however, different
approaches meet the above criteria with various strengths and deficiencies. From the perspective
of basic science, modulation of an isolated protein can help to determine its role in a chain of
events in which it participates. From a translational perspective, cellular switches can be used to
mimic pathological behavior, such as those involved in cancer, or to screen therapeutics for
participation in a targeted pathway. For example, pathways associated with aggressive cancers
may be switched from the “off” position to a pathologic state in the “on” position. These
switches enable control over in vitro and in vivo models of cancer and aid in the identification of
agents that interact with specific molecular pathways. While multiple switching approaches are
used in research, an ideal switch remains a goal on the horizon.
19

Figure 1.1 Cellular switches act at different points on protein expression. The central dogma of molecular biology is
evident: DNA is transcribed into mRNA, and mRNA is translated into protein. These three categories of
biopolymers interact with each other to do work within the cell. Representative cellular switches are indicated that
act on these processes.

1.2.1 Transcriptional Control
Cells naturally respond to their environment by altering gene transcription levels making
transcription an excellent place to design a cellular switch. Transcriptional control signals
increase or decrease the production of mRNA, which is related to the level of protein expression.
Subsequently, this transcriptional response can be magnified many-fold by downstream
effectors. Unfortunately, response to transcriptional regulation cannot be described as quick. It
can take upwards of 24 hours from transcriptional initiation for a mammalian gene to be fully
functional
72
. In contrast, when deactivating a target gene product, transcriptional approaches are
unable to influence the degradation rate of functional gene products. Despite the temporal delays
inherent in transcriptional switches, they can be robust, reversible, and accurate.

20
Many cell-switch approaches that target transcription rely on the interaction of a ligand
and a receptor, commonly resulting in dimerization that induces nuclear localization and binding
to a DNA-response element upstream of the gene of interest. The TetR systems are advantageous
over nuclear receptors in that they require the expression of only one control protein, TetR, to
modulate transcription. In contrast, nuclear receptors, as targeted by ecdysteroid, require the
constitutive expression of two proteins, VP16/EcR and RXR. Another advantage, TetR
approaches can either up-regulate or down-regulate in the presence of drug, while nuclear
receptors are currently limited to up regulation.

However, TetR has limitations when used in animal models. TetR has been found to
induce inefficient expression in certain tissues, and the drugs themselves can induce toxicity
73
.
The use of nuclear receptors, ecdysteroids in particular, offer advantages over other transcription
induction systems for in vivo studies. Steroids penetrate tissue and cellular barriers; therefore,
controlling cellular expression with a steroid ligand avoids some cellular penetration problems
observed with alternative approaches. Also, mouse studies have shown that ecdysteroid
pharmacokinetics are favorable over TetR since they have faster distribution and clearance
74
.
Lastly, ecdysteroid-controlled genes have low basal expression and high inducibility. While the
EcR switch has yet to be thoroughly developed for animal models, preliminary studies suggest
this approach may be superior for in vivo cellular switching
73
.

1.2.2 Translational Control
Protein translation is another node along the central dogma of modern biology that can be
targeted for cellular switching. Two common approaches include riboswitches and RNA
21
interference. Riboswitches are enzymatically active mRNA that regulate their own genetic code.
Currently, the most ubiquitous cellular switches are based on RNA interference, which use small
RNA molecules to target and destroy mRNA, reducing protein translation. Switches installed at
the level of protein translation have temporal advantages over transcriptional switching since
they act further down the gene expression pathway. Translational switches have the potential to
relay on and off signals more quickly because they directly target mRNA and avoid the cellular
transcriptional machinery. In addition, the translational machinery can increase or decrease
protein expression, even under constant transcription; therefore, direct targeting of translation
has broad potential for controlling the cellular levels of active proteins.

Using translation to control cellular function has advantages as well as pitfalls.
Riboswitches are unique because they do not require the constitutive expression of foreign
proteins to control expression. When using proteins to control genetic expression, such as tTA in
the Tet system, subtle increases or decreases in concentration may amplify changes in expression
of the target protein. Since riboswitches do not utilize cell proteins, they offer good control over
translation. Despite this, a major limitation of riboswitches is that they must be delivered by an
expression platform base. Additionally, only 6 aptamers have been identified that can be
engineered to function as riboswitches and only 2 of them, theophylline and Tet, have been used
as binding domains in creating more complex riboswitches
75
.
RNA interference has the advantage over other cellular switches in its ability to target
genes of known sequence. Setting up a functional RNAi system requires little more than
generating the desired oligonucleotide and administering it to cells. This circumvents the need to
do large-scale selection screens such as those required to generate riboswitches. The speed as
22
well as dose-dependent control of RNAi activity may be improved using the previously
described Tet responsive approaches
76
. Despite this, off-target effects remain problematic with
RNAi technology, which must be addressed when implementing and analyzing data from this
approach.

1.2.3 Post-translational control
Cellular switching at the post-translational level can be achieved by manipulating pre-
proteins in order to control their activity. One advantage of such a switch over a transcriptional
or translational modulator is its ability to operate on the scale of minutes as opposed to hours.
This speed reduces the time for the cell to compensate for the loss or addition of function with
confounding pathways. Also, it can avoid the potential toxicity that occurs due to prolonged
absence of essential functions, such as RISC. And furthermore, cellular switch at the protein
level may be more advantageous for study of cell cycle processes as they are temporally
contingent.

The most pronounced advantage of post-translational control for a cell switch system is
its ability to be temporally controlled. While transcriptional and translational control of protein
production can take upwards to 24 hours, protein dimerization and splicing can be successfully
accomplished within minutes
77
. Speedy switching is essential, as it enables the modulation of
essential genes without permitting excessive time for the accumulation of secondary and tertiary
cellular responses that may easily confound the primary effects of the target or even lead to cell
death. The rapamycin-FKBP strategy is rapid and controlled by a reversible interaction with a
small molecule; however, this approach still requires genetic manipulation of the target protein.
23
Similarly, the intein approach requires genetic engineering and can be drug mediated; however, a
major drawback of the intein strategy is that it is not reversible.

1.3 Environmentally Responsive Peptides
Environmentally responsive peptides (ERPs) are a group of biopolymers that undergo a
characteristic phase transition. These ERPs are based on 4 naturally occurring classes of proteins:
leucine-zippers, human collagen, human elastin, and silkworm silk
2
. When the fundamental
motif of these classes is repeated, biopolymers of higher ordered structures are generated. The
environmental cues that these ERPs respond to include temperature, pressure, pH, concentration,
and ionic strength. The addition of heat alone is sufficient to induce an ERP to phase transition.
Leucine zippers and collagen motifs disassociate with increased temperature, while elastin and
silk motifs associate under the same conditions. The reversibility of this phase transition also
varies amongst the four classes. Leucine zippers and elastin motifs will phase transition
reversibly, while collagen and silk motifs transition irreversibly. The class of ERP that
aggregates reversibly in response to heat is the elastin-like motif
11
.

1.3.1 Elastin-like Polypeptides
Elastin-like polypeptides (ELPs) are derived from the human extracellular matrix protein
tropoelastin, a precursor to elastin. ELPs are repetitive motifs of the hydrophobic domain of
tropoelastin. Due to their composition, ELPs are biodegradable
9
, biocompatible, and non-
immunogenic
8
. ELPs are comprised of the motif (Val-Pro-Gly-Xaa-Gly)
y
, with Xaa being
virtually any guest residue, and y denoting chain length. It is this pentapeptide motif that confers
the distinct reversible phase transition in ELPs. At a critical temperature, termed an inverse
24
transition temperature (T
t
), ELPs undergo a conformational change from single chained β-sheets
and random coils to insoluble Type II β-turn spirals. Below their T
t,
ELPs are soluble in solution
(Fig 1.2b). However, above this T
t
, ELPs become insoluble coacervates and form aggregates.
The temperature at which ELPs transition can be precisely controlled by varying the amino acid
guest residue as well as the chain length. Hydrophobic guest residues, such as leucine and
isoleucine, drive the ELP’s T
t
down, while hydrophilic guest residues, such as serine, increase
the ELP’s T
t
. Additionally, longer chain lengths result in lower T
t
s and shorter chain length
result in higher T
t
s. The production of ELPs with high molecular weights can be achieved by the
straightforward method of recursive directional ligation
78
(Fig 2.1a).
Elastin-like polypeptides are used in a variety of applications ranging from protein
purification, drug delivery, to hydrogel formation, all due to their temperature responsive
properties
52-54
. Genetic engineering allows for a facile method of ELP block copolymer
construction. Ligating in tandem two ELPs of varying hydrophobicities results in the formation
of stable protein nanoparticles
59
. The ELP with the more hydrophobic amino acids undergoes a
conformational change at a temperature below that of the second ELP block. This transitioned
ELP forms the core of the ELP based micelle, with the still soluble hydrophilic ELP block
forming the corona. This scheme has been used to load ELP micelles with therapeutics for drug
delivery applications
60-61
.

25

Figure 1.2 ELP cloning and characterization. (a) ELPs are encoded on a circular bacterial plasmid,
making cloning of desired lengths straightforward using recursive directional ligation. The serine library
is digested with 2 enzymes that directly flank the ELP coding region: XbaI and BamHI. Digests are run
on a Bio-Rad Experion Automated Electrophoresis System. (b) ELPs phase transition with the application
of heat. Soluble polymers become insoluble in solution and exhibit turbidity seen with the naked eye. (c)
ELPs with guest residue serine are run on a Beckman spectrophotometer over a range of temperatures and
monitored for change in absorbance. The abrupt change in turbidity signals an ELP transition.

The ELP’s ability to acutely change solubility in response to temperature makes it a good
candidate for an intracellular switch. Under physiological conditions, the only method of
modulating an ELP’s T
t
is with its length and guest residue (since variations in pH, pressure, and
salt concentrations would be toxic to the cell). An ELP library with a T
t
that is acceptable under
physiological conditions can be rationally designed. Urry et al.
79
successfully modeled a
predictor of ELP T
t
s based on their physical characteristics. For example, an ELP comprised of
all serine guest residues will have an inverse phase transition at 50
o
C. Alternatively, an ELP with
26
solely isoleucine residues will have a T
t
at 10
o
C. Using the rational behind these predictions, an
ELP library of mixed residues can be constructed that transitions within a 10 degree window of
37
o
C, the temperature at which cell cultures are most viable.

1.4 Clathrin-mediated Endocytosis
The transduction of particles across the cell membrane can be accomplished in one of two
ways: pinocytosis, the uptake of small molecules, and phagocytosis, the uptake of large particles.
The best characterized pinocytotic mechanism is clathrin-mediated endocytosis (CME). CME
transfers specific molecules used by the cell from the extracellular side to the intracellular
cytosolic side. It is utilized for the transport of iron via the transferrin receptor as well as the
epidermal growth factor (EGF) for hormone signaling via the EGF receptor (EGFR). CME can
best characterized as a receptor mediated process. When a ligand binds a cell surface receptor on
the extracellular side of the plasma membrane, a chain of events is triggered that utilizes CME
80
.
After ligand binding to the receptor, adaptor proteins found in the cytosol as well as clathrin bind
to the cytosolic side of the receptor. These proteins induce the inward budding of the plasma
membrane; this invagination is termed a clathrin-coated pit due to the clathrin that surrounds it.
The clathrin-coated pit then detaches from the membrane and enters the cytosol as a clathrin
coated vesicle. After budding from the membrane, clathrin and its adaptor proteins detach and
fuse with early endosomes which are either cycled back to the membrane, trafficked to
lysosomes, or taken to other cellular compartments.
A number of methods to halt CME are available. These include chemical inhibition
through wortmannin
81
, an inhibitor of PI3 kinase, as well a dynasore
82
, an inhibitor of dynamin, a
clathrin adaptor protein. Other methods include siRNA knockdown of clathrin
83
as well as the
27
overexpression of the dominant negative dynamin
84
. Moskowitz et al.
77
selectively inhibits CME
using a fusion construct. A 40 kD protein comprised of GFP and FKBP is attached to the N
terminus of the clathrin light chain (CLC) yielding GFP-FKBP-CLC. Despite being a fusion
protein, fusion protein expressing cells were still able to form clathrin lattices and perform CME.
Furthermore, there was no apparent dominant negative down regulation of CME. The addition of
FK1012, an analogue of rapamycin, caused the FKBP containing fusion protein to crosslink with
itself, effectively halting CME due to the sequestering of CLC. It was shown that this
crosslinking inhibited the internalization of transferrin as well as LDL, two known markers of
CME.

28
Chapter 2
A Tunable and Reversible Platform for the Intracellular Formation of
Genetically Engineered Protein Microdomains

2.1 Abstract
From mitochondria to the nuclear envelope, the controlled assembly of micro- and
nanostructures is essential for life; however, the level at which we can deliberately engineer the
assembly of microstructures within intracellular environments remains primitive. To overcome
this obstacle, we present a platform to reversibly assemble genetically engineered protein
microdomains (GEPMs) on the time scale of minutes within living cells. Biologically inspired
from the human protein tropoelastin, these protein polymers form a secondary aqueous phase
above a tunable transition temperature. This assembly process is easily manipulated to occur at
or near physiological temperature by adjusting molecular weight and hydrophobicity. We fused
protein polymers to green fluorescent protein (GFP) to visualize their behavior within the
cytoplasm. While soluble, these polymers have a similar intracellular diffusion constant as
cytosolic proteins at 7.4 μm
2
/s; however, above their phase transition temperature, the proteins
form distinct microdomains (0.1 −2 μm) with a reduced diffusion coefficient of 1.1 μm
2
/s.
Microdomain assembly and disassembly are both rapid processes with half-lives of 3.8 and 1.0
min, respectively. Via selection of the protein polymer, the assembly temperature is tunable
between 20 and 40 °C. This approach may be useful to control intracellular formation of
genetically engineered proteins and protein complexes into concentrated microdomains.

29
2.2 Introduction
The formation of synthetic subcellular compartments has long been of interest to
bioengineer cellular behavior
85-86
. There are numerous approaches to generate microparticles
outside of cells
87
, which can be loaded with cargo and incorporated into cells where they act as
intracellular microdomains
88-89
. However, these synthetic microparticles require cellular uptake
and disruption of the plasma membrane to reach the cytosol. Herein we characterize a method for
reversible intracellular assembly of genetically-encodable protein microdomains (GEPMs). This
approach would allow virtually any protein to become temperature sensitive and assemble into
structures, circumventing the need to perform large temperature mutant screens or the addition of
chemical modulators
90-91
. This triggered microdomain formation has potential applications
including protein sequestration, scaffold construction, and modulation of intracellular trafficking.
Additionally, the reversibility of this platform affords the ability to study the transitory effects of
such domains in a biological environment.

To direct GEPM assembly, protein polymers have been selected from the family of
elastin-like polypeptide (ELPs)
2,92
. Inspired from human tropoelastin, ELPs are composed from
the motif (Val-Pro-Gly-Xaa-Gly)
n
. As polypeptides, ELPs can be genetically encoded and
expressed inside living cells
2,93
. ELPs undergo temperature-dependent phase separation that is
rapid and reversible. Below a phase transition temperature (T
t
), ELPs are highly soluble. Above
T
t
, ELPs assemble into an aqueous two-phase system. T
t
can be genetically controlled by
modifying the identities of X and n
94
. Hydrophobic guest residues, X, such as valine and
isoleucine, have low T
t
s, while hydrophilic guest residues, such as alanine or serine, have higher
T
t
s. Similarly, ELPs of higher molecular weight, n, result in lower T
t
s. Importantly, these two
30
orthogonal parameters can be used to modify T
t
such that it occurs at physiologically relevant
temperatures and concentrations following expression in the cytoplasm. Previously, recombinant
DNA technology has produced stimuli-responsive polypeptides that form multimer associations,
also referred to as microdomains, that self-assemble extracellularly based on a pH-dependent
trigger
95
. Here, we expand the means with which to form biopolymer microdomain assemblies
by constructing a tunable and reversible self-assembling polypeptide comprised of an ELP that
forms intracellular microdomains (Figure 2.1). The potential of elastin-derived protein
polymers to mediate the reversible assembly of GFP microdomains is characterized and
visualized in real time (Fig 2.1b). Additionally, the adaptability of ELP chain length and guest
residue to be triggered in a variety of temperatures allows a broader temperature range for
GEPM formation (Fig 2.1c). Despite several decades of heterologous expression in E. coli
96
,
ELP expression has not previously been reported within mammalian cells, nor has the ELP
transition within mammalian cells been controlled and characterized. Understanding the
parameters of intracellular ELP assembly will aid in the design of GEPM switches that regulate
intracellular processes such as signaling and trafficking, as well as other cellular prostheses that
sort, sense, and modify their host cell. GEPM is the first platform, to our knowledge, to quickly
assemble and disassemble microdomains within the mammalian cytosol on the time scale of
minutes
97
.
31

Figure 2.1 Tunable assembly of genetically-encoded polymer microdomains (GEPMs). (a)
Schematic representation of triggered microdomain assembly inside living cells. Before heating, the
GFP-ELPs are soluble throughout the cytosol. After heating, the protein polymers assemble
microdomains enriched in the GFP fusion protein. Similar to ELP-mediated phase separation, this
process is reversible. (b) Demonstration of polymer phase separation across a valine library of ELPs
(Xaa=Val) (c) Correlation between transition temperatures for intracellular GFP-ELPs and ELPs in
free PBS based on a valine library (MW 19, 24, 28, and 38 kD). (R
2
=0.78; p=<0.0001; m= 2.72
[95%CI: 2.12 to 3.26]; b= -59.11 °C [95%CI: -77.89 to -40.48]) (d) Size distribution of polymer
organelles after assembly. Frequency based on n=20 cells from valine and V-A-A libraries. Frequency
distribution: 25%: 0.35, 50%: 0.57, 75%: 0.89
32
2.3 Materials and methods

2.3.1 Plasmid constructs
ELPs containing the sequence (Val-Pro-Gly-Xaa-Gly) were synthesized using plasmid
recursive directional ligation, as described previously
98
. Sequences included (Val-Pro-Gly-Val-
Gly) as well as (Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Ala-Gly). Each ELP
gene was restriction digested from its parent plasmid, purified using gel extraction (GE
Healthcare), and inserted into a modified pET-25b(+) expression plasmid (Novagen). The
modified plasmid was constructed by replacing the NdeI-BamHI cloning region of the pET-
25b(+) vector with a synthetic cassette encoding two opposing restricting enzyme sites (AcuI,
BseRI) flanked by an N-terminal methionine and C-terminal tyrosine. The modified vector was
linearized using BseRI and AcuI and then ligated with the ELP gene containing compatible
sticky ends. The product was transformed into chemically competent TOP10 cells (Invitrogen)
and plated on TB agar plates supplemented with 100 mg/L of ampicillin. The sequences were
verified using DNA sequencing (USC Norris Cancer Center). For the mammalian GFP fusions,
NT-GFP-pcDNA3.1 (Invitrogen) was modified to contain a BsgI restriction site downstream of
the GFP gene. The ELP gene segments, produced by digestion with BseRI and HincII, were then
ligated into the BsgI and PmeI site of the modified expression vector, termed MP1.

2.3.2 Cell culture
HEK-293 cells (human embryonic kidney cells) were obtained from American Type
Culture Collection (ATCC) and cultures in DMEM (Dulbecco's modified Eagle's medium)
medium (CellGro) supplemented with 10% (v/v) FBS (fetal bovine serum) and 5% CO
2
at 37
o
C.
33
Cells were seeded at a concentration of 3x10
5
on 35mm glass bottom dishes (MatTek) the day
before transfection. Cells were transfected in plain DMEM using Turbofect reagent (Fermentas).
Cells were incubated in DNA-transfection reagent for 6 h, washed with PBS, and cultured in
fresh complete medium for 48 hours prior to imaging.

2.3.3 Confocal microscopy and thermal control
Live cells were imaged using a Zeiss LSM 510 Meta NLO (Thornwood, NY) confocal
imaging system equipped with Argon, red HeNe, and green HeNe lasers and a Coherent
Chameleon Ti-Sapphire laser mounted on a vibration-free table. All images were acquired using
a Plan-Apochromat 63x oil immersion lens with a working distance of 0.19mm. For heating and
cooling experiments, cells were incubated in on an Instec HCS60 stage (Denver, CO) attached to
a temperature controller.

2.3.4 Fluorescence recovery after photobleaching (FRAP)
Cells were transfected and plated as described above. Transfectants were randomly
chosen and cells expressing high levels of GFP were excluded
99
. Spot photobleaching using
circular regions of interest (ROIs) 0.5-1.5um in diameter were selectively photobleached with
150 iterative rounds of illumination with a 30 mW Argon laser (488 nm) set to 100% power with
100% transmissionon a 63x oil-immersion lens. After bleaching, image acquisition was
performed with 2% transmission and using the same power. Four prebleach frames were
acquired for each experiment followed by continuous image acquisition for the ROI as well as
the surrounding cell for 90s postbleach. Images were taken at 100ms intervals. Due to the short
length of the experiment, problems associated with drifting out of focus were minimized
100
.
34
Fluorescence intensity measurements were exported from Zeiss LSM 510 imaging software.
Images were processed in Photoshop CS4 (Adobe, San Jose, CA) and recovery curves were
generated using Graphpad Prism 5 (La Jolla, CA).
The mobile fraction was calculated as: (F
∞
-F
post
/F
pre
-F
post
)(F
wholepost
/F
whole ∞
) where F
∞
is
the maximum fluorescence recovery after photobleaching of the ROI, F
post
is the fluorescence of
the ROI immediately following bleaching, F
pre
is the fluorescence immediately preceding
bleaching, F
wholepost
is the fluorescence of the whole cell immediately following bleaching and
F
whole ∞
is fluorescence of the whole cell at the end of the experiment
101
.
Diffusion coefficient was calculated as: ( βw
2
/4t
1/2
) where β is a parameter which depends
on percent bleach
101
, w is half the width of the laser beam at its point of focus, and t
1/2
is the time
required to recover half of the fluorescence intensity after the bleach.

35
2.4 Results
2.4.1 Tunable assembly of GEPMs
Plasmids encoding for ELPs of various guest residues and lengths had been previously generated
in house using recursive directional ligation
98
. To visualize the temperature-dependent formation
of GEPM within live cells, the ELP plasmid was engineered to have GFP coded at the N-
terminus of the gene (Fig 2.1a)
102
. At 28 kD, the GFP component comprises between 1/3 and 1/2
of the weight of the fusion protein (Table 2.1). To confirm the identity of the expressed
constructs, the lysates of transfected cells were analyzed by western-blot against GFP (Figure
2.2). Results of the GFP protein are consistent with native GFP. However, when fused to ELP,
there is an expected increase in molecular weight due to the addition of the ELP fusion.

Figure 2.2 GFP-ELP Expression in Hek293 cell culture.
Anti-GFP antibody western blot shows the addition of the ELP
polymer increases the molecular weight of the recombinant
GFP protein. This demonstrates that the appropriate GFP fusion
proteins are expressed in transfected mammalian cells. (left
column: molecular weight markers; right column: expected
molecular weight of GFP fusion proteins)
36

The effect of ELP length on the transition temperature and subsequent GEPM formation was
examined using live cell imaging on a Zeiss LSM 510 confocal microscope equipped with a
heating and cooling stage. The change of polymer T
t
as a function of ELP length, guest residue,
and concentration have previously been modeled on free ELP polymers
94
. Increased polymer
length results in a decreased transition temperature, which is a property of ELPs regardless of
guest residue. While the T
t
of an ELP in aqueous solution can be mathematically predicted, the
addition of a fusion protein as well as the presence of macromolecules in solution can affect the
predicted transition temperature
103
. We examined ELPs with guest residue valine, an ELP library
known to transition at physiologic temperatures, to characterize the effect the GFP fusion and
intracellular environment would have on the polymer’s T
t
(Fig 2.1b). Cells expressing the GFP-
ELP polymer were imaged on a glass-bottom dish while the temperature was increased from 10
o
C to 50
o
C at a rate of 1
o
C/ min, a rate slow enough to ensure an accurate measurement of the
transition. As expected, the T
t
decreased with increased polymer length (Fig 2.1c). However, the
intracellular T
t
was lower than that of the free polymer (Figure 2.3). This decrease in T
t
may be
attributed to the addition of GFP
103
, as well to intracellular polymer concentration. Additionally,
previous studies have indicated that the presence of co-solutes, such as BSA or salts, can lower
T
t
104-106
.
Table 2.1: Microdomain formation temperatures of thermally responsive polypeptides
Polymer
Molecular
Sequence
Molecular
Weight (kD)
*Intracellular
T
t
(
o
C)
GFP-ELP
T
t

**Free
ELP T
t

GFP-V48 GFP- (VPGVG)
48
47.5 45 - 38.9
GFP-V60 GFP- (VPGVG)
60
52.4 40 34.4 36.0
GFP-V72 GFP- (VPGVG)
72
57.2 33 - 33.5
GFP-V96 GFP- (VPGVG)
96
67.1 24 31.5 32.3
[* Transition temperature of GFP-ELP in cytoplasm
** Transition temperature of purified ELP suspended in phosphate buffered saline at 25 M]
37

GFP-ELP fusion proteins assembled GEPMs averaging 0.68 +/- 0.48 µm in diameter
(Fig. 2.1d). ELP aggregates measured less than 1 micron, similar in size to other intracellular
organelles such as endosomes which are approximately 0.5 µm
107
and are more monodisperse
than mitochondria which range from 1-4 µm
108
. There was no significant increase in ELP
aggregate size with increased polymer length. Also, aggregate size does not change with
prolonged incubation time.

2.4.2 Clustering of ELP microdomains is fast and reversible
Having demonstrated the temperature tunability of ELP-mediated intracellular assembly
(Fig 2.1b), we used kinetic studies to characterize the speed and reversibility of GEPM formation
(Figure 2.4). Additional kinetic results using polymers of different length and guest residue can
be found in Figure 2.5 and Figure 2.6. Beginning at 10
o
C, a temperature below the fusion
polymer’s T
t
, the temperature of the media bathing the GFP-ELP polymer was raised rapidly (5
min) to slightly above physiological temperature (40
o
C). Using the fluorescence intensity of the
fusion polymer’s GFP moiety as an indicator, the state of polymer aggregation is monitored with
Figure 2.3 Transition temperature of ELP is
slightly altered by the fusion to GFP. ELPs
expressed in E. coli were purified using inverse
transition cycling, assayed for change in turbidity
in phosphate buffered saline (PBS) across a
temperature ramp (1 °C/min) on a Beckman
Spectrophotometer, and the relevant phase
diagrams both followed a log-linear relationship
with concentration. At 25 uM, the addition of GFP
increases the transition temperature by ~3 degrees;
however, the slope of the concentration dependence
was greater for GFP-V96 (slope = -5.0 ± 1.2 °C
[log
10
(uM)]
-1
; mean ±95% CI) than for V96 alone
(slope = -1.9 ± 0.9 °C [log
10
(uM)]
-1
; mean ±95%
CI).
38
respect to time and temperature (Fig 2.4b). Within the first 5 minutes of the temperature ramp,
the polymeric aggregates begin to form. The surrounding cytosol decreases in fluorescence,
indicating the depletion of the soluble polymer. In 10 minutes GEPM formation is complete.
GEPM formation is defined as the self- assembly of the soluble GFP-ELP polymer into an
insoluble coacervate. Since the GFP-ELP polymer is sequestered in the GEPM, less is present as
diffuse monomers within the cytosol. A microdomain formation half-life of 1.2 min with a 95%
confidence interval of 1.0 to 1.5 min (n=6) was calculated. The half-life of polymer microdomain
formation has been observed to be below 3 minutes across all polymer lengths and guest residues
tested (Fig 2.5 and 2.6).

To demonstrate the polymer’s ability to transiently form GEPMs, immediately after
microdomain assembly, the cells were rapidly cooled down to 10
o
C. The temperature of ELP
disassembly is below than that of formation, 18.3 ± 2.1
o
C. We postulate that the GEPM formed
during the assembly process remains kinetically stable after cooling and requires lower
temperatures to re-solubilize. This effect has been reported in ELPs with the composition
VPAVG
109
. After cooling for 5 minutes, the microdomain had already begun to disassemble and
a corresponding increase in cytosolic fluorescence intensity is observed. Once the cooling phase
was complete, the fluorescence within the cell once again becomes uniform, demonstrating that
the GFP fusion polymer has re-solubilized. The half-life of GFP-V96 dissolution is 0.7 min (Fig
2.4d), making dissolution more rapid than assembly.

39

Figure 2.4 Clustering of ELP microdomains is fast and reversible. (a) ELP polymers of GFP-V96
were ramped from 10 to 40
o
C . Phase separation occurs at 34.6 ± 1.4 °C with a formation half-life of 1.2
min (95% CI 2.7,6.3) (n=6). (b) Fluorescence intensity with respect to time of phase separated GFP-ELPs
(GFP-V96) inside Hek293 cells. The increase in fluorescence intensity was measured at the location of
aggregation within the cytosol. The presence of GEPMs is indicated by the shaded region. (c) Half-life of
formation (d) Half-life of solubilization. (e) To rapidly re-solubilize the polymer organelles, the
temperature of the heating block was decreased to 10 °C. Resolubilization occurs at 18.3 ± 2.1 °C with a
half-life of 0.71 min (95% CI 0.45, 1.3)(n=6).

40

Figure 2.5 GFP-V72 microdomain formation is rapid and reversible during controlled heating and
cooling. (a) Hek293 cells transfected with GFP-V72 are quickly heated from 10
o
C to 45
o
C and then
cooled back down to 10
o
C. (b) The formation of microdomain is quantified using pixel intensity for the
heating portion of the experiment. (c) Half-life of GEPM formation is calculated as 0.75 95%CI [ (d)
Half-life of dissolution is 1.7 95% CI [1.14, 3.34].
41

Figure 2.6 GFP-2VA192 microdomain formation is rapid and reversible during controlled
heating and cooling. (a) Hek293 cells transfected with GFP-2VA192 are quickly heated from 10
o
C to
41
o
C and then cooled back down to 10
o
C. (b) The formation of microdomain is quantified using pixel
intensity for the heating portion of the experiment. (c) Half-life of GEPM formation is calculated as
2.4 min 95% CI [1.63, 4.22]. (d) Half-life of dissolution is ~1.7 min.
42

Figure 2.7.
Microdomain
formation and
temperature
fluctuations are
nontoxic to cells. Cells
transfected with GFP-
ELP and heated to
induce aggregation were
probed with ethidium-
homodimer (EthD-1)
(Invitrogen) as compared
to cells treated with
transfection reagent
alone (- control) and
cells killed with 70%
ethanol (+ control). No
cytotoxic staining is
observed in the GFP-
ELP cells.

43
2.4.3 Mobility of polymers depends on transition state
The rigidity of GEPMs within the cytoplasm was probed using fluorescence recovery
after photobleaching (FRAP). A circular region, ranging from 1-2 μm in diameter, was
irreversibly photo-bleached using a high-powered laser beam on the region of interest and the
fluorescence recovery was monitored until the recovery was complete
110
. A representative plot of
the fluorescence recovery versus time for GFP-V72 is shown for soluble and aggregated states
(Figure 2.8a and b). 3% of the total area of the cell is bleached before transition and 17% of the
GEPM GFP is bleached after transition, allowing for sufficient non-bleached polymer to be
present to allow fluorescence recovery. Prior to T
t
, fluorescence within the bleached area returns
to pre-bleached levels within milliseconds, indicating that the pool of GFP-ELP polymer is freely
diffusible. The mobile fraction of pre-transition cytosolic GFP-ELP fusion protein is 65.0 ± 4.8
% for valine library polymers, indicating the majority of cytoplasmic GFP-ELP is fluid and
diffuses freely
111
. Additionally, the cytoplasmic diffusion coefficient of soluble ELP polymers is
found to be 7.4 ± 1.4 µm
2
/sec, consistent with diffusion coefficients for soluble proteins of
similar molecular weight measured within the cytosol
112
.

Following GEPM formation at 40
o
C, the mobile fraction of the polymer decreases to
38.7 ± 3.9 % and the diffusion coefficient diminishes 7-fold to 1.1 ± 0.2 µm
2
/sec. While
fluorescence minimally recovers after bleaching, a large immobile fraction remains that does not
recover, indicating a loss in diffusibility (Fig 2.8d). The polymer itself may be diffusing within
the aggregate, but it is not readily diffusible with the remaining cytoplasmic polymer pool.
Previous studies have attributed decreased mobile fractions and decreased diffusion coefficients
to the formation of aggregates and complexes, thereby restricting polymer movement
113
.
44

Figure 2.8 Mobility of polymers depends on transition state. Soluble (a) and GEPM (b) GFP-ELPs
(V72, T
t
34
o
C ) are bleached using a circular beam of 1 μm diameter. Images include before
bleaching, directly after bleaching (t=0), and 60 seconds into the recovery period. Representation of
quantification of the fluorescence intensity within the bleached region of interest for soluble (c) and
GEPM (d) polymers. (e) Mobile fraction recovery for individual polymer constructs when soluble and
as GEPM. Bars represent the average value ± SD (n = 10 to 32 per group). Analysis by 2- way
ANOVA indicates that transition state significantly effects polymer mobility (*p = 2.10 x 10-7) while
ELP length does not. f) Diffusion coefficients for same constructs when in soluble and GEPM states.
A 2-way ANOVA analysis did not detect a dependence on ELP length, however the diffusion constant
was highly dependent on the transition state of the polymer (*p = 0.0035).
45
2.5 Conclusions
While the controlled assembly of structures within the cell is essential to life, our ability
to deliberately and genetically program the assembly of small structures inside living cells
remains primitive. Here, we describe for the first time a rational method for the programmable
and switchable assembly of polymeric aggregates in mammalian cells using protein polymers.
This approach utilizes the temperature dependent phase separation displayed by ELPs to allow
the formation of organelle-sized GEPMs inside the cytoplasm. It requires only the transfection of
the cell with a plasmid containing the ELP gene. This approach can be used to assemble one or
more functional proteins into cellular prostheses, which have the potential to mimic or alter
cellular functions. The approach combines the reversibility of the ELP-mediated phase
separation with the tunability of the transition temperature for these protein polymers. Live cell
studies utilizing FRAP demonstrated that fluorescence in these microdomains is less mobile than
for soluble proteins. By selectively activating microdomain formation, this work suggests that
these cellular prostheses have potential applications where assembly of functional proteins into
micro and nanostructures provides an avenue to regulate cellular function.

46
Chapter 3
Flipping the Switch on Clathrin-Mediated Endocytosis using Thermally
Responsive Protein Microdomains

3.1 Abstract
A ubiquitous approach to study protein function is to knock down activity (gene
deletions, siRNA, small molecule inhibitors, etc) and study the cellular effects. Using a new
methodology, this chapter describes how to rapidly and specifically switch off cellular pathways
using thermally responsive protein polymers. A small increase in temperature stimulates
cytosolic elastin-like polypeptides (ELPs) to assemble microdomains. We hypothesize that ELPs
fused to a key effector in a target macromolecular complex will sequester the complex within
these microdomains, which will bring the pathway to a halt. To test this hypothesis, we fused
ELPs to clathrin-light chain (CLC), a protein associated with clathrin-mediated endocytosis.
Prior to thermal stimulation, the ELP fusion is soluble and clathrin-mediated endocytosis
remains ‘on.’ Increasing the temperature induces the assembly of ELP fusion proteins into
organelle-sized microdomains that switches clathrin-mediated endocytosis ‘off.’ These
microdomains can be thermally activated and inactivated within minutes, are reversible, do not
require exogenous chemical stimulation, and are specific for components trafficked within the
clathrin-mediated endocytosis pathway. This temperature-triggered cell switch system represents
a new platform for the temporal manipulation of trafficking mechanisms in normal and disease
cell models and has applications for manipulating other intracellular pathways.

47
3.2 Introduction
The disciplines of synthetic biology
114
, chemical genetics
115
, and gene switches
116
all
rely on one fundamental and unifying concept: that control of protein-protein interactions
elucidates underlying mechanisms fundamental to cell biology. Within the past 50 years, an
array of molecular tools have been developed to deliberately control target protein activities.
117

Temperature sensitive mutants and chemical dimerizers
118
allow scientists to manipulate
endogenous proteins, while tetracycline-inducible promoters
119
and riboregulators
120
allow for
the exogenous control of genetic transcription. These inducible systems have been engineered to
respond to endogenous proteins, exogenous small molecules
121
as well as environmental sensors
such as pH
122
, light
123
, heat
55
, and glucose.
124
Unfortunately, major barriers for the synthetic
control of biological processes persist. The majority of existing methodologies control protein
activity at the genetic level, by placing the desired proteins for study under the control of
inducible promoters. While these systems are robust and afford high levels of induction control,
they lack the temporal sensitivity to rapidly modulate biologic processes.
118
For example,
standard tetracycline-inducible systems as well as RNAi knockdown require 24 to 48 hours
between input and measurable output of the system. This lag time can result in confounded data
due to compensatory mechanisms and off-target effects.
125-128
In addition, other limitations of
current programmable biologic systems include leaky promoters, delayed reversibility,
129
and
labor-intensive mutant screens and system optimization.

Herein, we describe a rapid, reversible post-translational protein switch that seeks to
address these problems. We propose to confer temperature sensitivity to a target process by
fusing one of its key effector proteins to an environmentally responsive protein polymer at the
48
genetic level.
55
This thermally sensitive elastin-like polypeptide (ELP) fusion is soluble at 31
o
C
and below, which allows the target protein complexes to assemble and disassemble as they
would in a normal cell. However, at 37
o
C this ELP phase separates, forming genetically
engineered protein microdomains (GEPMs)
55-56
enriched in the effector protein. The fusion of
the effector protein to the ELP causes other components of the target complex to be sequestered
in assembled microdomains. This inhibits their normal protein interactions, effectively shutting
‘off’ the target pathway. Additionally, the reversible nature of ELP phase separation allows of
the rapid return to the ‘on’ state with a temperature decrease. This strategy is conceivably
adaptable to a variety of target protein complexes.

As a proof-of-concept, we constructed a protein switch to exogenously control the
clathrin-mediated endocytosis pathway, a highly characterized mechanism for cellular
internalization.
130
Upon receptor stimulation by ligand, the clathrin-light chain (CLC) and
clathrin-heavy chain (CHC) are recruited from the cytosol to the plasma membrane to form a
basket-like triskelion around the budding vesicle. This clathrin-coated vesicle selectively sorts
the receptor and cargo to clathrin-coated pits that bud and traffic to early endosomes for further
sorting. We chose the CLC for fusion with our thermally responsive polypeptide for the
following reasons: i) it is essential for triskelion formation
130
; ii) its absence inhibits
internalization of G-protein coupled receptors
131
; and iii) it has been reported in fusion to green
fluorescent protein without loss of activity
77
(Figure 3.1). Triskelion formation requires clathrin
light and heavy chain interactions at the C-terminus, leaving the N-terminus accessible to the
cytosol.
132
Based on this information, we posited that the addition of a 38 kD soluble ELP to the
N-terminus of CLC would not disrupt function (Fig 3.1b).
49
To demonstrate the potential for this platform, this manuscript describes fusions between
ELP and CLC and their ability to inhibit receptor internalization upon formation of
microdomains. When cooled, the ELP microdomains dissolve, CLC fusion proteins return to a
diffuse cytosolic distribution, and receptor internalization returns to its previous level.

Figure 3.1 Triggered microdomain assembly in
the cytosol inhibits clathrin-mediated
endocytosis. a) Genes encoding three thermally
responsive ELPs (V96, A96, and V72) were fused
to the clathrin-light chain (CLC) gene. A myc
epitope was incorporated between the ELP and
CLC genes to enable immunofluorescence
detection of expressed proteins, called V96-CLC,
A96-CLC, and V72-CLC. b) Schematic of clathrin
triskelia composed of clathrin heavy chain (CHC)
and CLC with and without an ELP tag.
50
3.3 Materials and Methods
3.3.1 Plasmid Constructs
The CLC gene was inserted into the IRES-dsRED2 (Clontech, Mountain View, CA)
vector using PCR extension. The following primers were used to amplify CLC: Forward
(TAGCCTGACCTCGAGATGGCTGAGCTGG) and Reverse
(TCATCAGAATTCTCATCACACCAGCGGGGCCTG). The amplified sequence was digested
with NheI/ EcoRI and ligated into IRES-dsRED2 that had been digested with the same enzymes.
ELP genes, carried by a pET25b(+) plasmid (Clontech), were generated using recursive
directional ligation as previously described.
55
DNA sequences encoding ELPs were ligated
upstream of CLC after digesting the ELP plasmid with BseRI/ AcuI and the CLC plasmid with
BcgI. For plasmids containing the myc epitope, myc was inserted between ELP and CLC using
XhoI/ BlpI enzymes. The final open reading frames and encoded proteins are summarized in
Table 3.1 after confirmation using DNA sequencing (Retrogen, San Diego, CA).

51
Table 3.1 Gene and protein sequences of clathrin-light chain fusions with elastin-like polypeptides
V72-CLC
Open Reading
Frame of
Expressed Gene
ATGGGTTTGGGTGTTCCGGGCGTGGGTGTACCAGGTGTCGGTGTACCGGGTGTCGGCGTACCTGGCGTC
GGTGTCCCGGGTGTTGGTGTTCCGGGTGTAGGTGTTCCGGGCGTGGGTGTACCAGGTGTCGGTGTACCG
GGTGTCGGCGTACCTGGCGTCGGTGTCCCGGGTGTTGGTGTTCCGGGTGTAGGTGTTCCGGGCGTGGGT
GTACCAGGTGTCGGTGTACCGGGTGTCGGCGTACCTGGCGTCGGTGTCCCGGGTGTTGGTGTTCCGGGT
GTAGGTGTTCCGGGCGTGGGTGTACCAGGTGTCGGTGTACCGGGTGTCGGCGTACCTGGCGTCGGTGTC
CCGGGTGTTGGTGTTCCGGGTGTAGGTGTTCCGGGCGTGGGTGTACCAGGTGTCGGTGTACCGGGTGTC
GGCGTACCTGGCGTCGGTGTCCCGGGTGTTGGTGTTCCGGGTGTAGGTGTTCCGGGCGTGGGTGTACCA
GGTGTCGGTGTACCGGGTGTCGGCGTACCTGGCGTCGGTGTCCCGGGTGTTGGTGTTCCGGGTGTAGGT
GTTCCGGGCGTGGGTGTACCAGGTGTCGGTGTACCGGGTGTCGGCGTACCTGGCGTCGGTGTCCCGGGT
GTTGGTGTTCCGGGTGTAGGTGTTCCGGGCGTGGGTGTACCAGGTGTCGGTGTACCGGGTGTCGGCGTA
CCTGGCGTCGGTGTCCCGGGTGTTGGTGTTCCGGGTGTAGGTGTTCCGGGCGTGGGTGTACCAGGTGTC
GGTGTACCGGGTGTCGGCGTACCTGGCGTCGGTGTCCCGGGTGTTGGTGTTCCGGGTGTAGGTGTTCCG
GGCGTGGGTGTACCAGGTGTCGGTGTACCGGGTGTCGGCGTACCTGGCGTCGGTGTCCCGGGTGTTGGT
GTTCCGGGTGTAGGTGTTCCGGGCGTGGGTGTACCAGGTGTCGGTGTACCGGGTGTCGGCGTACCTGGC
GTCGGTGTCCCGGGTGTTGGTGTTCCGGGTGTAGGTGTTCCGGGCGTGGGTGTACCAGGTGTCGGTGTA
CCGGGTGTCGGCGTACCTGGCGTCGGTGTCCCGGGTGTTGGTGTTCCGGGTGTAGGTCGTCTCGAGGAG
CAGAAGCTGATCTCCGAGGAGGACCTGCTCGAGATGGCTGAGCTGGATCCGTTCGGCGCCCCTGCCGGC
GCCCCTGGCGGTCCCGCGCTGGGGAACGGAGTGGCCGGCGCCGGCGAAGAAGACCCGGCTGCGGCCTT
CTTGGCGCAGCAAGAGAGCGAGATTGCGGGCATCGAGAACGACGAGGCCTTCGCCATCCTGGACGGCGG
CGCCCCCGGGCCCCAGCCGCACGGCGAGCCGCCGGGGGGTCCGGATGCTGTTGATGGAGTAATGAATGG
TGAATACTACCAGGAAAGTAATGGTCCAACAGACAGTTATGCAGCTATTTCACAAGTGGATCGATTGCAGTC
AGAGCCTGAAAGTATCCGTAAATGGAGAGAAGAACAAATGGAACGCTTGGAAGCCCTTGATGCCAATTCTC
GGAAGCAAGAAGCAGAGTGGAAAGAAAAGGCAATAAAGGAGCTAGAAGAATGGTATGCAAGACAGGACGA
GCAGCTACAGAAAACAAAAGCAAACAACAGGGCAGCAGAAGAAGCCTTTGTAAATGACATTGACGAGTCGT
CCCCAGGCACTGAGTGGGAACGGGTGGCCCGGCTGTGTGACTTTAACCCCAAGTCTAGCAAGCAGGCCAA
AGATGTCTCCCGCATGCGCTCAGTCCTCATCTCCCTCAAGCAGGCCCCGCTGGTGTGA
Expressed Amino
Acid Sequence
MGLG(VPGVG)
72
RLEEQKLISEEDLLEMAELDPFGAPAGAPGGPALGNGVAGAGEEDPAAAFLAQQESEIAGIEN
DEAFAILDGGAPGPQPHGEPPGGPDAVDGVMNGEYYQESNGPTDSYAAISQVDRLQSEPESIRKWREEQMERL
EALDANSRKQEAEWKEKAIKELEEWYARQDEQLQKTKANNRAAEEAFVNDIDESSPGTEWERVARLCDFNPKSS
KQAKDVSRMRSVLISLKQAPLV
Molecular Weight
55.2 kD
Transition
temperature of
parent V72
(25 μM in PBS)
33.5
o
C
V96-CLC
Expressed Amino
Acid Sequence
MGLG(VPGVG)
96
RLEEQKLISEEDLLEMAELDPFGAPAGAPGGPALGNGVAGAGEEDPAAAFLAQQESEIAGIEN
DEAFAILDGGAPGPQPHGEPPGGPDAVDGVMNGEYYQESNGPTDSYAAISQVDRLQSEPESIRKWREEQMERL
EALDANSRKQEAEWKEKAIKELEEWYARQDEQLQKTKANNRAAEEAFVNDIDESSPGTEWERVARLCDFNPKSS
KQAKDVSRMRSVLISLKQAPLV
Molecular Weight
65 kDa
Transition
temperature of
parent V96
(25 μM in PBS)
31
o
C
A96-CLC
Expressed Amino
Acid Sequence
MGLG(VPGAG)
96
RLEEQKLISEEDLLEMAELDPFGAPAGAPGGPALGNGVAGAGEEDPAAAFLAQQESEIAGIEN
DEAFAILDGGAPGPQPHGEPPGGPDAVDGVMNGEYYQESNGPTDSYAAISQVDRLQSEPESIRKWREEQMERL
EALDANSRKQEAEWKEKAIKELEEWYARQDEQLQKTKANNRAAEEAFVNDIDESSPGTEWERVARLCDFNPKSS
KQAKDVSRMRSVLISLKQAPLV
Molecular Weight
62.3 kDa
Transition
temperature of
parent A96
(25 μM in PBS)
84
o
C
* Genes for V96-CLC and A96-CLC were constructed similarly to V72-CLC. Bold indicates the elastin-
like polypeptide sequence. Red indicates the myc epitope sequence. Underline indicates the human
clathrin light chain sequence.

52
3.3.2 Immunofluorescence
For fluorophore colocalization assays, cells were cooled at 4
o
C for 45 min and incubated
at specified temperatures for 45 min prior to fixation. Cells were washed with 50 mM
ammonium chloride before a 10 min permeabilization step using 0.1% Triton-X in phosphate
buffered saline (PBS). After washing 2x with PBS, cells were incubated for 1 hour at 37
o
C with
primary antibodies, washed, and incubated for another hour with secondary antibodies. Cells
were incubated with DAPI prior to mounting. Primary antibodies included chicken anti-myc
(Abcam, San Francisco, CA, ab19233), mouse anti-myc (Invitrogen, Carlsbad, CA, R950-25),
mouse anti-CLC (Abcam, ab24579), mouse-anti CHC (X22 hybridoma courtesy of Prof.
Okamoto
133
), rabbit anti-HA (Cell Signaling, Danvers, MA, C29F4), mouse anti-EEA-1
(Abnova, Taipei, Taiwan, H00008411-M03), LAMP1 (Abcam, ab24170), and Rab5 (Santa Cruz
Biotechnology, Santa Cruz, CA, sc-46692). All secondary antibodies were purchased from
Invitrogen and included Alexa 568 goat anti-chicken (A11041), Alexa 488 goat anti-mouse
(A11001), Alexa 488 goat anti-rabbit (A11008), Alexa 633 goat anti-rabbit (A21070). Confocal
images were captured on a Zeiss LSM 510 Meta NLO equipped with Argon, red HeNe, and
green HeNe lasers. Three-dimensional SIM images were acquired using a 60x oil-objective lens
on a GE DeltaVision OMX system equipped with 3 sCMOS cameras and 405, 448, and 568
excitation lines.

3.3.3 Receptor Internalization Assays
To assess receptor internalization, CHO cells were plated on glass coverslips and
transfected using Turbofect (Thermo Scientific, Waltham, MA) the following day with AngIIR
(N-terminal HA tagged) +/- V96-CLC (myc tagged between V96 and CLC). Due to ELP’s
53
transition’s dependence on concentration
59
, transfected cells were given 48 hours to reach
maximum ELP-CLC expression level prior to assay. 48 hours after transfection, cells were
incubated at 4
o
C for 45 min with rabbit anti-HA antibody (IgG) (C29F4, Cell Signaling, Boston,
MA) to label the receptor at the plasma membrane. After washing, cells were incubated at 37
o
C
for 45 min to allow for maximal ELP transition. For studies assessing the chemical inhibition of
receptor internalization, cells were treated with dynasore (50 μM) or monodansylcadaverine (300
μM). Angiotensin II (100 nM, Sigma-Aldrich, St. Louis, MO) was added for 30 min to stimulate
internalization of AngIIR. Cells were fixed with 4% paraformaldehyde in PBS for 10 min,
washed 1x with 50 mM ammonium chloride, and 1x with PBS before a 1 hr incubation at room
temperature with ~5 μg/mL Alexa488 goat anti-rabbit antibody (Invitrogen). Cells were then
incubated overnight at 4
o
C with ~2 μg/mL unlabeled goat anti-rabbit to block unlabeled surface
antibodies from subsequent probes. Cells were washed 2x with PBS and permeabilized with
0.1% Triton-X. After a PBS wash, cells were incubated for 1 hr at room temperature with ~5
μg/mL Alexa633 donkey anti-rabbit (Invitrogen). For cells expressing ELP CLC fusions,
immediately following permeabilization, cells were incubated at room temperature for 1 hr with
mouse monoclonal anti-myc antibody (Invitrogen) before probing with Alexa633 donkey anti-
rabbit (Invitrogen) and Alexa565 goat anti-mouse (Invitrogen). Cells were washed in PBS before
mounting.

3.3.4 Quantification of receptor internalization in single cells
To determine the amount of surface receptor internalized after stimulation, we used a single cell-
based method. This technique quantifies the amount of fluorescence emitted by the internalized
receptor relative to the total (internal and external) and presents it as a percentage of internalized
54
receptor
100
external internal
internal


. After a pre-incubation at 4
o
C for 45 min with rabbit anti-HA
antibody (Cell Signaling, C29F4), cells were stimulated with agonist at various time points and
conditions prior to PFA fixation. Prior to permeabilization with Triton-X-100, cells were
incubated for 1 hour with Alexa488 anti-rabbit secondary antibody. Once permeabilized, the
internalized receptor was stained with Alexa633 anti-rabbit secondary antibody. Each
experiment contained stimulated and un-stimulated control cells, which were used to determine
acquisition parameters on the confocal microscope. A 63x oil lens was used for these
experiments with an optical section of 4 μm. For each treatment parameter, 60 to 100 cells were
quantified for fluorescence using Zeiss LSM 510 Image Browser. Cells expressing high levels of
receptor (beyond the dynamic range) were excluded from the analysis.

3.3.5 Statistics
Unless otherwise noted, data are presented as means and 95% confidence intervals. The
significance of changes in receptor colocalization were determined a Student’s t-test. The
inhibition of receptor internalization due to ELP addition to CLC was first analyzed using 2-way
ANOVA for all groups. When a significant difference was found (p <0.05), a post hoc analysis
for each temperature and treatment was obtained using the Bonferroni t-test. Student’s t-tests
were performed between temperatures when comparing colocalization coefficients of antibodies.

55
3.4 Results
3.4.1 Design and characterization of a thermally responsive switch for clathrin-mediated
endocytosis
Intracellular microdomain assembly depends on the transition temperature of the free
ELP
55
. Therefore, we hypothesized that microdomain assembly and clathrin-mediated
endocytosis shutdown can be rationally designed based on prior ELP characterization. For an
ELP of the motif [Val-Pro-Gly-Xaa-Gly]
n
, as polymer chain length, n, increases, the assembly
temperature decreases. Based on previous intracellular ELP characterization
55
, three polymers
were selected for fusion with CLC (Table 3.1). Near physiological temperatures, V72-CLC and
V96-CLC (Xaa=Val, n=72 and 96 respectively) phase separate, while A96-CLC (Xaa=Ala, n =
96) remains soluble (Figure 3.2). After a 45 min incubation, the longer V96-CLC polymer
transitioned fully at 37
o
C, while V72-CLC only reached maximal microdomain assembly at 42
o
C (Fig 3.2). Both transition temperatures are ~5
o
C higher than for intracellular green
fluorescent protein fusions to V72 and V96
55
. The kinetics for V96-CLC assembly revealed that
a 45 min incubation at 37
o
C is sufficient to observe microdomains in more than 80 % of
transfected Chinese hamster ovary (CHO) cells (Fig 3.2C), and this finding is reconfirmed in
HEK293 cells (Figure 3.3). Additionally, V96-CLC cells incubated below 31
o
C for times in
excess of 45 min do not exhibit ELP transition, indicating an absence of a time limitation (data
not shown). At the temperatures explored, no microdomain assembly was observed for A96-
CLC; therefore, this fusion protein serves as a control for overexpression of a non-switchable
CLC fusion protein.
56

Figure 3.2 CLC microdomain assembly is tunable and rapid. ELPs fused to CLC were expressed in
CHO cells, incubated at various temperatures, fixed, and stained for the myc epitope (red). a)
Hydrophobicity and chain length were used to control microdomain formation. Hydrophobic ELPs V96
and V72 (red) co-expressed with AngIIR (green) resulted in microdomain formation at physiologic
temperatures while the more hydrophilic ELP A96 (red) did not. b) The temperature of microdomain
assembly depends on ELP length. V72-CLC and V96-CLC reach maximum assembly at 42 and 37
o
C
respectively. A96-CLC does not assemble at the temperatures evaluated. c) Kinetics of microdomain
formation in cells transfected with V96-CLC were assessed after shifting to 37
o
C. Cells were pre-
incubated at 4
o
C to ensure uniform solubilization prior to heating. Within 45 minutes, 80 % of cells
expressing V96-CLC formed microdomains. Mean ± 95% confidence interval (n=3).

Figure 3.3 ELP microdomain formation speed is retained across cell lines and polymer constructs a)
V96-CLC microdomain formation has the same kinetics in Hek293 cells as in CHO (Fig 2b). b)V72-
CLC GEPM assembly in CHO at 37
o
C has slower kinetics of assembly as compared to V96-CLC.

57
3.4.2 Cytosolic microdomains colocalize with early markers of clathrin-mediated endocytosis
Using confocal laser scanning microscopy and indirect immunofluorescence, we assessed
the intracellular effect of microdomain formation on proteins involved in clathrin-mediated
endocytosis. An antigenic myc epitope (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu) was
inserted between ELP and CLC to serve two functions: i) detect transfected cells; and ii)
characterize the formation of microdomains. A96-CLC, which does not assemble microdomains
at 25 or 37 °C, did not co-localize with CLC or CHC (Figure 3.4). In contrast, V96-CLC
assembles microdomains at 37 °C (Figure 3.5b). When soluble at 25 °C, colocalization of CHC
with V96-CLC remains low, whereas they were strikingly colocalized after microdomain
formation (67.0%) (Table 3.2). Next, total CLC was assessed for colocalization with
microdomains, which includes signal from both endogenous and expressed fusion proteins.
Similarly to CHC, significantly more CLC colocalized with V96-CLC at 37
o
C (40.8 %) relative
to at room temperature (9.7 %). A summary of co-localization studies performed in CHO cells
can be found in Table 3.1, and similar results were confirmed using HEK293 (Figure 3.6). These
results strongly suggest that during microdomain formation, V96-CLC binds and sequesters
other proteins associated with endocytosis. To probe this further, Rab5, an adaptor protein found
on clathrin-coated vesicles and EEA-1, a protein involved in vesicle docking to early endosomes,
were assessed for colocalization with V96-CLC. While the colocalization of Rab5 and EEA-1
with V96-CLC microdomains was not as robust as seen with CHC, there remained a significant
increase in overlap of the fluorescence signal upon microdomain formation at 37
o
C (Table 3.2).
However, this pattern was not observed with LAMP-1, a lysosomal marker. LAMP-1 colocalized
at the same background percentage when microdomains formed as in their absence. This
58
suggests that only proteins associated with clathrin-coated endocytotic vesicles incorporate into
these microdomains.

To further investigate their morphology, structured illumination fluorescence microscopy
was used to dissect a microdomain (Figure 3.7). This revealed that V96-CLC microdomains are
either hollow or impermeable to antibody probes. The V96-CLC appears to form a corona,
surrounding a core enriched in CHC, which is consistent with observations made using confocal
microscopy (Fig 3.5b and 3.6b). Thus, it appears that the microdomains are not homogenous
features in the cytosol, but adopt a structure of some complexity.

Table 3.2: CLC microdomains colocalize with markers of early endocytosis
ELP fusion Antibody Marker Percent colocalization at Significance
target 23 °C [%]
a
37 °C [%]
a
p value
CLC Clathrin coated vesicles 9.7 ± 16.2 40.8 ± 22.4 8.9x10
-7

CHC Clathrin coated vesicles 9.7 ± 8.2 67.0 ± 21.3 4.7x10
-14

EEA-1 Early endosomes 9.1 ± 8.5 42.6 ± 27.7 1.7x10
-8

Rab5 Early endosomes 10.4 ± 10.5 30.2 ± 23.1 8.3x10
-5

V96-CLC
(assembles at 37 °C)
LAMP-1 Lysosomes 8.3 ± 6.3 10.1 ± 8.9 n.s.
CLC Clathrin coated vesicles 10.1 ± 4.3 10.0 ± 5.9 n.s. A96-CLC
(does not assemble) CHC Clathrin coated vesicles 20.9 ± 9.6 16.2 ± 9.8 n.s.
a
CHO cells were transfected with an ELP fusion protein, incubated at 23 or 37 °C, fixed, stained using an anti-myc
antibody for the ELP and an antibody against the indicated target protein, imaged using confocal laser scanning
fluorescence microscopy and quantified for colocalization. Mean± SD (n=3), n.s. not significant, p>0.05.

59

Figure 3.4 Soluble A96-CLC does not co-localize with CLC or CHC. No puncta are visible at 25
o
C or
37
o
C, indicating A96-CLC remains soluble at those temperatures.

Figure 3.5 V96-CLC colocalizes with early markers of CME in CHO cells. (a) CLC (b) CHC (c)
EEA-1 and (d) Rab5 antibodies are used to stain V96-CLC expressing CHO cells at room temperature (25

o
C) and 37
o
C.

60

Figure 3.6 V96-CLC colocalizes with early markers of CME in Hek293 cells. (a) CLC (b) CHC (c)
EEA-1 and (d) Rab5 antibodies are used to stain V96-CLC expressing CHO cells at room temperature (25

o
C) and 37
o
C.

Figure 3.7. V96-CLC self-assembles into hollow spheres with CHC. Imaging on an OMX
reconstruction microscope elucidates the architecture of V96-CLC microdomains in CHOs. Myc staining
of V96-CLC (red), CHC (green), and DAPI staining of nucleus (blue). a) One slice through the interior of
the transfected cells shows 2 distinct CHC positive microdomains. b) YZ and XZ slices of the
reconstructed stack show CHC interior to V96-CLC.

61
3.4.3 Temperature triggered pathway modulation in mammalian cells
To validate that V96-CLC microdomain formation knocks down clathrin-mediated
endocytosis, CHO cells were transfected with V96-CLC and one of two receptors, either the
angiotensin II receptor (AngIIR) or M1 muscarinic receptor. G-protein coupled receptors
internalize via clathrin-mediated endocytosis; moreover, their agonist-dependent endocytosis is
strongly affected by CLC knockdown.
131
We used a single cell-based method to quantify the
amount of cargo endocytosed. This method allows us to measure the amount of internalized
receptor with respect to the total amount of receptor, internal and surface-associated, using
confocal microscopy (Figure 3.8a). Examination of AngIIR using antibodies established
observable receptor internalization after a 30 min agonist stimulation. We found that 60 % of
AngIIR was internalized after stimulation with 100 nM angiotensin, consistent with previously
published results.
131
At 31
o
C, cells transfected with V96-CLC internalized AngIIR similarly to
cells expressing only AngIIR (Fig 3.8b). However, at 37
o
C, when V96-CLC forms
microdomains, the degree of AngIIR internalization decreased significantly relative to cells
expressing only AngIIR (p<0.001). This effect was even more pronounced at 42
o
C (p<0.001)
(Fig 3.8c). Similar observations were confirmed using the M1 muscarinic receptor (Figure 3.9).

To compare the effectiveness of microdomain-mediated knockdown of endocytosis
knockdown relative to chemical inhibitors, we assayed receptor internalization in the presence or
absence of monodansylcadaverine and dynasore. Monodansylcadeverine inhibits clathrin-
mediated endocytosis while dynasore inhibits both clathrin-mediated and clathrin-
independent/lipid raft endocytosis pathways.
130
AngIIR transfected cells pre-treated with
dynasore and monodansylcadaverine exhibited reduced internalization of AngIIR (Figure
62
3.10a). In this model, pretreatment with monodansylcadaverine and dynasore had the same effect
on AngIIR internalization as cells expressing V96-CLC microdomains or un-stimulated cells.
Interestingly, pretreatment using chemical inhibitors of clathrin-mediated endocytosis had no
added effect on cells containing V96-CLC microdomains. Similar results were confirmed using
the M1 muscarinic receptor (Fig. 3.10b).

63

Figure 3.8 AngiotensinII Receptor Internalization is significantly reduced by microdomain
assembly. CHO cells were transfected with AngIIR, stimulated with (+Ang) or without (-Ang)
angiotensin (100 nM) for 30 min, and fixed. a) Surface AngIIR was labeled by immunofluorescence
(green), the cell membrane was permeabilized, and internalized AngIIR was then labeled (purple). Bar =
5 μm. b) Cells were then co-transfected with both V96-CLC and AngIIR, incubated with angiotensin
above and below the microdomain assembly temperature, and stained for AngIIR internalization. V96-
CLC expressing cells were identified by anti-myc antibodies (red). Microdomains were observed at 37
and 42 °C, at which temperature receptor internalization was significantly decreased. Bar = 5 μm. c)
AngIIR internalization with stimulation was quantified using image analysis at different temperatures for
cells with and without V96-CLC. At 37 and 42
o
C there was a significant decrease in receptor
internalization for cells with V96-CLC microdomains compared to those without (**p<0.0001). At 31
o
C,
V96-CLC remains soluble and does not affect receptor internalization. Mean ± 95% confidence interval
(n=3).

64
Temperature (
o
C)
% Internalization
31
o
C 37
o
C 42
o
C
0
20
40
60
80
100
M1R
M1R + V96-CLC
**

Figure 3.9 Internalization of the M1 muscarinic receptor is knocked down by V96-CLC
microdomains. Chinese hamster ovary (CHO) cells were transfected with the M1 muscarinic receptor
(M1R) with and without the V96-CLC fusion and incubated at different temperatures. Receptor
internalization was stimulated with 1 mM carbachol for 30 min prior to fixation. Cells were differentially
stained for internal and external receptor as described in the manuscript. At 37
o
C, there was a significant
decrease in receptor internalization in cells expressing V96-CLC compared to those without
(**p<0.0001). There was also a decrease in receptor internalization for cells with V96-CLC
microdomains at 37
o
C compared to soluble V96-CLC at 31
o
C. The receptor internalizes normally at 31
o
C, when V96-CLC remains soluble. Values indicate the mean ± 95% CI (n=3).

Figure 3.10 CLC-V96 microdomain assembly blocks clathrin-dependent internalization as
effectively as chemical inhibition. CHO cells were co-transfected with a) AngIIR or b) M1R with or
without V96-CLC. Differential GPCR internalization with and without stimulation is quantified using
immunofluorescence. When stimulated cells are pre-incubated with either monodansylcadaverine (MDC)
or dynasore (dyna), they yielded a similar reduction of internalization relative to un-stimulated cells.

65
3.4.4 Inhibition of GEPM mediated CME knockdown is reversible
Since ELP phase separation is reversible in vitro
55
, we assessed whether reversibility was
conferred to microdomain-mediated knockdown of clathrin-dependent endocytosis (Figure
3.11). Cells expressing both AngIIR and V96-CLC were preincubated at 4
o
C with the AngIIR
labeling antibody (Fig 3.11), as described in our knockdown experiment (Fig 3.8). Cells (T1)
were then incubated at 37
o
C for 45 min to induce intracellular microdomain formation. Two
subsets of these cells were incubated at room temperature for 45 min, to allow their
microdomains to solubilize. Among the resolubilized cells (T2), one set was incubated at 31
o
C
to assess receptor internalization upon stimulation, and the second set was returned to 37
o
C.
These reheated cells assembled microdomains for a second time, which re-inhibited
internalization. The results demonstrate that two rounds of heating and cooling causes no loss of
AngIIR internalization activity at 31 °C. When reheated to 37 °C, the microdomains once again
functionally block AngIIR internalization (Fig 3.11).

66

Figure 3.11 Resolubilizing V96-CLC microdomains confers function back to CME. a) CHO cells
transfected with V96-CLC and AngIIR are cycled from 31
o
C to 37
o
C, back down to 31
o
C, than back up to
37
o
C. b) Quantification of AngIIR internalization at different stages of V96-CLC microdomain formation.
The first transition knocks down CME at 37
o
C relative to non-V96-CLC expressing cells as well as
relative to the same cells at 31
o
C (**p<0.0001). The same amount of knockdown is observed after a
second transition. V96-CLC cells return to normal levels of internalization at 31
o
C, with no statistical
significance relative to non-transitioned cells also at 31
o
C. However, re-forming V96-CLC microdomains
knocks down CME again relative to non-expressing V96-CLC cells and relative to the same cells without
microdomains at 31
o
C (**p<0.0001). Values indicate the mean ± 95% CI (n=3).

67
3.4.5 ELP-CLC GEPMs are CME knock-down specific
The specificity of microdomain-mediated clathrin-mediated knock down was accessed by
looking at alternative routes of cellular internalization. Clathrin-mediated endocytosis is one of
many routes of cellular internalization. Extracellular molecules can enter cells via
macropinocytosis, caveolin-mediated endocytosis, as well as clathrin- and caveolin- free
endocytosis
134
. Caveolin-mediated internalization was assessed due being well characterized
with an array of commercially available ligands available for assay (Figure 3.12). Cell
internalization assays were performed as in Section 3.4.3 and 3.4.4, with the exception of an
additional, fluorescently tagged cholera toxin-B (CTxB) ligand. CtxB was added to the media at
the same time as angiotensin. There was no difference in uptake of CtxB between cells with or
without V96-CLC microdomains (p= 0.073).
A number of proteins not associated with clathrin-mediated endocytosis were also
assayed for their colocalization with V96-CLC microdomains (Figure 3.13). Markers of
caveolin-mediated endocytosis (Cav-1), late stage endocytic degredation (LAMP1, AP3), trans-
Golgi secretion (AP-1), protein folding and secretion (calnexin), and autophagy (LC3) were
assessed for colocalization with V96-CLC using confocal microscopy and found to have no
overlap.
68

Figure 3.12 Knock-down of CME with CLC microdomains does not affect a marker of caveolin-
mediated internalization. a) The uptake of cholera toxin B (CTxB), a ligand internalized via caveolin-
mediated internalization, was quantified in cells expressing AngIIR alone and AngIIR along with V96-
CLC. b) AngIIR internalization rates were quantified at 37
o
C after 30 min of AngIIR stimulation. AngIIR
internalization is knocked-down in cells expressing V96-CLC. . c) The same cells quantified for AngIIR
internalization in b), are quantified by total fluorescence for CTxB internalization. The knock-down of
clathrin-mediated endocytosis of AngIIR does not affect the caveolin-mediated internalization of CTxB,
which suggests V96-CLC microdomains minimally affect non-clathrin internalization pathways. Values
indicate the mean ± 95% CI (n=3).

69

Figure 3.13 Proteins not associated with CME do not associate with V96-CLC GEPMs. A variety of
trafficking associated proteins (Caveolin-1, AP-1, AP-3, LAMP-1, Calnexin, LC3) were assayed for their
association with V96-CLC microdomains at 37
o
C. None of the assayed proteins co-localized with V96-
CLC.

3.4.6 Proposed mechanism of ELP-CLC driven CME knock-down
There is more to the mechanism of CME shut-down than simple V96-CLC aggregation
into GEPMs. Unlike ELP aggregation in PBS, where the surrounding media is empty of
associating proteins, the aggregation of ELP-CLC constructs inside the cell are complicated by
proteins that associate with CLC.
The model proposed herein to describe the orthogonal cell switch assumes the system to
be in one of two states: ON or OFF. The ON state assumes the ELP portion of the ELP-CLC
fusion to be soluble because the ON state is below the ELP’s T
t
. ON indicates that the targeted
pathway, in this case clathrin-mediated endocytosis, functions normally. As the ELP in the ELP-
CLC fusion is soluble, it is more entropically favorable for CLC to direct the fusion protein’s
location. CLC associates with a number of internalization proteins, namely CHC, and is expected
to continue forming triskelion at the plasma membrane along with CHC below T
t
. Since ELP is
70
soluble during the ON state, it simply acts as a CLC tag, much like a fused GFP
77
, being taken to
the location that CLC drives it to go.

Figure 3.14 Mechanism of CME shut down. When CME is ON, ELP-CLC fusion are soluble and allow
CLC to be recruited to the plasma membrane for receptor-mediated endocytosis. Turning CME OFF is
done by increasing the temperature and sequestering proteins associated with internalization into ELP-
CLC microdomains in the cytosol.

Conversely, when the temperature is raised above the fused ELP’s T
t
, it becomes
entropically favorable for ELPs to aggregate with one another due to their switch to insolubility.
Because the entropy for ELPs to self-associate is higher than the entropy for CLC to continue
coating internalization vesicles, the ELP portion of the ELP-CLC fusion pulls CLC into cytosolic
microdomains. However, the non-covalent bonds CLC makes with other internalization proteins
are preserved when CLC is pulled into the ELP microdomain. The protein most closely
associated with CLC, CHC, has prominent staining within ELP-CLC microdomains.
Interestingly, a portion of EEA-1 and Rab5 also localize to ELP-CLC microdomains during the
71
OFF state. Both EEA-1 and Rab5 are markers of early endosomes. During receptor mediated
endocytosis, CLC and CHC coat the budding membrane and aid in the formation of clathrin-
coated vesicles at the plasma membrane. Prior to fusing with early endosomes, the clathrin-coat
encasing the vesicle is uncoated to allow vesicle fusion to the early endosome. CLC is not
expected to directly associate with the early endosome, yet markers for the early endosome are
found in ELP-CLC microdomains. A proposed hypothesis is that during ELP driven
microdomain assembly, proteins closely associated, yet not directly bound, to CLC will be
sequestered in the ELP-CLC microdomain as well. However, this would indicate that other
endocytosis markers would found in cytosolic microdomains, yet they are not. For example,
caveolin-1, a well characterized marker of caveolin mediated endocytosis is not pulled into the
ELP microdomains, perhaps indicating that the proteins pulled into the microdomains are not just
physically close to the plasma membrane, but must indirectly associate with the clathrin
mediated endocytosis pathway.
Clathrin is not solely located at the plasma membrane, it is also involved in sorting cargo
from the trans-Goli network out to the plasma membrane for secretion
135
. AP-1, an adaptor
protein for CLC at the trans-goli, was probed after ELP-CLC microdomain formation. While
proteins associated with CLC at the plasma membrane are sequestered in ELP-CLC
microdomains, no overlap between AP-1 and ELP microdomains was found during the cell’s
OFF state. There are two possible explanations for this. First, CLC is found at much lower
concentrations at the trans-Goli network, and the concentration was not high enough for ELP-
CLC to transition. Or second, while receptor mediated endocytosis of GPCRs was stimulated,
trans-Goli sorting was not, so there were no events driving CLC to coat vesicles, and
subsequently no associated proteins such as AP-1 found in ELP-CLC microdomains.
72
Figure 3.14 represents the proposed mechanism of ELP-CLC mediated shut-down of
CME. While in the ON state, ELPs are soluble and internalization occurs much as it would in the
absence of an ELP tag. Increasing the temperature past the ELP’s T
t
shifts the cell into the OFF
state, aggregating ELP-CLC fusion proteins. This aggregation sequesters CLC and CHC from
forming clathrin-coated pits, structures necessary for CME. The aggregation of ELPs also brings
proteins associated with early clathrin-mediated endocytosis such as EEA1 and Rab5 into the
microdomains. Proteins associated with late endocytosis and sorting such as LAMP1 and AP-1
are not pulled in, indicating that pathways associated with those proteins remain intact.
Additionally, other internalization pathways remain intact, such as the caveolin-mediated
pathway, further indicating that ELP-CLC mediated knock down of internalization is CME
specific.

73
3.5 Conclusions
Life scientists need better tools to elucidate a vast array of still unknown molecular
mechanisms. In this chapter, we have described a powerful new approach using a protein
polymer system that expands our arsenal of tools to probe cellular function in a rational,
immediate, and reversible manner.

Control of protein function is typically approached using one of two strategies. The first
targets protein transcription and translation prior to protein production.
116
The tetracycline
system places a gene of interest downstream of an inducible promoter, and gene activation or
inactivation occurs upon addition of a stimulus. However, the most significant drawback to
targeting protein prior to synthesis is the extended time required for the input to result in the
desired output.

The second, less-utilized method targets protein activity after the protein has been
synthesized. This approach allows for a higher level of temporal control compared to methods
targeting the transcriptional/ translational machinery. Variations of the FK506 molecule have
been adopted for just this purpose. Addition of a FK506 analogue stimulates the dimerization or
oligomerization of a protein tagged to the receptor of the FK506 analogue.
77,136
This system’s
output can be observed rapidly, yet its inactivation requires the addition of a competing analogue
or is entirely irreversible. Also, dimerization may increase, decrease, or leave the targeted protein
pathway entirely unaffected. Although there are many variations on this chemically inducible
system for the rapid control of protein interaction, there has until now been no technology that
can rapidly drive proteins to associate and lose function that is both rapid and reversible. Our
74
system, designed using genetically encodable protein polymers – ELPs – avoids the need for the
addition of small molecules to control the system. To demonstrate this technology, we fused an
effector gene for clathrin-light chain with that of a thermally responsive ELP, V96. V96 is a
protein polymer that, when expressed intracellularly, forms discreet microdomains above 35
o
C,
but is solubilized into individual polymers at room temperature.

The assembly of V96-CLC polymers into microdomains at 37
o
C was visualized using
confocal fluorescence microscopy. The addition of V96 to CLC conferred the phase transition
properties of the V96 polymer onto CLC. We were surprised to observe that additional proteins
were localized inside these microdomains. Clathrin-heavy chain, EEA-1, and Rab5, all markers
of early endocytosis formed puncta along with V96-CLC. However, Rab5 and EEA-1, proteins
involved in cargo sorting, associated with V96-CLC to a lesser degree than CHC. We
hypothesize that since Rab5 and EEA1 do not directly bind CLC, but interact via adaptor
proteins, not as many associate in the V96-CLC microdomains. Markers of late stage
endocytosis, such as LAMP1, do not enter microdomains at all, further supporting this
hypothesis (Fig 13.3).

Validation of clathrin-mediated endocytosis knockdown upon V96-CLC microdomain
formation was performed by measuring internalization rates of 2 G-protein coupled receptors:
AngIIR and the M1 muscarinic receptor. Agonist binding to these receptors stimulates
internalization, a process confirmed in the literature to occur by clathrin-mediated
endocytosis.
137-138
AngIIR internalization was reduced by 50% at 37
o
C and this effect was more
pronounced at 42
o
C, when slightly more thermal energy results in higher internalization of
75
AngIIR. Furthermore, microdomain-mediated inactivation of clathrin-mediated endocytosis
appears to be as effective as two chemical suppressants of this process, dynasore and
monodansylcadaverine.

In contrast to other intracellular switches, the assembly of ELP microdomains is rapidly
reversible.
55-56
After 2 cycles of heating and cooling, AngIIR continues to be internalized at 31
o
C and to be impaired for internalization at 37
o
C. The degree of internalization is consistent with
that seen after the first round of heating and cooling, indicating that V96-CLC cycles between
soluble and insoluble aggregate and turns clathrin-mediated endocytosis ‘on’ and ‘off.’

This chapter demonstrates that sequestration of a key effector protein, CLC, within an
organelle-like microdomain can inhibit the clathrin-mediated cell trafficking pathway. Although
these results validate our proof-of-concept study, a more thorough understanding of the
mechanisms of microdomain formation is necessary for the broad adoption of genetically
engineered protein microdomains as intracellular switches. For example, some residual
internalization occurs even after microdomain formation may be due to endogenous CLC still
present in the cell. While previous studies have shown that overexpressed CLC dominates
endogenous CLC and that the effect of endogenous CLC is negligible
77
, complete elimination of
endogenous CLC may augment the effect of these microdomains. Regardless of these
limitations, the apparent success of this approach opens the door for applications in the
controlled suppression of other cell signaling/trafficking pathways.

76
Chapter 4
An amphipathic α-helical peptide from apolipoprotein A1 stabilizes protein
polymer vesicles

4.1 Abstract
L4F, an alpha helical peptide derived from the lipid-binding domain of the ApoA1
protein, has potential applications in the reduction of inflammation involved with cardiovascular
disease as well as liver fibrosis. Despite its activity using in vivo models, recent clinical trials
evaluating L4F peptide alone revealed limited clinical efficacy due to a half-life of only 1.5
hours. To address this limitation, the stabilization of the amphipathic L4F peptide through fusion
to a high molecular weight protein polymer is here described. Comprised of multiple pentameric
repeats, elastin-like polypeptides (ELPs) are biodegradable protein polymers inspired from the
human gene for tropoelastin. Since L4F is only 2.2 kDa, the addition of a 74 kDa ELP is
intended to increase the hydrodynamic radius above the renal filtration cutoff. Dynamic light
scattering confirmed that the fusion peptide forms nanoparticles with a hydrodynamic radius of
approximately 49.5 nm, which is unexpectedly above that observed for the free ELP (~5.1 nm).
To further investigate the nanoparticle morphology, both regular and cryogenic transmission
electron microscopy revealed that these peptide nanoparticles have the morphology of vesicles.
On average, these vesicles are 43 nm in radius with lamellae 6.8 nm thick. To evaluate their
potential as therapeutic moieties, the L4F nanoparticles were then incubated with hepatic stellate
cells. Stellate cell activation leads to hepatic fibrosis. L4F nanoparticles suppressed hepatic
stellate cell activation in vitro. This study demonstrates the potential utility of protein polymers
to stabilize amphipathic peptides into vesicular nanoparticles with possible therapeutic activity.
77
4.2 Introduction
The prevalence of secondary diseases associated with obesity, such as non-alcoholic fatty
liver disease (NAFLD), are expected to rise with the high prevalence of obesity in Western
nations
139
. NAFLD, characterized by the deposition fat and the presence of inflammation, can
progress to hepatic fibrosis and cirrhosis
140
. A candidate for the prevention of progression of
NAFLD is the apolipoprotein A-1 mimetic D4F, which has been shown to prevent hepatic
fibrosis in murine models
141
. D4F and its natural enantiomer L4F have been extensively studied
in animal models related to lipid oxidation and inflammatory diseases. Based on the 18A
peptides, which were originally intended to displace apolipoproteins from HDL
142
, the 4F
peptides are amphipathic and create type A α-helices due to their 4 hydrophobic phenylalanine
residues. The 4F peptides bind oxidized lipids with an affinity 4-6 orders of magnitude higher
than ApoA-1
143
. 4F’s anti-inflammatory properties are attributed to their affinity for pro-
inflammatory oxidized lipids. L4F has been demonstrated to decrease inflammatory cytokines
including IL-6, TNF- α, and IL-1 β in obese mice
144-145
as well as inhibit activation of the
inflammatory transcription factor NF- κB in chronic kidney disease rat models
146
. D/L-4F’s
ability to decrease systemic inflammation is the basis for its exploration in inhibiting the
localized inflammation and activation of hepatic stellate cells and the progression of liver
fibrosis.

While D4F is anti-fibrotic in vivo
141
, we chose to develop the L-amino acid – L4F –
peptide for three reasons. First, unlike D4F, the L4F peptide can be directly engineered onto an
ELP protein polymer to increase its hydrodynamic radius and possibly extend its half-life, reduce
the dose, and maintain efficacy. Recent clinical trials with L4F have shown that the peptide has a
78
short half-life in humans of about 1-2 hours
147
, which suggests that therapy would benefit from a
higher hydrodynamic radius. Secondly, using D-amino acids chronically can cause high tissue
accumulation due to impaired breakdown
148
. Lastly, when administered subcutaneously, the L
form of the 4F molecule was found to be just as effective at treating atherosclerosis as the D
form
147,149
. It is therefore possible that D4F’s anti-fibrotic mechanism would be conserved in its
L4F enantiomer.

However, since L4F is a biologic molecule made of natural amino acids, it is subjected to
proteolysis. More importantly, L4F is only 2.2 kDa in size, which means that it could be rapidly
cleared via renal filtration. To circumvent these two issues, we propose the addition of a high
molecular weight elastin-like polypeptide (ELP) to modulate the biodistribution of the peptide.
ELPs are derived from the human gene for tropoelastin and are highly repetitive polypeptide
chains of the amino acid sequence (Val-Pro-Gly-Xaa-Gly)
n
, where Xaa can be substituted for
virtually any amino acid
55
. ELPs are known to phase separate above a transition temperature, T
t
,
which can be tuned by selection of Xaa and n. ELPs are emerging as a platform to display and
manipulate the molecular weight of drugs, partly because they are biodegradable, biocompatible,
and non immunogenic
11
. Since they are genetically encoded, they lend themselves to direct
fusion with biologically functional proteins. The addition of ELPs to low molecular weight
peptides has been shown to both increase the half-life of the peptide as well as decrease systemic
clearance
150
.

In this study, we report the surprising formation of L4F ELP fusion proteins into 49.5 nm
nanoparticles at 37
o
C, significantly below the transition temperature of the parent ELPs. We also
79
demonstrate L4F nanoparticles ability to inhibit the activation of hepatic stellate cells in vitro.
We expect these data to act as a springboard for the development of a high molecular weight
ApoA-1 mimetic peptide with anti-inflammatory activity.

80
4.3 Materials and Methods
4.3.1 Construction of L4F ELP fusions
A DNA sequence encoding for the peptide L4F followed by a thrombin cleavage site and
ELP insertion site (IDT Technologies, Coralville, IA) was cloned into a pET25b+vector
(Clonetech, Mountain View, CA). A forward primer
(TATGGATTGGTTCAAAGCGTTTTATGATAAAGT GGCGGAAAAATTCAAAGAAGCG
TTCGGTCTGGTTCCGCGTGGTTCTGGTTACTGATCTCCTCG) and a reverse primer
(GATCCGAGGAGATCAGTAACCAGAACCACGCGGAACCAGACCGAACGCTTCTTTG
AATTTTTCCGCCACTTTATCATAAAACGCTTTGAACCAATCCA ) were annealed, and
ligated into a pET25b+ vector digested with NdeI/ BamHI to generate a 2 base pair overhang
created by digestion of at an amino terminal BseRI cut site. Various ELP genes
59
were ligated
downstream of the L4F encoding sequence using BseRI/BamHI cut sites in both L4F and ELP
plasmids to digest and 1 μL T4 DNA ligase (Invitrogen, Carlsbad, CA) to ligate, resulting in N-
L4F-thrombin-ELP-C (Table 1). The resulting fusion protein constructs were expressed in BLR
E.Coli and purified using the ELP-mediated phase separation
151
.
Table 4.1 Recombinant protein polymers examined during this study
label
*
encoded amino acid sequence MW
[kDa]
**
T
t

[
o
C]
Property at
37
o
C
***
slope
[
o
C /
log
10
( μM)]
y-intercept
[
o
C]
A192 G(VPGAG)
192
Y 73.7 61.9 soluble -9.2
[-9.9 to -8.6]
74.9
[74.0 to 75.8]
L4F-A192 MDWFKAFYDKVAEKFKEAFGLVPRGS
G(VPGAG)
192
Y
76.6 45.8 nanoparticle -2.6
[-3.6 to -1.7]
49.6
[48.2 to 50.9]
V2A192 G(VPGVG VPGAG VPGAG)
64
Y 75.4 47.2 soluble -5.8
[-6.7 to -4.9]
55.9
[54.6 to 57.1]
L4F-V2A192 MDWFKAFYDKVAEKFKEAFGLVPRGS
G(VPGVG VPGAG VPGAG)
64
Y
79.0 34.3 coacervate -1.3
[-1.6 to -1.0]
36.2
[35.8 to 36.6]
*Genes encoding for L4F-A192, V2A192, and L4F-V2A192 were constructed similarly to A192.
**The observed transition temperature at 25 μM in PBS.
***The slope, b, and Y-intercept, m, were derived from the log-linear regression analysis for transition temperature
vs concentration fit to the equation T
t
= m Log
10
[Conc] + b. mean [95% CI]

81
4.3.2 Optical characterization of the ELP phase diagram
The phase behaviors of ELPs were characterized as a function of molecular weight and
concentration by measuring the solution turbidity at 350 nm of protein polymer as a function of
temperature. 300 μL of protein polymers in phosphate buffered saline (PBS) were observed in a
Beckman Tm microcell at a constant ramp rate of 1
o
C min
-1
and measurements were captured 3x
min
-1
by a UV visible spectrophotometer (DU800 Spectrophotometer, Beckman Coulter, CA).
The maximum first derivative of the curve was defined as the transition temperature, T
t
.
4.3.3 Dynamic Light Scattering
A Dynapro plate reader (Wyatt Technology Inc., Santa Barbara, CA) was used for all
hydrodynamic radius and polydispersity measurements. Polymers were prepared at 25 μM
concentrations in PBS, filtered through 0.2 μm cellulose acetate filters, and centrifuged at 4
o
C,
1200 rpm to remove air bubbles. Mineral oil was added to the top of the sample to prevent
evaporation. Polymers were observed over a range of temperatures from 4 to 60
o
C in 1
o
C
increments.

4.3.4 Regular and cryogenic transition electron microscopy
TEM and Cryo-TEM images were captured on an FEI Tecnai 12 TWIN Transmission
Electron Microscope, operating at 100 kV for TEM and 80 kV for cryo-TEM. TEM samples
were pipetted onto a carbon-coated copper grid (Electron Microscopy Sciences, Hatfield, PA).
Filter paper was used to wick away excess solution. 10uL of 2 wt% aqueous uranyl acetate was
used to stain samples. For cryo-TEM, samples were pipetted onto lacey carbon coated TEM
grids (LC325-Cu, Electron Microscopy Sciences) pre-treated with plasma air to make the lacey
carbon film hydrophilic. Samples were vitrified by plunging them into a liquid ethane reservoir
82
precooled with liquid nitrogen. Both TEM and Cryo-TEM images were acquired with a 16 bit 2k
x 2k FEI Eagle bottom mount camera (Hillsboro, OR). Images were processed with ImageJ
(NIH, Bethesda, MD). Nanoparticle size was averages from three areas of view with more than
50 particles per image for TEM and 20 particles for Cryo-TEM. Bilayer thickness was averaged
across three points along the circumference of the vesicle. Numbers are presented as averages ±
95% confidence interval.

4.3.5 α-Smooth Muscle Actin Assay
Primary mouse hepatic stellate cells (HSCs) were isolated by collagenase/pronase
digestion and Stractan density gradient centrifugation before being cultured on plastic for 3 days
with or without L4F-A192
141
. HSCs were fixed with 10% formaldehyde. Primary antibody
staining was done using a mouse monoclonal anti- α-SMA antibody (1:100, Sigma), followed by
a fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody (Sigma). Images
were acquired using a Nikon PCM-2000 confocal microscope.

83
4.4 Results
4.4.1 Characterization of L4F ELP fusions
To develop a soluble ELP carrier for delivery of the L4F peptide, two ELPs were
evaluated (Table 4.1). Both A192 and V2A192 have the same number of 192 pentameric repeat
units, resulting in similar molecular weights. L4F was ligated at the N-terminus of the ELP of
interest (Figure 4.1a). ELPs reversibly phase separate in response to heating (Fig 4.1b);
however, the goal of this project was to determine if fusion to the amphiphilic L4F peptide is
sufficient to mediate the assembly of a nanoparticle of much higher hydrodynamic radius (Fig
4.1c). All four ELP constructs were purified using the ELP-mediated phase separation, which
resulted in highly pure samples with expected molecular weights (Fig 4.1d).

84

Figure 4.1 Fusion of an amphipathic peptide and an ELP designed to assemble nanostructures. a)
The amphipathic L4F peptide was appended at the amino terminus of two ELP genes called V2A192 and
A192 to drive nanoparticle assembly and increase hydrodynamic radius. b) ELPs undergo temperature-
mediated phase separation called coacervation, which can be used for purification. c) The amphipathic
peptide L4F (triangle) mediates assembly of nanostructures that are sterically stabilized by ELPs and
these structures also phase separate in response to heating. d) A library of purified protein polymers
(Table 4.1) were evaluated for identity and purify using SDS-PAGE and stained with copper chloride.

Next, fusion proteins were characterized for their phase behavior using optical density
measurements with respect to temperature and concentration. Below their phase transition
temperature, ELPs are soluble in water and have low optical density. Above their transition
temperature, there is a sharp increase in solution turbidity (Figure 4.2a-d). As has been observed
previously
59
, the transition temperature depends on the logarithm of concentration (Fig 4.2e,f).
This increase signifies the phase transition, or transition temperature of the ELP. Above this
temperature, the ELP become insoluble and forms microparticles called coacervate
152
. An ELP’s
transition temperature is highly contingent on the hydrophobicity of the guest residue, Xaa, in the
85
ELP sequence, Val-Pro-Gly-Xaa-Gly
153
. Guest residues that are more hydrophilic have higher
transition temperatures relative to residues that are more hydrophobic. For example, an ELP with
alanine guest residues, Xaa, will have a higher transition temperature relative to an ELP with
valine guest residues
154
.

A high molecular weight ELP, A192, with the hydrophilic the guest residue alanine phase
separates between 58 and 70 °C

(Fig 4.2a). The addition of the 2.2 kDa L4F fusion to the N-
terminus of the ELP has a surprisingly large effect on the transition temperature. L4F-A192, at
25 μM, phase separates above 47 °C, which is 14

°C below the parent ELP. Similarly, at 25 μM,
V2A192 phase separates above 46

°C, and again the L4F peptide decreases the transition
temperature to 35

°C, below body temperature (Fig 4.2d). This depression of the phase transition
temperature for L4F-V2A192 will prevent this formulation from remaining soluble at
physiological temperatures; however, L4F-A192 is an excellent candidate for solubility during
systemic administration or in vitro evaluation in cell culture.
86

Figure 4.2 Fusion of the L4F peptide depresses the ELP transition temperature. Two ELPs of the
same length and different transition temperatures were appended to L4F and observed using optical
density as a function of temperature and concentration. a) Unmodified A192 phase separates near 60 °C.
b) Addition of L4F to A192 depressed the transition temperature to about 45

°C. c) Unmodified V2A192
phase separates around 47

°C. d) Addition of L4F to V2A192 decreases the transition temperature to
about 34

°C. e-f) The concentration-temperature phase diagrams for all constructs follow a log-linear
relationship. L4F suppressed both the magnitude and the slope (Table 1) of the line for both e) A192 and
f) V2A192.

87
4.4.2 L4F mediates the assembly of ELP nanoparticles
Having observed the significant depression in the transition temperature upon fusion to
the amphipathic L4F peptide, we speculate that this short peptide may possess the ability to drive
self-association of the fused ELP into nanostructures. L4F ELP fusions assemble nanoparticles
prior to undergoing bulk phase transition. When assaying the hydrodynamic radius using
dynamic light scattering, both L4F-A192 and L4F-V2A192 form nanoparticles that are stable up
to a temperature of 46 and 34
o
C respectively. Both ELP monoblocks, A192 and V2A192, that
lack the L4F domain, do not exhibit any signs for assembling into nanoparticles (Figure 4.3a,c).
Monoblock ELPs are not expected to have a large hydrodynamic radius because they remain
unimeric until they begin to phase separate. This L4F nanoparticle assembly is attributed to its
amphipathic nature. When in an alpha helical secondary structure, L4F orients 4 hydrophobic
phenylalanine to one face. Since A192 and V2A192 do not assemble into nanostructures by
themselves, the assembly of these polymers into nanostructures upon fusion to the L4F peptide
suggests that these domains interact with each other to sequester hydrophobic residues away
from the aqueous environment. Therefore, it is the L4F region of the fusion protein that is
hypothesized to be driving this assembly, as well as sitting in the inner core of the nanoparticle
(Fig 4.1c).

Investigation of L4F nanoparticle size distribution at 37
o
C further supported the
difference in radius between L4F ELP fusions and plain ELPs. A192 and V2A192 controls have
low hydrodynamic radii, both measuring only 5 nm. In contrast, both L4F nanoparticles have a
much higher average radius. The particle radius for L4F-A192 is at 49.5 nm. Similarly, L4F-
V2A192 also has a hydrodynamic radius of 41.5 nm.
88

Figure 4.3 Fusion to the L4F peptide mediates nanostructure assembly. Dynamic light scattering was
used to characterize the thermal stability of the fusion proteins (25 μM, PBS). a) The hydrodynamic
radius for L4F-A192 is greater than for A192 even below the ELP transition temperature. b) Similarly, the
hydrodynamic radius for L4F-V2A192 is greater than V2A192 even below the transition temperature.
Both c) L4F-A192 and d) L4F-V2A192 form a population of nanoparticles with a hydrodynamic radius
near 50 nm at 37 °C.

To further investigate the morphology of these peptide nanoparticles, the L4F-A192
nanoparticles were subsequently imaged using transmission electron microscopy (TEM) (Figure
4.4). These images support the polydispersity and median sizes measured by DLS. To verify that
the imaged samples were representative of the total solution and also the filtered solution
89
evaluated by DLS, both unfiltered and filtered samples were evaluated. For regular TEM
imaging, the TEM samples were negatively stained using 2 % uranyl acetate. Unfiltered L4F-
A192 yielded a radius of 41.1 +/- 16.4 nm (Fig 4.4a). Filtered through 0.2 μm pores, this radius
decreases to 27.9 +/- 11.2 nm. When imaged by cryo-TEM, unfiltered L4F-A192 nanoparticles
averaged 36.3 +/- 12.9 nm (Fig 4.4c), while filtration results in a radius of 43.3 +/- 16.8 nm.

Additionally, cryo-TEM images reveal the wall thickness of the observed vesicular
structures is consistently 8.4 ± 1.3 nm,, a value that is close to twice the size of the individual
fused proteins . Collectively, our results suggest that the L4F amphipathic peptide promotes the
assembly of the fused proteins into spherical, bilayered vesicles.

90

Figure 4.4 Nanostructures formed by L4F fusions are peptide vesicles. TEM and Cryo-TEM were
used to characterize purified L4F-A192 that was a-b) unfiltered and c-d) filtered through a 0.2 μm filter.
a,c) TEM images of negatively stained L4F-A192 revealed evidence of vesicles. Unfiltered, vesicles
averaged 41.1 ± 16.4 nm and filtered vesicles are 27.9 ± 11.2 nm. b,d) Similarly, cryo-TEM revealed the
presence of unilamellar vesicles with an average radius of 36.3 ± 12.9 nm unfiltered and 43.3 ± 16.8 nm
when filtered. Consequently, the average membrane thickness of unfiltered particles is 8.4 ± 1.3 and 0.2
um filtered particles is 6.8 ± 0.7 nm (n=18,8). The scale bar indicates 200 nm.

91
4.4.3 L4F nanoparticles prevent hepatic stellate cell activation
Having demonstrated that the L4F peptide mediates the assembly of nanoparticles
stabilized by ELP, it was unclear if the peptide would remain accessible to cellular targets. To
evaluate this possibility, we selected hepatic stellate cells as an in vitro model for assessing L4F
activity. Quiescent hepatic stellate cells have a compact morphology with cytoplasmic lipid
droplets that are lost with activation. When cultured on plastic, hepatic stellate cells
spontaneously activate and secrete extracellular matrix proteins. Murine hepatic stellate cells
(HSCs) were cultured on plastic for 3 days in the presence or absence of L4F-A192 and A192
alone. Control hepatic stellate cells show morphologic evidence of activation and stain positive
for α-smooth muscle actin, a known marker of stellate cell activation
155
(Figure 4.5a). Co-
culture with 10 μM A192 ELP does not appear to inhibit stellate cell activation (Fig 4.5b).
However, HSCs cultured in the presence of 10 μM L4F-A192 retain the compact morphology
and cytoplasmic lipid droplets characteristic for quiescence cells (Fig 4.5c). Thus, it appear that
despite the involvement of the L4F peptide in the stabilization of peptide vesicles, they retain
their intrinsic ability to act on a biological target associated with their anti-inflammatory
properties.
92

Figure 4.5 L4F-A192 nanoparticles inhibit activation of hepatic stellate cells (HSCs). Murine HSCs
were seeded on plastic for 3 days then stained for α-smooth muscle actin. a) Without treatment, HSC
seeded on plastic lose their compact morphology and cytoplasmic lipid droplets and stain positively for α-
smooth muscle actin. b) 10 μM A192 added to the media resulted in minimal inhibition of HSCs
activation c) 10 μM L4F-A192 treated HSCs exhibit characteristic compact morphology for quiescent
stellate cells.

93
4.5 Discussion
Unexpectedly, the addition of a high–molecular-weight protein polymer to the relatively
short L4F peptide leads to the stabilization of nanoparticles. As protein polymers, ELPs were
utilized due to their potential biocompatibility and biodegradability
156
. In addition, the
temperature sensitivity of ELPs has recently been exploited to create micelles for the
encapsulation of chemotherapeutics
60-61
. However, these schemes rely on a temperature triggered
self-assembly of a hydrophobic ELP block to create the internal portion of the micelle
59
. In
contrast, the L4F ELP fusion assembles nanoparticles without the need to use heat as a trigger.
The same amphipathic characteristics of L4F that make it a potent binder of oxidized lipids, also
drives the hydrophobic regions of L4F to self-assemble in the absence of lipid membranes or a
thermal stimulus. Most surprisingly, the addition of a large hydrophilic ELP tail to these
interacting L4Fs results in stable, nanoscale vesicles. DLS measurements indicate that L4F ELP
fusions form nanoparticles that are stable up to the transition temperature of the fused ELP (Fig
4.3). L4F-V2A192 retains a 41.5 nm radius up to 45
o
C, above which a larger hydrodynamic
radius is observed owing the imminent phase separation of the ELP V2A192. This is consistent
with L4F-V2A192’s increase in optical density observed on a UV-Vis spectrophotometer (Fig
4.2). Due to the greater hydrophilic nature of A192 over V2A192, the L4F-A192 fusion remains
stable at temperatures slightly higher than L4F-V2A192. L4F-A192 retains a hydrodynamic
radius of 49.5 nm up to 50
o
C, again consistent with its increased in turbidity demonstrated with
spectrophotometry.

Nanoscale vesicles composed from amphiphilic copolypeptides have previously been
reported
89,157-158
. To achieve diameters below 100 nm, these authors performed multi-step
94
extrusion to the decrease vesicle diameter from a few micrometers down to the nanoscale. We
present an alternate approach to the formation of nanoscale vesicles. The formation of ~100 nm
diameter particles from L4F ELP fusions does not require extrusion. Additionally, the majority
of literature describing peptide-derived vesicles has used chemically synthesized polymers as
precursors. In contrast, this manuscript uses biological synthesis of protein polymers to produce
vesicles that self-assemble.

The presence of L4F ELP fusion nanoparticles was confirmed using both regular TEM
and Cryo-TEM. Cryo-TEM images of L4F-A192 show peptide vesicles with a 43.3 nm radius.
This radius is consistent with the hydrodynamic radius of L4F-A192 measured using DLS.
Interestingly, Cryo-TEM images also captured what appears to be a unilamellar polypeptide
membrane. Unfiltered vesicles have a membrane thickness of 8.4 nm and filtered vesicles have a
similar membrane thickness of 6.8 nm. Greater than the thickness of a phospholipid bilayer (4-5
nm)
159
, this membrane thickness is consistent with the thickness of a bilayer composed from the
L4F peptide attached to a sterically-stabilizing ELP
160
.

Having discovered that the L4F peptide fusion results in the assembly of peptide vesicles,
we next ascertained whether or not the peptide would be sufficiently accessible to retain its
known biological activity, namely the potential to reduce inflammation resulting from oxidized
lipids
141
. In this report, nanoparticle assembly has surprisingly little effect on the potential
biological activity of L4F. To demonstrate this, we employed a well-characterized assay for the
activation of hepatic stellate cells, which HSCs are seeded on plastic and observed for changes in
cellular morphology. In vivo activated HSCs deposit of collagen involved with liver fibrosis;
95
furthermore, when plated on tissue culture polystyrene HSCs also adopt an activated
morphology. Inactivated HSCs maintain a compact morphology, characteristic to quiescent
stellate cells. However, in the absence of an inhibitor, HSCs seeded on plastic lose their compact
morphology and cytoplasmic lipid droplets and increase in α-smooth muscle actin. Similar to
past reports
141
, the addition of L4F-A192 nanoparticles to the media inhibits the activation of
HSCs. This indicates that despite the addition of a 74 kDa ELP and its assembly into 49.5 nm
radius nanoparticles, the proposed anti-fibrotic activity of L4F is retained.

Many peptides display potent biological activity, however, issues such as low molecular
weight hinder their direct use as therapeutics
161
. For example, a 22 kDa recombinant human
growth hormone is shown to be therapeutically efficacious in vitro
162
. However, by increasing
the molecular weight with the addition of a 20 kDa PEG side chain, the efficacy and half-life of
the recombinant protein increased in vivo compared to recombinant protein alone while retaining
high in vitro bioactivity. The authors proposed that addition of PEG increased the molecular
weight of human growth hormone and aided in avoiding rapid renal clearance. Herein we
proposed a similar scheme. L4F, a potent therapeutic molecule for atherosclerosis, has a
molecular weight of only 2.2 kDa, suggesting a short in vivo half-life due to high renal clearance.
The addition of a 74 kDa ELP would increase the molecular weight of L4F significantly whilst
maintaining the bioactivity seen in L4F alone. Unexpectedly, fusions to the ELP protein
polymer not only increased the hydrodynamic radius, but also lead to the assembly of
nanoparticles with a vesicular morphology. To the best of our knowledge, this is the first report
of a protein polymer fused to a biologically active peptide domain (L4F) that both assembles
vesicles and maintains the therapeutic potential of its active domain
163
.
96

Herein we present a scheme for the assembly of stable, potent nanoparticles driven by the
amphipathic nature of L4F, an apolipoprotein A1 derived peptide. We genetically engineered a
L4F peptide with two high molecular weight ELP protein polymer tails. At body temperature,
L4F-A192 self assembles into nanoparticles with a hydrodynamic radius of 49.5 nm. This self-
assembly is driven by the amphipathic nature of L4F. Using cryogenic TEM, we observed the
formation of unilamellar vesicles with a membrane thickness of 6.8 nm. Despite forming
nanoscale vesicles, L4F retained its anti-inflammatory properties as reported by a hepatic stellate
cell activation assay. Because they assemble vesicles of about 100 nm in diameter, this L4F ELP
fusion strategy has the potential to decrease the renal clearance for the peptide, serve as an
assembly platform for other amphipathic peptides, and enable co-encapsulation of soluble
therapeutics into the aqueous interior of the vesicle.

97
Chapter 5
Conclusions

5.1 Significance
At their disposal, a vast “molecular toolbox” is available to probe protein function within
a complex biologic system
164
. These molecular cell switches target desired proteins and signaling
pathway for activation or deactivation, useful processes when mimicking disease states. For
example, if protein A is hypothesized to be instrumental in cell proliferation, then selectively
removing protein A from the system and observing the downstream effects would elucidate
whether that protein is implemented in that process. Additionally, while screening a compound
for potential efficacy on a specific pathway, , cell switches are invaluable controls that can be
used to target that pathway and determine whether the drug being screened is acting in its
hypothesized manner.

This molecular toolbox gives scientists a broad array of options with which to study
protein and cellular activity with the aim of elucidating the mechanisms of disease. A number of
these tools are easily adaptable to new systems, such as siRNA, where the researcher need only
design a genetic sequence and transfect their target cells to observe the desired protein knock
down effects. There is an even larger array of tools tailored to specific systems that researchers
can re-engineer for their own applications. However, these designer systems are created for
specific applications and repurposing them can be challenging. A number of additional problems
arise while using molecular switches to target cellular processes at the transcriptional and
translational levels. The largest drawback to these systems is time. Transcriptional and
98
translational genetic switches function by increasing or decreasing the cellular production of a
protein. Not only does this take 24 to 48 hours to take effect, and bring with it a host of potential
off-target and compensatory mechanisms
127
, but it also precludes the ability to study a protein’s
activity dynamically and in real time. Additionally, these systems do not offer rapid reversibility.
Once produced by translational machinery, the protein must be degraded by natural degradation
pathways before it is removed from the system.

Molecular switches targeting endogenous proteins post-translation offer tight temporal
control not found in genetic switches targeting protein production at transcriptional and
translational levels. Novel systems using chemical inducers of dimerization have been developed
to address these temporal issues
77,165
. In short, a ligand binding domain is attached to protein of
interest. The addition of a dimerizing ligand will cause homo or hetero dimerization of the
attached protein. This induced dimerization can activate or inactivate the target pathway. The
most commonly used dimerization paradigm uses rapamycin as the dimerizing ligand and
FKBP12 and the FRB as the dimerizing domains. While this system can induce dimerization in a
matter of minutes, it still requires the addition of exogenous drugs to activate dimerization. This
addition requires cell permeable drugs as well as drugs that do not have endogenous cellular
targets, as is the case with rapamycin and the mTOR pathway
166
.

To address the need for easily adaptable and rapid methods for controlling cellular
activity, this thesis describes an ELP based scheme that can orthogonally direct protein function.
ELPs are part of a class of “smart materials” that rapidly respond to environmental changes. This
response results in a self assembled ELP molecular structures. We hypothesized that ELP
99
structures can be used to sequester a protein of interest, inhibiting it from functioning normally,
effectively working as a “knock out”. Herein ELP’s thermal responsiveness is exploited to create
thermally triggered intracellular switches. A proposed improvement on current cell switch
technologies, we proposed that a robust intracellular switch would possess the following criteria:
i. Adaptable, easy to implement to a variety of systems
ii. Rapid, avoid activation of compensatory systems
iii. Reversible, easily returned to baseline
iv. Specific, minimize off-target effects

5.2 Conclusions
To create an intracellular switch with these properties, first, ELP’s were assessed for their
ability to undergo thermally triggered self assembly in the mammalian cellular cytosol. Prior to
our intracellular ELP transition experiments, ELP self assembly had solely been characterized
outside the cell, and only limitedly been observed within E. Coli
97
. It is straightforward to
engineer ELP self-assembly at physiologic temperatures by varying the ELP’s molecular weight
and guest residue, making ELPs prime candidates for a programmable and switchable
intracellular structure. The ability to rationally design an ELP’s T
t
to occur at a given
temperature is conserved from observed T
t
s in PBS to observed T
t
s in the cytosol (Fig 2.1). This
correction allows for the easy identification of optimal ELPs for this application. Since ELPs are
genetically encodable, screening a library of ELPs for an optimal formulation that transitions at
physiologic temperatures was straightforward and identified a number of lengths and guest
residues for further study. Intracellularly, ELPs were characterized for self-assembly and
mobility. With temperature stimulation, an ELP self assembles with a half-life of 3.8 min (Fig
100
2.5 and 2.6), and de-assembles equally quickly. This intracellular ELP assembly prompted us to
coin the term genetically engineered protein microdomain, or GEPM.

To test the hypothesis that ELPs can knock-out protein function due to the self-assembly
of the CLC-ELP fusion, an ELP, V96, identified as transitioning at physiologic temperatures,
was attached to the N-terminus of the clathrin-light chain domain (Fig 3.1b). At 31
o
C, prior to
V96 driven self assembly, V96-CLC fusions retain standard CLC function and allow receptor
mediated GPCR internalization to occur. After temperature stimulated self-assembly, V96-CLC
microdomains sequester CLC and inhibit CME from internalizing ligand stimulated GPCRs.
This is a microdomain driven process because A96-CLC, a control fusion that assembles at
temperatures above those tested, remained soluble in the cytosol and did not inhibit GPCR
internalization at 37
o
C. V96-CLC knockdown of CME is shown to be as effective as known
chemical inhibitors of CME, dynasore and monodansylcadaverine. Unlike chemical inhibitors of
internalization, V96-CLC knocks out CME reversibly and can rapidly cycle from active to
inactive simply by decreasing and increasing the temperature (Fig 3.11). Also, this knock-down
of internalization is specific to CME. Studies using cholera toxin-B, a standard marker for
caveolin-mediated endocytosis, continued to internalize after V96-CLC microdomain formation
and CME knock down. Colocalization assays of fluorescently labeled antibodies specific to
components of caveolin-mediated internalization, such as caveolin-1, did not show overlap with
V96-CLC microdomains, further supporting that V96-CLC acts specifically on CME with
limited off-target effects.
The experiments herein presented describe a novel, intracellular switch capable of rapid,
reversible control over a tagged protein. This system successfully addresses the 4 criteria we
101
described as needing improvement in current intracellular switches. Since one need only tag a
protein with an ELP to confer temperature responsiveness to that protein, this system is easily
adaptable to any protein that can continue to function normally with an added tag. Target
proteins can be sequestered into ELP microdomains and effectively knocked out in a time scale
of minutes, making our system rapid. Additionally, ELP phase transitions are rapidly reversible,
allowing our system to knock out pathways and knock them back in rapid fashion, a
characteristic unique to ELP based molecular switches. Finally, our system does not knock out
universal protein function, but knocks out the specific pathway being targeted by the ELP fusion.
All four of these characteristics make temperature dependent ELP switches a unique tool for the
dynamic study of intracellular biologic functions.

This thesis also describes novel ELP assemblies outside the cytosol by characterizing the
mechanism and application of ELP vesicles using L4F-ELP fusion proteins. L4F is poised to be a
promising therapeutic for the inhibition of inflammatory diseases, however, first, it must be
formulated to avoid rapid renal clearance. Attaching a 38 kD ELP was expected to increase the
molecular weight of L4F and increase L4F’s half life in the blood. Unexpectedly, attachment of
the 38 kD ELP resulted in vesicular assemblies with a 50 nm radius. These structures formed
below the attached ELP’s T
t
, indicating the structure formed as a result of the L4F moiety. This
is consistent with the amphipathic structure of L4F. Stellate cell assays show that these L4F-ELP
vesicles retain L4F’s anti-inflammatory activity despite the proposed structure where L4F is
sitting within the lamella layer of the vesicle. Further studies are required to reveal whether the
L4F-ELP vesicle will indeed have the postulated extended half-life in vivo.
102
The emerging field of protein engineering is generating novel biomaterials in the form of
protein polymers. Recombinant technologies afford precise sequence control and composition of
materials. Elastin-like polypeptides have emerged form this field as tools with a variety of
biomedical applications. This thesis seeks to expand the body of knowledge on ELPs as well as
describe two novel applications of these polymers.

5.2 Proposed Improvements
The work presented in this dissertation has promising applications to the field of
orthogonal protein control and synthetic biology, however, two improvements have been
identified prior to the wide-spread adoption of the system. The largest hindrance in the
development of ELPs as rapid intracellular switches is the low transfection efficiency of ELP
constructs, seen in both GFP-ELP as well as ELP-CLC plasmids. Using FACS analysis of GFP-
ELP transfected cells, a correlation between ELP T
t
and transfection efficiency was found. The
lower an ELP’s T
t
, the less transfected cells were observed 48 hours post transfection. This
problem becomes particularly apparent when adapting an ELP intracellular switch from an
individual cell assay to a 96-well format whose output will be automated by a plate reader and
not an individual researcher. The amount of transfected cells expressing the desired construct is
greatly outnumbered by the amount of cells not expressing the construct, making it difficult to
distinguish between the effects of ELP intracellular switches and endogenous proteins in non-
transfected cells. Other members in our lab have successfully overcome this low ELP
transfection issue by implementing a viral vector for transduction
56
. Viral vector transduction of
ELP into cells results in more cells expressing the ELP construct. Additionally, selecting a
single, stable clone from this transduction will overcome the issue of varying levels of construct
103
expression between individual cells. Since T
t
is heavily influenced by ELP concentration, having
a homogenous cell population with uniform construct concentrations would provide tighter
control and prediction over the exact temperature of microdomain formation.

The second suggested improvement identified during the course of these studies is the
effect of endogenous, untagged proteins. We do no expect endogenous, untagged CLC to affect
the ability of V96-CLC to knockdown CME. Previous reports
77
of tagged CLC transfectants
have indicated that the amount of tagged CLC outweighs untagged endogenous CLC by a ratio
of 9:1. Since each clathrin-coated pit requires multiple CLC domains, statistically, there will not
be a clathrin-coated pit that does not have a large portion of tagged ELP-CLC present. While the
necessity of multiple CLCs to construct one clathrin-coated pit works in this system’s favor,
attaching an ELP to another protein that does not require the interaction of multiple proteins
might yield a high background from untagged proteins that are still able to function normally. To
overcome this, siRNA knockdown of the endogenous, untagged protein may be required for
complete knock-down in non-CME systems.

Figure 5.1 ELP transfection
efficiency is dependent on
transition temperature of ELP.
ELP libraries with lower transition
temperatures (ie Valine, red) have
a low rate of expression in
mammalian Hek293 cells than
ELP libraries with comparatively
higher transition temperatures (ie
Alanine, blue). Slope 0.07 ± 0.02,
Y-intercept -0.65 ± 0.78, R
2
= 0.44.

104
5.4 Future Applications
The development of novel tools for the orthogonal control of intracellular proteins grant
biologists the ability to pick and choose which molecular biology assay would most suit their
application needs. To this end, the mechanism of the system herein proposed could be extended
beyond simply switching proteins OFF by sequestering them in ELP microdomains, but also
switching protein activity ON by inducing dimerization due to ELP assembly. For example, a
protein that requires dimerization to be activated, could be attached to an ELP. Once the ELP has
been thermally stimulated to assemble, it would bring the attached protein into close proximity to
other ELP tagged proteins, effectively stimulating dimerization and subsequent activation. The
EGF receptor has been identified as a promising candidate for a future ELP-inducted activation
pilot study. In the absence of ligand, EGFR is in monomeric form at the plasma membrane.
Binding of an EGF ligand to the EGF receptor induces EGFR dimerization and internalization. It
is hypothesis that ELP induced aggregation of EGF receptor could induce EGFR dimerization
and internalization in a ligand independent manner.
The molecular toolbox has greatly expanded within the past decade; however it still
requires future development to achieve its full potential. To fully harness protein activity, there
must be an increase in the complexity of system design. For example, the coordinated control of
multiple pathways within a single cell would allow for the study of model systems that more
closely mimics in vivo states. This would require multiple molecular switches to be implemented
within a single cell. The work presented herein seeks to add to the growing number of molecular
tools available for these purposes will the ultimate goal of a fully controllable biologic system.

105
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Asset Metadata

Creator Pastuszka, Martha K. (author)

Core Title Flipping the switch on protein activity activity: elastin-like polypeptides assemble into cell switches and vesicles

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology and Toxicology

Publication Date 07/15/2014

Defense Date 05/22/2014

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

Tag bioengineering,cell biology,clathrin,confocal microscopy,elastin‐like polypeptide,fusion protein,nanoparticles,OAI-PMH Harvest,polymers,receptor mediated internalization

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Mackay, John Andrew (committee chair), Hamm-Alvarez, Sarah F. (committee member), Okamoto, Curtis Toshio (committee member)

Creator Email martha.past@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-439984

Unique identifier UC11287020

Identifier etd-PastuszkaM-2681.pdf (filename),usctheses-c3-439984 (legacy record id)

Legacy Identifier etd-PastuszkaM-2681.pdf

Dmrecord 439984

Document Type Dissertation

Format application/pdf (imt)

Rights Pastuszka, Martha K.

Type texts

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...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA