Toward Photo-control of Peptide Structure in Vivo

by

Lei Chi

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Graduate Department of Chemistry University of Toronto

© Copyright by Lei Chi 2010

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Toward Photo-control of Peptide Structure in Vivo

Lei Chi

Doctor of Philosophy

Department of Chemistry University of Toronto

2010

Abstract

An ability to manipulate the activity of a specific protein inside living cells offers

exciting prospects for the study of protein function in vivo. Azobenzene derivatives

introduced as intramolecular bridges have been demonstrated to reversibly

photoregulate secondary structures and functions of peptides and proteins in vitro. My

overall goal is to create a generally-applicable process for the reversible photocontrol of

protein-protein interactions within the complex environment of a living cell. Results of

studies toward this aim are presented.

A blue-green absorbing (~480 nm) azobenzene derivative cross-linker was

designed that reversibly controlled the helical content of attached peptides with a half-

life of the cis state of ~50 ms. This rapid photoswitch may prove useful as a tool for

probing dynamic processes in biochemical systems using light.

The effect of cross-linker position (N-terminus, middle, C-terminus) on a photo-

switchable 32-residue helical peptide was studied. Although the activation energies for

thermal cis – trans relaxations were not the same, linker position did not affect the

change in helix content. This work provides useful information for the effective

photoregulation of much longer helices such as occur in coiled-coils.

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Fluorescently labeled, cross-linked, modified Fos/Jun peptides with and without

cell-penetrating peptide (CPP) tags were prepared for the purpose of photocontrolling

peptide-peptide interactions in vivo. One of the peptides showed a degree of

photocontrol of helicity. Cell uptake of CPP-tagged peptides was demonstrated.

However, overall peptide behavior was dominated by undesired aggregation.

A simple reporter, a cross-linked peptide bearing an environmentally sensitive

fluorophore at a key site, was designed for detecting photoswitching in vivo.

Photoisomerization of the cross-linker caused changes in the local chemical

environment and changes in fluorescence intensity of the environmentally sensitive

dyes in vitro. However, no change in fluorescence was observed in the living systems

we investigated.

Conclusions and suggestions for further work aimed at achieving the overall goal

stated above are discussed.

.

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Acknowledgments

First and foremost I would like to thank my supervisor, Professor Andrew Woolley.

Drew, thank you for guiding me and encouraging me over the years with your patience

and knowledge. Thank you for always being there to support me when my project did

not go well, and thank you for the time you spent editing my thesis. Without you this

degree would have been impossible for me. I don’t think I could have ever found a

better graduate supervisor. I would also like to offer my sincere gratitude to Anna for

always being so kind and warm. Without you the life in lab wouldn’t have been half as

fun.

I owe my deep gratitude to Professor Deborah Zamble and Professor Mark Nitz.

Thanks for suffering through my committee meetings and inspiring me with many

valuable advices over the years. Thanks for correcting my thesis carefully while you

were engaged in many more important things. Without you this thesis would not have

been possible.

I also want to acknowledge the past and present members of the Woolley lab,

Jack, Oleg, Darcy, Tyler, Stacy, Fuzhong, Andrew, Joe, Vitali and Lila, who have helped

me along the way and made my graduate life enjoyable. Jack, thanks for always being

ready to help and caring about me. Oleg, thanks for synthesizing the cross-linkers for

me, without you my project would have been at least twice as difficult. Darcy and Tyler,

thanks for leading me through the experiments, and always being patient. Stacy, thanks

for being so kind and giving me valuable suggestions on thesis writing and defense, and

thanks for your presents and birthday cakes over the years. They made me feel at home

in a foreign country. Fuzhong and Andrew, thanks for your valuable suggestions on my

project. Joe, thanks for helping me with the Fos/Jun project, you were the best

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undergrad student I’ve ever seen. Vitali and Lila, thanks for your support and friendship.

Furthermore, I would also like to thank the members of the Zamble and Nitz lab

(especially Yanjie, Shiela Joanna and Harini) for helping me with lab instruments as well

as their friendship. To my friends outside the confines of the University walls, you know

who you are but you probably don’t know the positive impact you had towards the

completion of this degree – thank you.

No project could be done without the assistance of collaborators. I would like to

acknowledge the support of the great researchers I have worked with. Melissa Chang

and Professor Jean Gariépy, thanks for your help with CHO cell culture. Locksum Wong

and Professor Vincent Tropepe, thanks for your help with Zebrafish embryo

microinjection. Erich Damm and Professor Rudolf Winklbauer, thanks for our help with

Xenopus embryo microinjection. I’d also like to thank the late Professor Jerry Kresge for

the use of his flash photolysis apparatus.

Thanks to the Department of Chemistry and University of Toronto, who have

provided the financial support I have needed to produce and complete my thesis.

Thanks to everybody who was important to the successful realization of thesis, as well

as expressing my apology that I could not mention personally one by one.

Finally, I would like to show my gratitude to my parents and my husband Kun.

Mom and Dad, thanks for your love and support through out my life. You have been

proud of me and I hope you still will be. Kun, no word can express my appreciation to

your dedication, love and persistent confidence in me. You have been nothing but

supportive through out my bad days. I could not have endured the past years without

you.

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Table of Contents Acknowledgments........................................................................................................... iv

Table of Contents............................................................................................................ vi

List of Abbreviations........................................................................................................ xi

List of Tables................................................................................................................. xiii

List of Figures ............................................................................................................... xiv

List of Schemes ........................................................................................................... xxii

List of Appendices........................................................................................................xxiii

Chapter 1 Introduction ..............................................................................................1

1.1 The α-helix ............................................................................................................1

1.2 Controlling the α-helical Conformation ..................................................................2

1.2.1 Stabilizing α-helical Peptides with Covalent Bonds between Side-chains .........................................................................................................3

1.2.2 Control of α-helical Peptides with Non-Covalent Interactions between Side-chains.................................................................................................5

1.3 Reversible Control of α-helical Peptides with Light ...............................................7

1.3.1 Spiropyran and its Application to the Control of α-helices ..........................7

1.3.2 Azobenzene .............................................................................................11

1.3.3 Using Azobenzene to Control α-helices....................................................12

1.4 Applications of Photocontrol of α-helices Using Azobenzene .............................17

1.4.1 Photocontrol of Coiled-coil Interactions ....................................................17

1.4.2 Photocontrol of DNA Binding by Azobenzene Cross-linked α-helical Peptides ...................................................................................................19

1.5 Development of Azobenzene Cross-linkers ........................................................20

1.6 References..........................................................................................................22

Chapter 2 A Blue-Green Absorbing Cross-linker for Rapid Photo-switching of Peptide Helix Content .........................................................................................26

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2.1 Abstract ...............................................................................................................26

2.2 Introduction .........................................................................................................27

2.3 Experimental Procedures ....................................................................................29

2.3.1 Synthesis of the Cross-linker ....................................................................29

2.3.2 Peptide Cross-linking................................................................................32

2.3.3 UV/Vis Spectra and Photoisomerization...................................................33

2.3.4 Circular Dichroism Measurements............................................................34

2.3.5 Molecular Modeling ..................................................................................35

2.4 Results and Discussion.......................................................................................36

2.4.1 Design and synthesis of the cross-linker ..................................................36

2.4.2 UV-Vis spectra: pH and solvent dependence ...........................................38

2.4.3 Kinetics of Isomerization...........................................................................42

2.4.4 Conformational analysis of the cross-linker and effects of photoisomerization ...................................................................................43

2.5 Summary.............................................................................................................47

2.6 Acknowledgements .............................................................................................47

2.7 References..........................................................................................................48

Chapter 3 Effect of Cross-linker Position on Photocontrol of Helix Content....50

3.1 Introduction .........................................................................................................50

3.1.1 Intrinsic Helix Propensity ..........................................................................50

3.1.1.1 Helix-coil Transition .....................................................................51

3.1.1.2 Helix Capping ..............................................................................51

3.1.1.3 Helix Propensity Prediction..........................................................53

3.1.1.4 Effects of C vs. N-terminal Sequence Changes...........................53

3.2 Materials and Methods........................................................................................54

3.2.1 Peptide Synthesis.....................................................................................54

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3.2.2 Peptide Cross-Linking and Purification.....................................................55

3.2.3 Circular Dichroism Measurements............................................................56

3.2.4 UV/Vis Analysis and Photo-isomerization.................................................57

3.3 Results ................................................................................................................57

3.3.1 Design and Synthesis of FK22A, FK22B, FK22C.....................................57

3.3.2 UV/Vis Analysis of Cross-linked FK22 Peptides.......................................60

3.3.3 Thermal Relaxation of Cross-linked FK22 Peptides .................................62

3.3.4 Circular Dichroism Analysis ......................................................................64

3.3.5 Circular Dichroism Melting Curve – Comparison of Stabilities..................66

3.4 Discussion...........................................................................................................67

3.5 References..........................................................................................................72

Chapter 4 Attempted Photo-control of the Fos/Jun Heterodimer.......................75

4.1 Introduction .........................................................................................................75

4.1.1 The Coiled-coil Motif.................................................................................76

4.1.2 Fos and Jun..............................................................................................79

4.1.3 FRET ........................................................................................................80

4.1.4 Using Fluorescein-Rhodamine as a FRET pair to Analyze Jun-Fos Dimerization and DNA Binding .................................................................82

4.1.5 Cell Penetrating Peptides .........................................................................84

4.1.6 CHO Cells ................................................................................................86

4.2 Materials and Methods........................................................................................87

4.2.1 Peptide Synthesis and Labeling ...............................................................87

4.2.2 Peptide Cross-Linking, Cysteine Capping and Purification.......................89

4.2.3 UV/Vis Analysis ........................................................................................91

4.2.4 Circular Dichroism Analysis ......................................................................92

4.2.5 Fluorescence Measurements ...................................................................92

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4.2.6 Cell Uptake Experiments and Fluorescence Imaging ...............................93

4.3 Results ................................................................................................................94

4.3.1 Peptide Design .........................................................................................94

4.3.2 Peptide Synthesis, Labeling and Cross-linking.........................................98

4.3.3 Cell Uptake Experiments ..........................................................................99

4.3.4 UV/Vis Analysis and Isomerization Measurements ................................101

4.3.5 CD Analysis............................................................................................104

4.3.6 Fluorescence Measurements .................................................................106

4.4 Discussion.........................................................................................................110

4.5 References........................................................................................................113

Chapter 5 Attempted Fluorescence Imaging of Azobenzene Photo-switching In Vivo ...................................................................................................118

5.1 Introduction .......................................................................................................118

5.1.1 Chemical Environment Change at Val, Aib of the JRK Peptide..............118

5.1.2 Environmentally Sensitive Dyes .............................................................119

5.1.3 Microinjection..........................................................................................121

5.2 Materials and Methods......................................................................................123

5.2.1 Peptide Synthesis and Purification .........................................................123

5.2.2 Peptide Cross-Linking and Purification...................................................124

5.2.3 Fluorescent Dye Labelling of Cross-Linked Peptide ...............................124

5.2.4 UV/Vis Analysis and Photo-isomerization...............................................129

5.2.5 Circular Dichroism Measurement ...........................................................130

5.2.6 Fluorescence Measurements .................................................................131

5.2.7 Fluorescence Imaging and In Vivo Experiments.....................................132

5.2.8 Microinjection..........................................................................................133

5.3 Results ..............................................................................................................134

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5.3.1 Design and Synthesis of Fluorescent Dye Labelled Cross-Linked Peptides .................................................................................................134

5.3.2 UV Spectra of Fluorescently Labeled Cross-linked Peptides .................136

5.3.3 Fluorescence Intensity Changes Upon irradiation ..................................138

5.3.4 Isomerization Study of Fluorescein Labeled Peptide 2f..........................141

5.3.5 CD of Peptides 2a and 2f .......................................................................142

5.3.6 Fluorescence Imaging of Peptide 2f in Microinjected Zebrafish and Xenopus embryos...................................................................................144

5.3.7 Fluorescence Study of Peptide 2f in Xenopus embryo Cell Lysate ........146

5.4 Discussion.........................................................................................................149

5.5 References........................................................................................................150

Chapter 6 Conclusion and Future Directions..........................................................153

Appendices ..................................................................................................................157

Copyright Acknowledgements......................................................................................172

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List of Abbreviations

Acm Acetamidomethyl

ACN Acetonitrile

AP-1 Activator protein-1

Ala Alanine

ATP Adenosine triphosphate

bZIP Basic-leucine zipper

Coumarin 3-(4-maleimidophenyl)-7-(diethylamino-4-methyl) coumarin

Cys Cysteine

CD Circular dichroism

DIPEA Diisopropyl ethylamine

DMF Dimethylformamide

DMSO Dimethylsulfoxide

DTT Dithiothreitol

EDTA Ethylenediaminetetraacetic acid

ESI-MS Electrospray ionization mass spectrometry

FRET Fluorescence resonance energy transfer

Gly Glycine

GSH Glutathione

HPLC High perfromace liquid chromatography

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Ile Isoleucine

LED Light-emitting diode

Leu Leucine

MALDI-MS Matrix assisted laser-desoprtion ionization mass spectrometry

MS Mass spectrometry

MTS Methane thiosulfonate

NHS N-hydroxy sucinimide

NMR Nuclear magnetic resonance

SbTMU O-(N-succinimidyl)-N,N,N’,N’-bis-(tetramethylene)-uronium

hexafluorophosphate

SPPS Solid phase peptide synthesis

TFA Trifluoroacetic acid

TFE Trifluoroethanol

THF Tetrahydrofuran

TLC Thin layer chromatography

Tris Tris(hydroxymethyl)aminomethane

Trp Tryptophan

Tyr Tyrosine

UV Ultraviolet

Val Valine

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List of Tables

Table 2.1 Isomerization data of FK-11, JRK-7, and azobenzene glutathione derivative42

Table 3.1 Percentage of cis-form at photostationary state under 370 nm irradiation .....61

Table 3.2 Activation energy and half-life of cross-linked peptides in different buffers ....62

Table 3.3 Helix contents of sx-FK22 peptides in different states at 20 oC......................66

Table 3.4 Helix content of FK22 and dark-adapted sx-FK22 peptides ...........................67

Table 4.1 Cell-penetrating peptide (CPP) sequences. ...................................................85

Table 5.1 Conditions for fluorescence spectra and time scans....................................131

Table 5.2 Photo-switching dependent fluorescence parameters .................................141

Table 5.3 Light induced cis-to-trans isomerization study of 2f samples in different buffers.....................................................................................................................................148

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List of Figures

Figure 1.1 Structure of an α-helix.....................................................................................2

Figure 1.2 A. Structure of modified PTHrP(7-34)NH2 with a lactam bridge at i, i + 4. B.

Structure of a hexapeptide containing two lactam bridges...............................................4

Figure 1.3 A. i, i + 7 disulfide bond formation in a short peptide. B. Modified disulfide

bridged N-terminal fragment of PTHrP.............................................................................5

Figure 1.4 Photoisomerization of spiropyran compounds in aqueous solution (R = Alkyl

groups).............................................................................................................................7

Figure 1.5 Schematic illustration of the coil/α-helix transition occurring for spiropyran-

modified poly (L-glutamic acid) in HFP. (Adapted with permission from reference [30].

Copyright 2001 American Chemical Society.)..................................................................9

Figure 1.6 Synthetic route for the spiropyran cross-linker..............................................10

Figure 1.7 Schematic representation for the reversible photoregulation of helical

structures on the cross-linked peptides. (Reproduced with permission from reference

[31]. Copyright 2006 American Chemical Society.)........................................................10

Figure 1.8 Structural changes of azobenzene ...............................................................12

Figure 1.9 Chemical structure and photoisomerization of the azobenzene cross-linker

developed by Kumita et al. (Reproduced with permission from reference [47]. Copyright

2000 National Academy of Sciences.) ...........................................................................14

Figure 1.10 (A) JRK peptide (acetyl-EACARVAibAACEAAARQ-amide) cross-linked

between Cys residues spaced i, i + 7. (B) CD spectra of trans (s) and cis (- - -) JRK-X.

(Reproduced with permission from reference [52]. Copyright 2005 American Chemical

Society.) .........................................................................................................................14

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Figure 1.11 Histogram showing the distribution of Cys-Cys (S-S) distances (0.2 Å

intervals) expected for the noncross-linked JRK peptide under the conditions of the CD

experiment [overall helix content, 25% (-)]. The dotted line shows the expected

distribution for noncross-linked JRK, where the overall helix content is 5% (e.g., at high

temperature). The total number of structures was ~100 000 in each case. Green line

(the distance range allowed by a cis cross-lnker); blue line (the distance range allowed

by a trans cross-linker); orange line (the distance range allowed by a linker in the

inversion transition-state structure); red line (the distance range allowed by a rotational

transition state). Some representative linker structures are shown above. (Reproduced

with permission from reference []. Copyright 2005 American Chemical Society.) ..........16

Figure 1.12 Schematic diagram of photo-controlled DNA binding by a bZIP peptide

dimer. (Reproduced with permission from reference [53]. Copyright 2000 Wiley

InterScience.).................................................................................................................17

Figure 1.13 Models showing the photocontrol of DNA binding by AZO(i, i + 7) GCN4-

bZIP. In the trans conformation, the cross-linker induces a bend in the helix, which can

be expected to destabilize zipper formation and consequently DNA binding (left). In

contrast, the cis conformation of the cross-linker is compatible with zipper formation and

thus DNA binding (right). (Reproduced with permission from reference [55]. Copyright

2006 American Chemical Society.) ................................................................................18

Figure 1.14 Azobenzene derivative cross-linkers with methanethiosulfonate (MTS)

reaction groups. (Reproduced with permission from reference [52]. Copyright 2005

American Chemical Society.) .........................................................................................21

Figure 2.1 Primary sequences of the cross-linked peptides examined in this study: (a)

FK-11 (azo-linker 1 reacted via cysteine residues spaced i, i + 11); (b) JRK-7 (azo-linker

1 reacted via cysteine residues spaced i, i + 7); (c) cross-linked glutathione.................37

Figure 2.2 UV-Vis spectra of the cross-linked FK-11 peptide (~30 µM) in aqueous buffer

at series of pH values. ...................................................................................................38

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Figure 2.3 UV-Vis spectra of the cross-linked FK-11 peptide (~70 µM) in aqueous buffer

pH 7 with added methanol (A) or DMSO (B). Spectra for the glutathione-linked azo

compound (~35 µM) in aqueous buffer pH 7 with added methanol (C) or DMSO (D)....40

Figure 2.4 UV-Vis spectra of the cross-linked JRK-7 peptide (~50 µM) in aqueous buffer

pH 7 with added methanol (A) or DMSO (B). .................................................................41

Figure 2.5 (A) Trans and cis forms of the cross-linker structure used for molecular

dynamics simulations. Bonds labelled a-d were constrained as described in the methods

section (B). Probability distributions of distances between S atoms (circled in (A))

derived from the molecular dynamics simulations (cis (- - -), trans (___); distances

between Cys S atoms in ideal α-helices spaced i, i+7 and i,i+11 in the primary sequence

are indicated; (C) Some representative structures and S-S distances observed during

the simulations. Cis forms show a wider range of S-S distances. ..................................44

Figure 2.6 Molecular models of trans cross-linked FK-11 (A) and cis cross-linked JRK-7

(B) in a helical conformation...........................................................................................45

Figure 2.7 Time-dependent circular dichroism (CD) signal at 225 nm of (A) cross-linked

FK-11 (~25 µM, 1 cm cell) and (B) cross-linked JRK-7 (~100 µM, 0.1 cm cell) observed

after conversion of a percentage of each peptide to the cis isomer. In (A), relaxation

from cis-to-trans is associated with an increase in peptide helix content. In (B),

relaxation from cis-to-trans is associated with an decrease in peptide helix content.

Solvent conditions: 75% methanol in 10 mM sodium phosphate buffer, pH 7.0, 10 oC..46

Figure 3.1 Cross-linked FK22 peptides models and sequences (α-helix conformation of

each peptide was built with HyperChem.)......................................................................59

Figure 3.2 UV Spectra of Cross-linked FK22 Peptides. .................................................61

Figure 3.3 Arrhenius plots of cis-trans thermal relaxation of cross-linked peptides. A)

sx-FK22A, B) sx-FK22B, C) sx-FK22C, in 10 mM pH 7.0 sodium phosphate buffer; D)

sx-FK22A, E) sx-FK22B, F) sx-FK22C, in 6 M pH 7 urea buffer ....................................63

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Figure 3.4 CD spectra of FK22 peptides. A) Dark adapted sx-FK22A (85 μM), sx-

FK22B (90 μM), and sx-FK22C (80 μM); B) sx-FK22A, C) sx-FK22B, D) sx-FK22C in

dark-adapted, irradiated states, and calculated 100% cis state. Samples were measured

at 20 oC in 10 mM pH 7.0 sodium phosphate buffer. .....................................................65

Figure 3.5 Thermal melting curve of FK22 and sx-FK22 peptides .................................67

Figure 3.6 A) N-terminus and B) C-terminus backbone hydrogen bonds showing the

local environment. At the N-terminus, the first hydrogen bond is formed between the

C=O of the capping group and the N-H of the 4th residue of an α-helix. The C-terminus

forms the last hydrogen bond between the C=O of the 13th residue and the NH2

capping group (The sample peptide has 16 residues in total.) (Adapted with permission

from reference [22]. Copyright 1993 American Chemical Society.)................................69

Figure 4.1 Model of a fluorophore-labeled, cross-linked Fos/Jun heterodimer undergoing

FRET. ............................................................................................................................76

Figure 4.2 Helical wheel diagram of a parallel leucine zipper composed of helix A and B

(looking down the helix axis from the N- to the C-terminus). Heptad positions on helix A

are labeled a to g. Heptad positions on helix B are labeled a’ to g’................................77

Figure 4.3 Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun

bound to DNA (Reproduced from protein data bank, PDB ID: 1FOS)............................80

Figure 4.4 Fluorescently labeled Fos-Jun-AP-1 complex. Coordinates for Fos, Jun, and

DNA were derived from the crystal structure of the complex, with additional amino acids

modeled in alpha-helical conformation. The C-terminal cysteines were further modified

with acetamidofluorescein and acetamido-tetramethylrhodamine. Fluorophores are

shown oriented away from the leucine zipper; however, the structure is not minimized;

this depiction represents one of many possible conformations that the complex may

assume. Fos is shown as a purple ribbon with its appended cysteine in purple CPK

representation. The rhodamine label is shown in pink CPK representation. Jun is shown

as a blue ribbon with its appended cysteine in blue CPK representation. The fluorescein

label is shown in yellow CPK representation. DNA is shown in gray. (Reproduced with

permission from reference [30]. Copyright 2001 American Chemical society.) ..............84

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Figure 4.5 Sequences of synthesized peptides. , resin ............................................87

Figure 4.6 Schematic representation of desired and competing complexes for the Fos-

Jun coiled coil. The helical wheel diagram looks down the axis of the α-helices from N-

to C-terminus, and illustrates the attractive and repulsive forces which prevent and

maintain specificity in the cases of JunW and JunWCANDI, respectively. Black represents

residues found in all peptides; red represents library options; residues circled in green

represent residues selected in JunW, JunWCANDI; residues circled in magenta represent

selections that are unique to JunWCANDI. (Reprodued with permission from reference

[57]. Copyright 2007 American Chemical Society.)........................................................96

Figure 4.7 Molecular models of cross-linked JunWCANDI (blue) and Fos (red) in A) cis

form, peptides adopt helical conformations and form a leucine zipper; B) trans form,

helix is destabilized, peptides become random coil and can not form a leucine zipper.

These models are hypothetical. .....................................................................................97

Figure 4.8 Fluorescence microscope images of CHO cells incubated with A) FL-CPP-

JunWCANDI, B) FL-JunWCANDI with FITC (480 nm excitation) filtersets; and C) Rhd-CPP-

Fos, D) Rhd-Fos with Cy3 (545 nm excitation filterset)................................................100

Figure 4.9 UV Spectra of fresh FL-JunWCANDI, FL-CPP-JunWCANDI (A) and fresh Rhd-

Fos, Rhd-CPP-Fos (B). Normalized old Rhd-Fos samples before and after reduction

with TCEP, as well as freshly prepared Rhd-Fos (C). Normalized freshly prepared Rhd-

Fos, old Rhd-Fos sample reduced with TCEP for 4 days and iodoacetamide capped

Rhd-Fos (IA-Rhd-Fos) (D). All samples above were measured in 10 mM, pH 7.0

phosphate buffer. Extinction coefficients for TAMRA bound to homodimeric Escherichia

coli ribosomal protein L7/L12 (E). Dimer a and monomer b were measured in the

standard buffer 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 100 mM KCl, and 3 mM

dithiothreitol. (Reprodued with permission from reference []. Copyright 1996 American

Chemical Society). .......................................................................................................103

Figure 4.10 left, dark adapted and irradiated sx-FL-CPP-JunWCANDI (5 min of irradiation,

sample displayed no change after 5 min); right, sx-Rhd-Fos irradiated for 5, 10 and 15

xix

min. All samples were irradiated with a high intensity 365 nm UV LED described in

4.2.3, and measured in 10 mM, pH 7.0 phosphate buffer. ...........................................104

Figure 4.11 CD Spectra of uncross-linked 48.6 μM FL-JunWCANDI and 42.6 μM Rhd-Fos

in pH 7.0 sodium phosphate buffer at 25 oC; ...............................................................105

Figure 4.12 CD spectra of 72 μM dark-adapted sx-FL-JunWCANDI (solid line) and

irradiated sx-FL-JunWCANDI (dashed line). Conditions 100 mM NaCl, 50 mM sodium

phosphate buffer, pH 7.0, 20 oC; irradiation used 370 nm light for 5 min, ~20 mW

source. .........................................................................................................................106

Figure 4.13 Fluorescence emission scans of (A) 0.63 μM fresh FL-JunWCANDI titrated

with Rhd-Fos up to 6 eq.. (B) 2.5 μM TCEP reduced 0.63 μM FL-JunWCANDI titrated with

Rhd-Fos up to 6 eq.. (C) 2.14 μM IA-FL-JunWCANDI titrated with IA-Rhd-Fos up to 4 eq.

Ex = 495 nm. Black lines represent pure peptides prior to titration. Arrows indicate the

trend of spectral change upon addition of Rhd-Fos. Intensity changes due to dilution

have been corrected.. Emission of due to direct excitation of rhodamine at 495 nm was

subtracted from all the spectra. Buffer conditions: 100 mM NaCl, 50 mM sodium

phosphate, pH7.0.........................................................................................................108

Figure 4.14 Fluorescence emission spectra of iodoacetamide capped JunWCANDI-Fos

pairs. (A) IA-FL-CPP-JunWCANDI – IA-Rhd-Fos (red solid line) and IA-FL-JunWCANDI – IA-

Rhd-CPP-Fos (blue line). (B) IA-FL-CPP-JunWCANDI – IA-Rhd-Fos measured after

various period of time. Concentration: 0.5 μM each, 1:1 ratio at 20 oC. Buffer condition:

100 mM NaCl 50 mM sodium phosphate pH 7.0 .........................................................110

Figure 5.1 Chemical structures of environmentally sensitive dyes...............................119

Figure 5.2 Developmental stages of A) the Zebrafish embryo; B) the Xenopus embryo

.....................................................................................................................................122

Figure 5.3 Structures of the fluorescent labelled photo-switchable peptides ...............125

Figure 5.4 Schematic diagram of fluorescence excitation beam causing designed photo-

switch with fluorescent reporter to isomerize from cis to trans. This process is

xx

associated with an emission fluorescence intensity change of the environmentally

sensitive fluorophore....................................................................................................135

Figure 5.5 UV spectra of dark-adapted and irradiated peptides 2a-2f and IA-JD-FL. ..137

Figure 5.6 Fluorescence emission spectra of dark-adapted peptide 2a-2e and IA-JD-FL, and time scans after irradiation. ...................................................................................139

Figure 5.7 The absorbance and fluorescence response of peptide 2f upon relaxation from cis to trans (indicated by the arrow). Increasing absorbance at 370 nm due to cis-

to-trans isomerization of the photo-switch (left, thermal) is accompanied by increasing

fluorescence at 520 nm (right, induced by 450 nm excitation beam). ..........................140

Figure 5.8 Time scans of irradiated peptide 2f with fixed slit width, different excitation wavelengths (left) and fixed excitation, emission wavelengths and different excitation slit

widths (right). ...............................................................................................................142

Figure 5.9 CD Spectra of dark-adapted and irradiated peptide 2a and 2f at 20 oC in 10 mM pH 7.0 sodium phosphate buffer ...........................................................................142

Figure 5.10 Fluorescence imaging results of peptide 2f in 10 mM, pH 7.0 sodium phosphate buffer. One of the 2f samples was irradiated with 365 nm LED prior to imaging to isomerize trans to cis. Imaging beam at fluorescein excitation wavelength

triggers cis to trans isomerization, associated with fluorescein emission intensity

change. Left: time scans of both irradiated and non-irradiated (prior to imaging) samples

without neutral density filters, imaging lamp was turned on after the 9th data point; Right:

cis-to-trans isomerization constant K vs. standardized final fluorescence intensity

imaged with beams through different neutral density filters (to alter the intensity of

excitation beam)...........................................................................................................144

Figure 5.11 Fluorescence imaging of Zebrafish embryos injected with peptide 2f at different stages. A. 1-2 h, imaging lamp was turned on at 1.3 second; B. 4 h, imaging

lamp was turned on after the 4th data point ..................................................................145

xxi

Figure 5.12 Fluorescence imaging of Xenopus embryos injected with peptide 2f and imaged at the 4-cell stage, imaging lamp was turned on at 10 second........................146

Figure 5.13 Fluorescence study of peptide 2f in Xenopus embryo cell lysate. Left: emission scans of 2f samples of equal concentrations in different buffers; Right: time scans of irradiated 2f samples in different buffers........................................................147

Figure 5.14 Stability study of peptide 2f in Xenopus embryo lysate without yolk proteins.....................................................................................................................................148

xxii

List of Schemes

Scheme 2.1....................................................................................................................37

Scheme 2.2....................................................................................................................39

xxiii

List of Appendices Appendix 1: NMR Spectrum of Methanethiosulfonic Acid S-{2-[Ethyl-(4-{4-[ethyl-(2-

methanesulfonylsulfanyl-ethyl)-amino]-phenylazo}-phenyl)-amino]-ethyl} Ester (blue-

green absorbing cross-linker).......................................................................................157

Appendix 2: MS of of Methanethiosulfonic Acid S-{2-[Ethyl-(4-{4-[ethyl-(2-

methanesulfonylsulfanyl-ethyl)-amino]-phenylazo}-phenyl)-amino]-ethyl} Ester (blue-

green absorbing cross-linker).......................................................................................158

Appendix 3: MS of cross-linked FK-11 peptide ............................................................159

Appendix 4: MS of cross-linked JRK-7 peptide............................................................160

Appendix 5: MS of glutathione-linked azo compound ..................................................161

Appendix 6: MS of FK22A............................................................................................162

Appendix 7: MS of FK22B............................................................................................163

Appendix 8: Ms of Rhd-Fos .........................................................................................163

Appendix 9: MS of Rhd-CPP-Fos ................................................................................164

Appendix 10: MS of FL-JunWCANDI...............................................................................164

Appendix 11: MS of IA-Rhd-Fos ..................................................................................165

Appendix 12: MS of IA-Rhd-CPP-Fos..........................................................................165

Appendix 13: MS of IA-FL-JunWCANDI ..........................................................................166

Appendix 14: MS of IA-FL-CPP-JunWCANDI..................................................................166

Appendix 15: MS of sx-Rhd-Fos ..................................................................................167

Appendix 16: MS of JRK-Dpr .......................................................................................167

Appendix 17: MS of peptide 2a ....................................................................................168

xxiv

Appendix 18: MS of peptide 2b....................................................................................168

Appendix 19: MS of peptide 2c ....................................................................................169

Appendix 20: MS of 2d ................................................................................................169

Appendix 21: MS of peptide 2e ....................................................................................170

Appendix 22: MS of peptide 2f.....................................................................................170

Appendix 23: MS of peptide IA-JD-FL..........................................................................171

1

Chapter 1 Introduction

1.1 The α-helix Proteins are built from regular local folds of the polypeptide chain called the

secondary structure. The α-helix is the most abundant secondary structure. Around 30%

of amino acid residues are found in α-helices1. The α-helix structure was first described

by Pauling and co-workers in 19512,3 to have 3.7 residues per turn with a pitch of 5.5 Å.

This structure was then quickly supported by an X-ray study of hemoglobin4. In 1966, a

refined crystal structure of an α-helix was reported5, and this has become the standard

definition of an α-helix: ~3.6 residues per turn with torsion angles of Φ = -57o and Ψ = -

47o, and a pitch of 5.5 Å (Figure 1.1). Side chains spaced i, i + 4, i + 7, i + 11, i + 14, i +

21 are therefore on the same side of an α-helix.

The core of an α-helix is tightly packed, minimizing the exposure of the

hydrophobic regions to water5. The peptide C=O bond of the nth residue points along

the helix towards the N-H group of the (n+4)th residue5. This results in a strong

hydrogen bond that stabilizes the α-helix structure. Furthermore, the dipole moments of

the amide bonds all point in the same direction (towards the C-terminus of the α-helix),

giving it a significant dipole moment that is positive towards the N-terminus and

negative towards the C-terminus. The initial few residues at the N-terminus and the final

few residues at the C-terminus are unique because their amide groups have unsatisfied

H-bonding donors or acceptors. The nature of these groups can have significant effects

on the helix structure and stability 6.

2

Figure 1.1 Structure of an α-helix.

1.2 Controlling the α-helical Conformation As the most prevalent secondary structure element observed in proteins, the α-

helix is essential for the folding and biological functions of proteins7,8,9. Control of the

stability of helices is therefore of particular interest to researchers. Numerous

approaches have been reported for the control of the α-helicity of peptides including the

formation of disulfide or amide bonds, introduction of ion pairs and metal chelates, and

light irradiation with an installed azobenzene or spiropyran cross-linker.

N-terminus

C-terminus

3.6 residues/turn

3

1.2.1 Stabilizing α-helical Peptides with Covalent Bonds between Side-chains

Early studies on the control of α-helical peptides focused on constraining the

peptides to an α-helical conformation. One feasible type of approach is to form covalent

bonds between side-chains of residues separated in the sequence.

For example, Chorev et al. cyclized parathyroid hormone related protein amide

(PTHrP(7-34)NH2) via covalent bond formation between the ε-amino of Lys13 and the

α-carboxyl of Asp17 (i, i + 4) (Figure 1.2A)10. The lactam ring appeared to lock the

peptide in the bioactive conformation or a closely related structure, because it was

found to be 5~10 fold more potent than the parent peptide. A similar study was carried

out on analogues of growth hormone releasing factor (GRF)11. Three analogues of

GRF1-29 (cyclo8-12, cyclo21-25, and cyclo8-12, 21-25), as well as the linear peptide, were

synthesized and extensively studied with circular dichroism (CD) and nuclear magnetic

resonance (NMR). The results showed that i, i + 4 lactam bridges were able to stabilize

a local α-helical conformation, and could be of general use in the design of active,

stable peptides. Stabilization of α-helical structure by the formation of i, i+4 lactam

bridges was also demonstrated for the neuropeptide Y (NPY) 12 . Introducing two

overlapping lactam bridges into a peptide stabilized the conformation even further. A

hexapeptide containing two Lys (i)-Asp (i + 4) lactam bridge was synthesized and

demonstrated by NMR and CD to form a rigid α-helix in water and water/ trifluoroethanol

(TFE) (Figure 1.2B)13.

4

Lys13 Asp17

OHN

Boc Lys Lys Ala Asp Asp OPac

HN C

O

HN C

O

Figure 1.2 A. Structure of modified PTHrP(7-34)NH2 with a lactam bridge at i, i + 4. B. Structure of a hexapeptide containing two lactam bridges.

Another general method to stabilize an α-helix is disulfide bond formation.

Jackson et al. synthesized four short peptides containing a two-turn α-helix stabilized by

a single intramolecular disulfide bond bridging the i and i + 7 residues (Figure 1.3A)14.

CD experiments showed that although the Acm protected forms were reasonably helical

due to high alanine content, the disulfide linked peptides displayed ~100% helicity at

0oC and were able to retain >48% helicity at 60 oC. A study of a modified N-terminal

fragment of parathyroid hormone related protein (PTHrP) showed that the i, i + 3

disulfide bridged form (Figure 1.3B) contained one turn of the helix in water, whereas

the Acm protected form had no ordered structure at all in an aqueous environment15.

Hence the i, i + 3 disulfide bridge introduced, rather than merely stabilized an α-helical

conformation.

1

1

1 = HN S NH

OO

I2

A B

A

5

Ala Val Ser Gly DCys Gln Leu LCys His Asp Lys Gly NH2S S

Figure 1.3 A. i, i + 7 disulfide bond formation in a short peptide. B. Modified disulfide bridged N-terminal fragment of PTHrP.

Other covalent side-chain bridges can also stabilize α-helices. In one study, Glu

residues at positions i and i + 7 were linked by α,ω-diaminoalkanes in peptides of

arbitrary sequence16. Another approach introduced an i, i + 4 hydrazone link (N-N=CH-

CH2CH2) on the side-chains of N-termini of short peptides to replace a putative

hydrogen bond17. A metathesis-based approach was also applied and found effective at

i, i+7 positions with 11 carbons in the metathesized cross-link18.

These approaches demonstrated stabilization of an α-helical conformation and

provided useful tools for the study of structure and biological function of peptides and

proteins. However, their applications are limited to synthetic peptides due to the

requirement for unnatural amino acids or on-resin ring closure.

1.2.2 Control of α-helical Peptides with Non-Covalent Interactions between

Side-chains

Non-covalent interactions, such as ion pairs, or metal chelates were applied and

found effective to either stabilize or destabilize α-helical conformations.

Marqusee et al. inserted three Glu- - Lys+ salt bidges spaced at i, i + 4 positions

in a 17-residue alanine peptide and stabilized the α-helix19. In another attempt involving

B

6

ionic interactions, four glutamic acids were placed on a 14-residue peptide at i, i + 4, i +

7, and i + 11 positions, so that the binding of linear polyamines (e.g. spermine) could

induce the α-helical conformation. Upon addition of spermine, the helicity of the peptide

increased from 19% to 38%20.

Incorporating metal chelates can also affect the α-helicity of peptides. One

approach utilized model α-helical peptides containing two Cys residues in various

sequential arrangements. With i, and i + 1, i + 2, or i +3 arrangements, the addition of

As(III) caused helical destabilization, whereas i, i + 4 Cys residues stabilized α-helices

when bound to As(III)21. In another approach, Futaki et al. designed a redox switch

based on Fe(III) metal chelates22. By placing a pair of iminodiacetic acid derivatives of

lysine (Ida) residues at i, i + 2 positions in a 17-residue model peptide, a significant

decrease in the helical content was observed by the addition of Fe(III), whereas Fe(II)

had no influence on the stability of the helix. Redox control of the helical structure was

achieved by reducing Fe(III) to Fe(II) using Na2S2O4 followed by the subsequent

reoxidation using air. This approach was then applied to control leucine zipper formation

between Fos/Jun derived peptides22.

Promising as the metal chelates approach is, its applications are also limited to

synthetic peptides because of the incorporation of the unnatural amino acid Ida. This

system may not be a good choice as a reversible switch in vivo, because it requires the

continuous addition of metal ions and EDTA to trigger the switch, which is hard to

perform inside cells. Moreover, high concentrations of metal ions and EDTA are

poisonous to cells23, 24.

7

1.3 Reversible Control of α-helical Peptides with Light Light provides a clean trigger for the control of helix stability. In contrast to the

metal chelates approach, light avoids introducing additional foreign substances into the

system. With photoswitching chromophores such as spiropyran and azobenzene

derivatives, reversible optical control of helical conformations is feasible. Light also

offers the possibility of probing and manipulating proteins with high temporal and spatial

resolution within the complex environment of a living cell25,26.

1.3.1 Spiropyran and its Application to the Control of α-helices

Photochromism of spiropyran compounds involves two photoisomers, the neutral

spiro form and the zwitterionic merocyanine form, which are characterized by large

differences in geometry and polarity (Figure 1.4). Therefore, their interconversion may

strongly affect the structure of the attached macromolecules.

N

R

O

NO2

Figure 1.4 Photoisomerization of spiropyran compounds in aqueous solution (R = Alkyl groups).

The first photoresponsive polypeptide, poly(L-glutamic acid) containing

spiropyran units in the side chains was reported by Ciardelli and co-workers in 1989.27

Poly(L-glutamic acid) ( vM = 250k) was labeled with 41 mol % spiropyran and the

460 ~ 600 nm

Dark

8

resulting product was only soluble in hexafluoro-2-propanol (HFP), where it exhibited an

intense photochromism. At room temperature in the dark, the polypeptide chain adopted

an essentially random coil conformation and yielded colored solutions, owing to the

presence of the merocyanine forms on the side chains; irradiation with visible light or

just exposure to sunlight caused formation of α-helix and the complete bleaching of the

solutions, as a result of the formation of the colorless spiro form. This was confirmed by

13C NMR and CD study. On dark adaptation, the helix content progressively decreased,

and the original disordered conformation was restored. It was also found that the spiro

species content in the dark-adapted solution changed with the HFP

(hexafluoropropanol)/DCE (dichloromethane) composition, so irradiation at selected

solvent compositions made it possible to control the intensity of the photo-response.

However, the usage of organic solvent instead of aqueous solution limits the potential

application of this method to biological systems.

Although the photoinduced conformational change of poly(L-glutamic acid) were

extensively described,27, 28 the driving forces responsible for it remained unknown until

the work of Angelini and co-workers.29 Their study suggested that interactions between

the photochromic side chains induced the conformational change (Figure 1.5). In the

dark, the merocyanine units have a strong tendency to form dimeric species; as a result,

the macromolecules are forced to adopt a disordered structure. When the side chains

are photoisomerized to the spiro form, such dimers are destroyed, and the

macromolecules assume the helical structure.

9

Figure 1.5 Schematic illustration of the coil/α-helix transition occurring for spiropyran-modified poly (L-glutamic acid) in HFP. (Adapted with permission from reference [30]. Copyright 2001 American Chemical Society.)

The above examples show that when introduced into poly(L-glutamic acid)

systems, spiropyran was able to induce random coil/α-helix transition upon exposure to

sunlight or ambient indoor lighting. However, as mentioned above, the usage of organic

solvent greatly restricts the biological applicability. This drawback was overcome by

Fujimoto et al. in 2006.31 They developed a new photochromic cross-linking agent

containing a spiropyran skeleton, which was synthesized according to Figure 1.6. This

cross-linker was then introduced onto lysine residues spaced at i, i + 7 at the N-terminus

of a synthetic 14-residue peptide. At room temperature in 100 mM phosphate buffer, the

native peptide took a random-coiled structure, while the helical content of cross-linked

peptide increased up to 63% after exposure to room light (>460 nm). The value

decreased to 48% at the spiropyran-merocyanine equilibrium state in the dark for 3 h

(Figure 1.7). This switching behavior could be repeated more than four times under the

same conditions as mentioned above without substantial loss of the photoresponse.

Sunlight

Dark

10

R' = (CH2)2CO2N

O

O

N

Me Me

R'

O NO2

R'

Figure 1.6 Synthetic route for the spiropyran cross-linker

Figure 1.7 Schematic representation for the reversible photoregulation of helical structures on the cross-linked peptides. (Reproduced with permission from reference [31]. Copyright 2006 American Chemical Society.)

The beauty of this system is that the switching occurs in aqueous solution (100

mM phosphate buffer) when visible light (>460 nm) is applied, which has no influence

on other biomolecules and tissues. This expands the possibilities for photochemical

control of biological recognition events in vivo. However, the half-life for thermal

relaxation of the spiropyran cross-linker occurs over hours31, a lot longer than some

naturally occurring photoswitchable proteins, which reset in the dark in seconds32,33.

Spiropyran gets photobleached easily, while azobenzene is very fatigue-resistant.

N,N’-disuccinimidyl

11

Moreover, spiropyran and merocyanine are not symmetrical, leading to two isomers

upon cross-linking, which makes it difficult to study their photo-control ability.

Merocyanine has E, Z conformations, resulting in more isomers, which complicate the

structural study even more.

1.3.2 Azobenzene

Azobenzene and its derivatives are aromatic compounds composed of two

phenyl rings linked by a -N=N- double bond, and different chemical functional groups

extending from the phenyl rings34.

Azobenzene has two isomers, the planar trans form (Figure 1.8 left), with a

distance of 9.0 Å between the para carbon atoms, and the twisted cis form (Figure 1.8

right), with a distance of 5.5 Å35. The photo-isomerization of azobenzene occurs on

picosecond timescales36. Absorption of a photon leads to an excited state, in which the

barrier for isomerization is substantially smaller than in the ground state, thus decay can

lead to either the trans or cis ground state. Therefore, irradiation of an azobenzene

solution leads to a photostationary state that is predominantly trans or cis depending on

the wavelength of light used for irradiation. In addition to the photoisomerization, the cis

form will thermally relax to the more stable trans form37.

12

Figure 1.8 Structural changes of azobenzene

Azobenzene derivatives are robust, and relatively easy to synthesize38 . The

photoisomerization occurs with high quantum yield in the range of 330-450 nm (where

proteins are transparent), and is very fatigue-resistant. 39 Due to these favourable

properties, we chose azobenzene compounds as photo-switches to control peptide

secondary structure.

1.3.3 Using Azobenzene to Control α-helices

The first applications employing azobenzene to photoinduce random coil/α-helix

transitions were reported in early 1980’s.40-42 Azobenzenes were introduced into the

side chains of high-molecular-weight poly(L-glutamic acid). Irradiation at 350 nm

produced trans-to-cis isomerization of the azo side chains. The back reaction to the

trans form was obtained by irradiation with 450 nm light or dark adaptation. In organic

solvents, such as trifluoroethanol or trimethyl phosphate, or aqueous solutions at

physiological pH, light did not induce any variation in the main-chain structure while in

9.0 Å 5.5 Å 300 nm < λ < 400 nm

400 nm < λ, or heat

trans cis

13

aqueous solution at critical pH values,42 or in the presence of surfactants, 43 the

azobenzene modified peptide switched from random coil to up to 30% α-helix upon

exposure to 350 nm light. The conformational change was completely reversed when

the sample was dark-adapted or irradiated at 450 nm. The driving force of the coil/helix

transition was considered to be the polarity change of azo units upon isomerization,

which caused a change in location of the macro-molecules relative to the micelles

formed by the surfactants43.

When attached to the side chains of polypeptides, azobenzene was also found to

be capable of reversing the helical sense of poly-(L-aspartate).44-46 The CD of poly-(L-

aspartate) containing a high mol % of azo units changed from positive (left-handed

helix) to negative (right-handed helix) after irradiation in 1,2-dichloroethane.44 In solvent

mixtures, a photoinduced reversal of the helix sense occurred even for polypeptides

containing small amounts of azo units.45

Our lab has been seeking to find general strategies for the photo-control of

peptide and protein structure – and thereby activity, with azobenzene derivatives. We

chose to tether both ends of the azobenzene moiety to form a cross-link between two

sites within a peptide or protein. Intramolecular cross-links may restrict the flexibility of

azobenzene, allowing more effective induction of local conformational changes in the

attached system upon photo-isomerization.

Our first design was aimed the control of helix content in a simple peptide

system47. An i to i + 7 cysteine residue spacing was selected due to its compatibility with

a cis conformation and lower compatibility with a trans conformation of the linker. The

designed azobenzene derivative cross-linker (Figure 1.9) incorporated two α-

14

iodoacetamido groups to permit intramolecular cross-linking via reaction with cysteine

side-chains, and contained a minimum number of single bonds between the para

position of the benzene rings and iodoacetamido groups, to minimize its flexibility and

maximize the conformational switching effect. In accordance with the design, the

photoisomerization of the azobenzene cross-linker from the trans to the cis form caused

a large increase in the helix content of the peptide, in water (Figure 1.10). Thermal- or

photoisomerization from cis to trans returns the peptide to its disordered form.

Photoswitching can occur many hundreds of times without fatigue.

Figure 1.9 Chemical structure and photoisomerization of the azobenzene cross-linker developed by Kumita et al. (Reproduced with permission from reference [47]. Copyright 2000 National Academy of Sciences.)

Figure 1.10 (A) JRK peptide (acetyl-EACARVAibAACEAAARQ-amide) cross-linked between Cys residues spaced i, i + 7. (B) CD spectra of trans (s) and cis (- - -) JRK-X. (Reproduced with permission from reference [52]. Copyright 2005 American Chemical Society.)

15

Further studies explored the behavior of short peptides containing the same

azobenzene cross-linker between cysteine residues at positions i, i + 4 or i, i + 11 in the

sequence.48 It was found that trans-to-cis photoisomerization significantly increased the

helix content in the i, i + 4 case (FK-4) and significantly decreased the helix content in

the i, i + 11 case (FK-11). Being able to choose whether the dark-adapted cross-linker

stabilizes a helix (i, i + 11) or destabilizes it (i, i + 4; i, i + 7) enables greater control of

the target system because the dark-adapted state of azobenzene is >99% trans

isomer,49 whereas the percentage of cis isomer that can be achieved upon irradiation is

typically 70-90% depending on the system. This fact sets intrinsic limits on the extent of

the photocontrol of protein activity that may be possible using azobenzene. If the trans

form is active, a ~5-fold change in the concentration of the active species is possible

(e.g., from ~100% trans in the dark to 20% trans upon irradiation). However, if the cis

form is the active form, a much larger change in the concentration of the active species

is possible (e.g., from

16

the cross-linker causes conformational changes in the peptide51. This analysis has

shown that the effect of cross-linker photoisomerization on the equilibrium helix content

of an attached peptide can be predicted by comparing the range of S-S distances in the

intrinsic conformational ensemble of the peptide with the range compatible with the

chemical structure of the linker (Figure 1.11). This analysis provided a framework for the

rational application of this and related intramolecular cross-linkers to the control of

peptide and protein structure.

Figure 1.11 Histogram showing the distribution of Cys-Cys (S-S) distances (0.2 Å intervals) expected for the noncross-linked JRK peptide under the conditions of the CD experiment [overall helix content, 25% (-)]. The dotted line shows the expected distribution for noncross-linked JRK, where the overall helix content is 5% (e.g., at high temperature). The total number of structures was ~100 000 in each case. Green line (the distance range allowed by a cis cross-lnker); blue line (the distance range allowed by a trans cross-linker); orange line (the distance range allowed by a linker in the inversion transition-state structure); red line (the distance range allowed by a rotational transition state). Some representative linker structures are shown above. (Reproduced with permission from reference [52]. Copyright 2005 American Chemical Society.)

17

1.4 Applications of Photocontrol of α-helices Using Azobenzene In addition to short α-helical peptides, azobenzene derivatives have been used to

control more complex structures composed of α-helices as well as the DNA binding of

peptides.

1.4.1 Photocontrol of Coiled-coil Interactions

Caamaňo and co-workers used a dibromoacetyl derivative of azobenzene to link

two basic regions of the basic leucine zipper (bZIP) transcriptional activator GCN4 via

cysteine residues in hopes of photo-regulating DNA binding activity53. Upon photo-

isomerization from trans to cis, the DNA binding affinity for the cross-linked peptide

increased 60-70 fold (Figure 1.12), however, the binding was not reversible.

Figure 1.12 Schematic diagram of photo-controlled DNA binding by a bZIP peptide dimer. (Reproduced with permission from reference [53]. Copyright 2000 Wiley InterScience.)

Another study on GCN4 with the same goal was performed by Kumita et al. A

modified sequence incorporating two cysteine residues with i, i + 7 spacing was

18

produced, and the modified peptide was cross-linked54 . Although irradiation of the

peptide produced changes in helicity, aggregation occurred leading to loss of

reversibility. Subsequently, a modified GCN4-bZIP DNA-binding protein was produced

with a sulfonated azobenzene chromophore introduced between Cys residues at

positions 262 and 269 (S262C, N269C) within the zipper domain55. As predicted, the

trans form of the chromophore destabilized the helical structure of the coiled-coil region

of GCN4-bZIP, leading to diminished DNA binding relative to wild type. Trans-to-cis

photoisomerization of the chromophore increased helical content and substantially

enhanced DNA binding (Figure 1.15). The system was observed to be readily

reversible; thermal relaxation of the chromophore to the trans state and concomitant

dissociation of the protein-DNA complex occurred with τ1/2 ~10 min at 37oC.

Figure 1.13 Models showing the photocontrol of DNA binding by AZO(i, i + 7) GCN4-bZIP. In the trans conformation, the cross-linker induces a bend in the helix, which can be expected to destabilize zipper formation and consequently DNA binding (left). In contrast, the cis conformation of the cross-linker is compatible with zipper formation and thus DNA binding (right). (Reproduced with permission from reference [55]. Copyright 2006 American Chemical Society.)

19

1.4.2 Photocontrol of DNA Binding by Azobenzene Cross-linked α-helical

Peptides

MyoD is a muscle-specific transcription factor that has a basic helix-loop-helix

(bHLH) structure and binds DNA upon dimerization of the HLH domain. It contacts the

major groove of the DNA target sequence through its basic domain with limited

specificity in vitro56. Guerrero et al. replaced Met 116 and Ser 123 (i and i + 7 spacing)

located on the water-exposed face of the recognition helix (basic domain) of MyoD with

cysteine residues and subsequently incorporated an azobenzene derivative cross-

linker57. The helicity and the activity of this photoMyoD could be controlled by light

pulses. In the irradiated state with the cross-linker predominantly in the cis

configuration, significant stabilization of the recognition helix was observed. Compared

to the dark-adapted photoMyoD, irradiation generated increases in DNA-binding by

more than two orders of magnitude.

Another example involves HDH-3, a 18-residue polypeptide based on the

recognition helix of the Q50K engrailed homeodomain, which binds to the major groove

of DNA via helix-3 within a helix-turn-helix motif58. An azobenzene cross-linker was

installed onto the mutant HDH-3 through two cysteine residues in an i, i + 11 spacing.

The dark-adapted HDH-3 adopted a mainly α-helical structure, whereas the helical

content of the irradiated HDH-3 was reduced significantly and the peptide resembled

that of the largely unstructured, unalkylated peptide. Furthermore, the dark-adapted

peptide bound to its natural DNA target sequence with much higher affinity than a

mutant DNA sequence, despite lacking helices-1 and -2 and the N-terminal arm of

Q50K engrailed. This DNA-binding affinity and specificity of HDH-3 could be controlled

20

externally by light. The authors claimed that the dark-adapted HDH-3 is the most

specific designed DNA binding miniature homeodomain reported to date, and contribute

the specificity to the preorganization of the helix induced by the cross-linker.

These studies demonstrated the photocontrol of the structures and DNA binding

of miniature proteins, and opened a way to investigate cellular processes through

reversible photocontrol of transcription.

1.5 Development of Azobenzene Cross-linkers To achieve greater control of peptide/proteins, a further series of cross-linkers

were designed and studied.59, 60 Zhang et al. reported the design and synthesis of a

water-soluble, sulfonated version of an azobenzene-based thiol reactive cross-linker59.

The sulfonated compound was shown to cause a similar degree of conformational

control on a model peptide helix system as its nonsulfonated counterpart but could be

introduced without the need for any organic cosolvent.

Rates of thermal cis-trans isomerization can be manipulated by altering the

chemical structure of the linker. To increase the rate of thermal reversion, Pozhidaeva

et al. designed derivatives in which enhanced delocalization of the para amino group

lone-pair electrons would be possible. High reactivity and selectivity toward Cys

residues was maintained by introducing methanethiosulfonyl groups.60 These groups

also permitted attachment of the cross-linker to peptides and proteins via disulfide

linkages that can be subsequently cleaved, if desired, using reducing agents. When

used to cross-link the FK-11 peptide described above, these linkers enabled

21

photocontrol of peptide helical content in a manner similar to the original iodoacetamide-

based cross-linker but with a large range of thermal stabilities observed for the cis forms

from 11 s to 43 h at 25 oC (see below). These cross-linkers thus broadened the range of

reagents available for reversible photo-control of peptide and protein conformation.

Figure 1.14 Azobenzene derivative cross-linkers with methanethiosulfonate (MTS) reaction groups. (Reproduced with permission from reference [52]. Copyright 2005 American Chemical Society.)

22

1.6 References

1. Barlow, D. J.; Thornton, J. M., Helix geometry in proteins. J. Mol. Biol. 1988, 201, 601-19. 2. Pauling, L. & Corey, R. B. Two hydrogen-bonded spiral configurations of the polypeptide chain. J. Am. Chem. Soc 1950, 72, 5349. 3 . Pauling, L. & Corey, R. B. Atomic coordinates and structure factors for two helical configurations of polypeptide chains. Proc. Nat. Acad. Sci. USA 1951, 37, 235-240 4. Perutz, R. R.; Liquori, A. M.; Eirich, F., X-ray and solubility studies of the haemoglobin of sickle-cell anaemia patients. Nature 1951, 167, 929-31. 5. Arnott, S.; Wonacott, A. J., Atomic co-ordinates for an alpha-helix: refinement of the crystal structure of alpha-poly-l-alanine. J. Mol. Biol. 1966, 21, 371-83. 6. Presta, L. G.; Rose, G. D., Helix signals in proteins. Science 1988, 240, 1632-41. 7. Cox, J. A., Calcium-Calmodulin Interaction and Cellular Function. J. Cardiovasc. Pharmacol. 1986, 8, 48-51. 8. Brennan, R. G.; Matthews, B. W., Structural Basis of DNA - Protein Recognition. Trends Biochem. Sci. 1989, 14, 286-90. 9. Mercier, G. A.; Osman, R.; Weinstein, H., Role of primary and secondary protein-structure in neurotransmitter receptor activation mechanisms. Protein Eng. 1988, 2, 261-70. 10. Chorev, M.; Roubini, E.; Mckee, R. L.; Gibbons, S. W.; Goldman, M. E.; Caulfield, M. P.; Rosenblatt, M., Cyclic parathyroid-hormone related protein antagonists - Lysine-13 to aspartic-acid 17 [I to (I + 4)] side-chain to side-chain lactamization. Biochemistry 1991, 30, 5968-74. 11. Fry, D. C.; Madison, V. S.; Greeley, D. N.; Felix, A. M.; Heimer, E. P.; Frohman, L.; Campbell, R. M.; Mowles, T. F.; Toome, V.; Wegrzynski, B. B., Solution structures of cyclic and dicyclic analogs of growth-hormone releasing-factor as determined by 2-dimensional NMR and CD spectroscopies and constrained molecular-dynamics. Biopolymers 1992, 32, 649-66. 12. Bouvier, M.; Taylor, J. W., Probing the functional conformation of neuropeptide-Y through the design and study of cyclic analogs. J. Med. Chem. 1992, 35, 1145-55. 13. Bracken, C.; Gulyas, J.; Taylor, J. W.; Baum, J., Synthesis and nuclear-magnetic-resonance structure determination of an alpha-helical, bicyclic, lactam-bridged hexapeptide. J. Am. Chem. Soc. 1994, 116, 6431-32. 14. Jackson, D. Y.; King, D. S.; Chmielewski, J.; Singh, S.; Schultz, P. G., General-approach to the synthesis of short alpha-helical peptides. J. Am. Chem. Soc. 1991, 113, 9391-92. 15. Pellegrini, M.; Royo, M.; Chorev, M.; Mierke, D. F., Conformational consequences of i,i+3 cystine linkages: Nucleation for alpha-helicity? J. Pept. Res. 1997, 49, 404-14. 16. Phelan, J. C.; Skelton, N. J.; Braisted, A. C.; McDowell, R. S., A general method for constraining short peptides to an alpha-helical conformation. J. Am. Chem. Soc. 1997, 119, 455-60.

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17 . Cabezas, E.; Satterthwait, A. C., The hydrogen bond mimic approach: Solid-phase synthesis of a peptide stabilized as an alpha-helix with a hydrazone link. J. Am. Chem. Soc. 1999, 121, 3862-75. 18. Schafmeister, C. E.; Po, J.; Verdine, G. L., An all-hydrocarbon cross-linking system for enhancing the helicity and metabolic stability of peptides. J. Am. Chem. Soc. 2000, 122, 5891-92.

19. Marqusee, S.; Baldwin, R. L., Helix stabilization by Glu- ... Lys+ salt bridges in short peptides of denovo design. Proc. Nat. Acad. Sci. U.S.A. 1987, 84, 8898-902. 20. Tabet, M.; Labroo, V.; Sheppard, P.; Sasaki, T., Spermine-induced conformational-changes of a synthetic peptide. J. Am. Chem. Soc. 1993, 115, 3866-68. 21. Cline, D. J.; Thorpe, C.; Schneider, J. P., Effects of As(III) binding on α-helical structure. J. Am. Chem. Soc. 2003, 125, 2923-29. 22. Futaki, S.; Kiwada, T.; Sugiura, Y., Control of peptide structure and recognition by Fe(III)-induced helix destabilization. J. Am. Chem. Soc. 2004, 126, 15762-69.

23. Hershko, C., Mechanism of iron toxicity and its possible role in red-cell membrane damage. Semin. Hematol. 1989, 26, 277-85.

24 . Hugenschmidt, S.; Planasbohne, F.; Taylor, D. M., On the toxicity of low-doses of tetrasodium-ethylenediamine-tetraacetate (Na-EDTA) in normal rat-kidney (NRK) cells in culture. Arch. Toxicol. 1993, 67, 76-78. 25. Volgraf, M.; Gorostiza, P.; Numano, R.; Kramer, R. H.; Isacoff, E. Y.; Trauner, D., Allosteric control of an ionotropic glutamate receptor with an optical switch. Nat. Chem. Biol. 2006, 2, 47-52.

26 . Banghart, M.; Borges, K.; Isacoff, E.; Trauner, D.; Kramer, R. H., Light-activated ion channels for remote control of neuronal firing. Nat. Neurosci. 2004, 7, 1381-86. 27. Ciardelli, F.; Fabbri, D.; Pieroni, O.; Fissi, A., Photomodulation of Polypeptide Conformation by Sunlight in Spiropyran-Containing Poly(L-Glutamic Acid). J. Am. Chem. Soc. 1989, 111, 3470-3472.

28. Fissi, A.; Pieroni, O.; Ciardelli, F.; Fabbri, D.; Ruggeri, G.; Umezawa, K., Photoresponsive Polypeptides - Photochromism and Conformation of Poly(L-Glutamic Acid) Containing Spiropyran Units. Biopolymers 1993, 33, 1505-1517. 29. Angelini, N.; Corrias, B.; Fissi, A.; Pieroni, O.; Lenci, F., Photochromic polypeptides as synthetic models of biological photoreceptors: a spectroscopic study. Biophys. J. 1998, 74, 2601-10.

30. Pieroni, O.; Fissi, A.; Angelini, N.; Lenci, F., Photoresponsive polypeptides. Acc. Chem. Res. 2001, 34, 9-17. 31. Fujimoto, K.; Amano, M.; Horibe, Y.; Inouye, M., Reversible photoregulation of helical structures in short peptides under indoor lighting/dark conditions. Org. Lett. 2006, 8, (2), 285-287.

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32. Lanyi, J. K., Bacteriorhodopsin. Annu. Rev. Physiol. 2004, 66, 665-688. 33. Genick, U. K.; Soltis, S. M.; Kuhn, P.; Canestrelli, I. L.; Getzoff, E. D., Structure at 0.85 angstrom resolution of an early protein photocycle intermediate. Nature 1998, 392, (6672), 206-209.

34. Kumita, J. R. Reversible control of peptide structure and activity by an azobenzene photo-switch. 2003, Ph. D. Thesis 35. Brown, C. J., A refinement of the crystal structure of azobenzene. Acta Cryst. 1966, 21, 146-152.

36. Wachtveitl, J., Nagele, T., Puell, B., Zinth, W., Kruger, M., Rudophbohner, S., Oesterhelt, D., and Moroder, L., Ultrafast photoisomerization of azobenzene compounds. J. Photochem. Photobiol., A Chem. 1997, 105, 283-288 37. Rau, H., “Azo Compounds.” Photochromism: Molecules and Systems; Durr, H. and Bousas-Laurent, H. eds.; Elsevier: New York, 165-192.

38. Molecular Switches, ed. B. L. Feringa, Wiley-VCH, Weinheim, 2001, and references cited therein.

39. Rau, H. In Photochemistry and Photophysics; Rabek, J. F., Ed.; CRC Press Inc.: Boca Raton, FL, 1990; Vol. 2, 119-141.

40. Pieroni, O.; Houben, J. L.; Fissi, A.; Costantino, P., Reversible Conformational-Changes Induced by Light in Poly(L-Glutamic Acid) with Photochromic Side-Chains. J. Am. Chem. Soc. 1980, 102, 5913-5915. 41. Houben, J. L.; Fissi, A.; Bacciola, D.; Rosato, N.; Pieroni, O.; Ciardelli, F., Azobenzene-Containing Poly(L-Glutamates) - Photochromism and Conformation in Solution. Int. J. Biol. Macromol. 1983, 5, 94-100. 42. Ciardelli, F.; Pieroni, O.; Fissi, A.; Houben, J. L., Azobenzene-Containing Polypeptides - Photoregulation of Conformation in Solution. Biopolymers 1984, 23, 1423-1437. 43. Pieroni, O.; Fabbri, D.; Fissi, A.; Ciardelli, F., Photomodulated conformational-changes of azo-modified poly(l-glutamic acid) in micellar systems. Makromol. Chem., Rapid Commun. 1988, 9, 637-640. 44 . Ueno, A.; Anzai, J.; Osa, T.; Kadoma, Y. Light-induced conformationalchanges of polypeptides. photoisomerization of azoaromatic polypeptides. Bull. Chem. Soc. Jpn. 1979, 52, 549-554.

45. Ueno, A.; Takahashi, K.; Anzai, J.; Osa, T. Photocontrol of polypeptide helix sense by cis-trans isomerization of side-chain azobenzene moieties. J. Am. Chem. Soc. 1981, 103, 6410-6415.

46. Ueno, A.; Adachi, K.; Nakamura, J.; Osa, T. Photoinduced conformational changes of azoaromatic polyaspartes containing octadecyl side chains. J. Polym. Sci., Polym. Chem. 1990, 28, 1161-1170.

47. Kumita, J. R.; Smart, O. S.; Woolley, G. A., Photo-control of helix content in a short peptide. Proc. Nat. Acad. Sci. U.S.A. 2000, 97, 3803-8.

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48. Flint, D. G.; Kumita, J. R.; Smart, O. S.; Woolley, G. A., Using an azobenzene cross-linker to either increase or decrease peptide helix content upon trans-to-cis photoisomerization. Chem. Biol. 2002, 9, 391-7. 49. Dias, A. R.; Minas da Piedade, M. E.; Martinho Simoes, J. A.; Simoni, J. A.; Teixeira, C.; Diogo, H. P.; Meng-Yan, Y.; Pilcher, G. Enthalpies of formation of cis-azobenzene and trans-azobenzene. J. Chem. Thermodyn. 1992, 24, 439-447. 50. James, D. A.; Burns, D. C.; Woolley, G. A., Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylalanine residues. Protein Eng. 2001, 14, 983-91.

51. Burns, D. C.; Flint, D. G.; Kumita, J. R.; Feldman, H. J.; Serrano, L.; Zhang, Z.; Smart, O. S.; Woolley, G. A., Origins of helix-coil switching in a light-sensitive peptide. Biochemistry 2004, 43, 15329-38.

52. Woolley, G. A., Photocontrolling peptide alpha helices. Acc. Chem. Res. 2005, 38, 486-93. 53. Caamano, A. M.; Vazquez, M. E.; Martinez-Costas, J.; Castedo, L.; Mascarenas, J. L., A light-modulated sequence-specific DNA-binding peptide. Angew. Chem. Int. Ed. 2000, 39, 3104-07.

54. Kumita, J. R.; Flint, D. G.; Woolley, G. A.; Smart, O. S., Achieving photo-control of protein conformation and activity: producing a photo-controlled leucine zipper. Faraday Discuss. 2003, 122, 89-103; discussion 171-90.

55. Woolley, G. A.; Jaikaran, A. S.; Berezovski, M.; Calarco, J. P.; Krylov, S. N.; Smart, O. S.; Kumita, J. R., Reversible photocontrol of DNA binding by a designed GCN4-bZIP protein. Biochemistry 2006, 45, 6075-84. 56. Buskin, J. N.; Hauschka, S. D., Identification of a myocyte nuclear factor that binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene. Mol. Cell. Biol. 1989, 9, 2627-40.

57. Guerrero, L.; Smart, O. S.; Weston, C. J.; Burns, D. C.; Woolley, G. A.; Allemann, R. K., Photochemical regulation of DNA-binding specificity of MyoD. Angew. Chem. Int. Ed. 2005, 44, 7778-82.

58. Guerrero, L.; Smart, O. S.; Woolley, G. A.; Allemann, R. K., Photocontrol of DNA binding specificity of a miniature engrailed homeodomain. J. Am. Chem. Soc. 2005, 127, 15624-9. 59. Zhang, Z. H.; Burns, D. C.; Kumita, J. R.; Smart, O. S.; Woolley, G. A., A water-soluble azobenzene cross-linker for photocontrol of peptide conformation. Bioconjug. Chem. 2003, 14, 824-829.

60. Pozhidaeva, N.; Cormier, M. E.; Chaudhari, A.; Woolley, G. A., Reversible photocontrol of peptide helix content: adjusting thermal stability of the cis state. Bioconjug. Chem. 2004, 15, 1297-303.

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Chapter 2 A Blue-Green Absorbing Cross-linker for Rapid Photo-

switching of Peptide Helix Content

This chapter is published work, reproduced with permission from: Chi, L;

Sadovski, O; Woolley, G. A.; Bioconjug. Chem. 2006, 17, 670-676. Copyright 2006

American Chemical Society.

(Oleg Sadovski performed the synthesis of the cross-linker described here.

Andrew Woolley performed all the molecular modeling. I performed the peptide cross-

linking and purification as well as all the biophysical characterization of the cross-linked

peptides)

2.1 Abstract Azobenzene derivatives can be used to reversibly photo-regulate secondary

structure when introduced as intramolecular bridges in peptides and proteins. Here we

report the design, synthesis, and characterization of a disubstituted N,N-dialkyl

azobenzene derivative that absorbs near 480 nm in aqueous solution, and relaxes with

a half-life of ~50 milliseconds at room temperature. The wavelength of maximum

absorbance and the rate of thermal relaxation are solvent dependent. An increase in the

percentage of organic solvent leads, in general, to a blue-shift in the absorbance

maximum and a slowing of the relaxation rate. In accordance with the design, the

27

thermal relaxation of the azobenzene cross-linker from cis to trans causes an increase

in the helix content of one peptide where the linker is attached via Cys residues spaced

at i, i + 11 positions, and a decrease in helix content of another peptide with Cys

residues spaced at i, i + 7. This cross-linker design thus expands the possibilities for

fast photo-control of peptide and protein structure.

2.2 Introduction Reversible optical control of protein structure and function offers the possibility of

probing and manipulating individual proteins within the complex environment of a living

cell1, 2. Azobenzene, in particular, has been a popular choice as a photo-switching

chromophore due to its robust photo-physical properties and relative ease of synthesis3,

4. However, there is no wavelength at which only one azobenzene isomer absorbs; the

absorption spectra of trans and cis forms overlap extensively. Thus, irradiation typically

produces photostationary states composed of at most ~80% cis or ~95% trans isomers5,

6. This fact limits the degree of photo-switching that is possible; e.g. the fraction of cis

isomer can be changed from ~5% to 80% (16-fold) or trans from ~20% to 95% (~5-

fold)6. Thermal isomerization, in contrast, yields >99.99% trans isomer7. Therefore, if

thermal isomerization is used to reset the switch, a much greater fold change in the cis

isomer is then possible.

The half-life for thermal cis-to-trans isome