STRUCTURAL STUDIES OF AZOBENZENE-MODIFIED PROTEINS

Darcy C . Bums

A thesis subrnitted in confomity with the requirements

For the degree of Master of Science

Graduate Department of Chemistry

University of Toronto

O Copyright by Darcy C. Burns 2001

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Structural Studies of Azobenzene-modified Proteins

By Darcy C . Burns

Master of Science, Department of Chernistry

University of Toronto

200 1

ABSTRACT

Photoregdation is a powerfùi tool, since it provides a non-invasive method of

reversibly controlling bio logical activit ies. The photochrome phenylazophenyIalanine

(PAP) has k e n tested for its abiIity to reversibly photoregdate two biologicd processes:

enzyme activity and ion charnel gating. In an effort to judge PAP as a candidate for

photoregdation, we have initiated studies to detemine the conformations of PAP

residues at specinc sites in RNase S and gramicidm A. PAP has been incorporated at

position 7 (PAP7) and at position 1 0 ( P M I O) in separate S-peptide samples, and

incorporated at position 1 (PAPI) in gramicidin A samples. Standard NMR techniques in

combination with molecular modelling are bemg used to assess the structure and

dynamics of azobenzene in each system and to gauge the effectiveness of photocontrol of

each system.

First, and foremost 1 would like to extend my gratitude to Dr. WooIley . . .

Thank-you Dmew This thesis has been extremely chdenging at tirnes (17ve never been

tested Iike this before), and you have kept me on track throughout every trial and every

triiulation. Imagination, critical thinking, intelligent experimentation, md carefùl

observation: each of these ski& are important to quality research, and T have k e n able

to irnprove upon aiI of them under your tutelage. Also appreciated has k e n your

generosity . . . . San Diego, New Jersey, the CSCCE, and Christmas dinners immediately

corne to mind. Overall, you have provided me with a learning experience beyond my

imagination and have definïtely proven to be an excellent mentor and guide durnig my

t h e in Toronto. Once again - thank you.

Linda, Andrew, Janet, Tyler, Dom, Christine, Vitali, Ananda. . . . you have all

kept me honest, educated, and entertained during the Iast few years. Hopehlly we'll be

able to share many more squash matches, pints O' beer , and L u c b Dragon lunches in the

coming years. Jack - don7t thuik that I would ever Ieave you out. 1 especially thank you

for good advice, constant encouragement, and one great tie. Tmly you are the "expert" m

the iab, 1 have very much enjoyeci and appreciated the camaraderie of everyone in the

lab.

I have been extremely lucb by having supportive roommates during the past two

years. Thank you Sheldon, Nevin, and James, for heIping to maintain my focus (and

sanity), yet a h helping me to let loose fiom time to t h e . It has defbitely k e n nice to

be able to corne home to a happy house each and every ni&. Cheers.

A b , thanks to my mother, father and brother for all of the love, care, and support

that a son and brother would ever want. Another thesis translated to more computer

problems, and where wodd 1 be without technical support (probably still recovering

back-up files). Thanks Dad.

Cindy, 1 love you, I can't begin to express how lucky 1 feei to have you by my

side. 1 know that 1 could not have accomplished any of this work without your love, and

encouragement and 1 can't begin to tell you how gratefd 1 am to you for burning the

rnidnight oil with me and for listening to more chemistry and biochemistry than a

zoologist should ever have to- Together we have corne through two degrees and five

great years. 1 can hardiy wait for the many great years ahead of us to do ld .

There are m q others who 1 would wish to thank, however, in the interest of

space 1 WU simply Say that 1 appreciate alI of the weil-wishing, and support offered by

my fiiends. It is often your kind words that keep my spirits uplifted.

With Love,

Darcy O

This thesis is dedicated to my father (CharIes M- Burns), mother (Iris A- Burns), and

brother (Justin T- Burns).

"IntelZigence is I ike hming 4-wheel dm>e . . . . itjusî enables you to get stuck in more

remote places"

- mon.

TABLE OF CONTENTS

ABSTRACT ACKNOWLEDGEMENTS TABLE OF CONTENTS LlST OF FIGURES LlST OF TABLES

i i iii vi ix

xii

CHAPTER 1 GENERAL INTRODUCTION 1

1 .l PHOTOISOMERIC MOLECULES 2 GENERAL DESCRIPTION OF PHOTOISOMERIC MOLECULES 2 AZOBENZENE 2

1.2 AZOBENZENE-PHOTOREGULATED SYSTEMS PEPTLDES OLIGONUCLEOTrDES ENZYMES ION CHANNELS

CHAPT ER 2 AZOBENZENE-REGULATED RNASE A ACTlVlTY II

2.1 INTRODUCTION GENEEUIL. DESCRIPTION of RNase A and RNase S RNase A MECHANISM RNase A and RNase S STRUCTURE EXPERIMENTAL OUTLINE: CHAPTER 2

2.2 MATERIALS AND RlETHODS SYSNTHESIS AND PURlFICATION OF PEPTIDES NMR SPECTROSCOPY UV-VIS SPECTROSCOPY MOLECULAR MODELLING

2.3 RESULTS S-PEPTIDE S ynthesis, mirification and NMR Spectroscopy

PAP S-PEPTIDE ANALOGS W-VIS and NMR Spectroscopy

RNaseSANDRNaseA W-VIS and NMR Spectroscopy

PAP-RNase S MUTANTS UV-VIS and NMR Spectroscopy Mo iecular Mo deliing

2.4 DISCUSSION S-PEPTIDE PAP S-PEPTIDE ANALOGS

W-VIS Spectroscopy of PAP Peptides NMR Spectroscopy of PAP Peptides

PAP-RNase S MUTANTS UV-VIS Spectroscopy of PAP-RNase S Mutants NMR Spectroscopy of PAP-RNase S Mutants Modelling and Kinetics of RNase S Mutants

CHAPTER 3 AZOBENZENE-REGULATED GRAMlClDlN A CHANNELS

3.1 INTRODUCTION GENERAL DESCRIPTION of GRAhaCIDIN MEEHANISM OF ION TRANSPORT AZOBENZENE-PHOTOREGULATED GRAMICIDIN A EXPERIMENTAL OUTLINE: CHAPTER 3

3.2 MATE-S AND Ml3T'HODS MOLECULAR MODELLING

3 3 RESULTS MOLECULAR MODELLING: POTENTIAL ENERGY SURFACES MOLECULAR MODELLING: ELECTROSTATLC SURFACES

3.4 DISCUSSION GRAMICIDIN MOLECULAR MODELLING

3.5 REFERENCES

APPENDICES

APPENDIXA SINGLE POINT POTENTLAL ENERGY EXTRACTION

SPqarse.pl ELECTROSTAmC ENERGY EXTRACTLON

vii

APPENDIX B nmrPIPE PROCESSING SCHEME FOR DQCOSY SPECTRA

APPENDIX C HYPERCHEM TEMPLATE FILES

Formyl group Ethanoliunine residue

HYPERCHEM PARAMETER FILES amberspe-PAP-txt amberben-PAP-txt ambembd_PAP.txt arnberstr-PAP-txt

APPENDIX D CALCULATEON FOR AMOUNT OF S-PEPTIDE :S-PROTEIN COMPLEX

LIST OF FIGURES

Page

CEAPTER 1 GENlERAlL INTRODUCTION 1.1 Azobenzene UV-VIS spectrum and energy diagram

1.2 Mechanism of cis/trans thermal isomerïzation in azobenzene

CHAPTER 2 AZOBENZENE-REGULATED RNase S

fiinuclease A bound by ATTA-DNA substrate

Putative mechaniSm for cIeavage of poly(A) by mase A, showïng

substrate binding subsites

Structure of phenylazophenylalanme (PM), showing torsion angles

PrÏmary amho acid sequence of rnodifïed S-peptide (S-peptide-2)

1 D 'HNMR of deuterated reduced S-peptide-2, and deuterated met(0)-

S-peptide-2

1D presaturation 'HNMR of reduced S-peptide-2

2D wgTOCSY of S-peptide-2

2D wgDQCOSY of S-peptde-2

W-VIS spectra and recovery plots of ixans- and cis-PAP7 (A), and

trans- and cis PAPl O (l3)

ID 'HNMR of deuterated trans-PAP 1 O

ID 'HNMR of deuterated (A) trans- and (B) ck-PM7

'H DQCOSY of deuterated trans and cis-PAP7

W-VIS spectra of S-protein

1D 'HNMR of S-protein

'K DQCOSY of RNase A

UV-VIS spectra of trans and cis-PAP7 (A) and recovery (B) of trans-

P M 7

W-VIS spectra of tram and cis-PAP 10 (A) and recovery (B) of tram-

P M 1 0

1 D 'HNMR of deuterated tram-PAP7-RNase S (A) and deuterated cis-

PAP7-RNase S (ES)

ID 'HNMR of deuterated tram-PAP 10-RNase S (A) and deuterated

cis-PAPI O-RNase S (B)

DQCOSY spectrum of deuterated trans-PAP7-RNase S

DQCOSY spectnun of deuterated trans-PAP7-RNase S and deuterated

cis-PAP7-RNase S

DQCOSY spectnim of deuterated tram-PAPl O-RNase S

DQCOSY spectrum of deuterated tram-PAPl0-RNase S and deuterated

cis-PAP 1 O-RNase S

Recovery of tram-PAP 1 0-RNase S fkom cis-P AP 10-RNase S as

followed by DQCOSY NMR

Potential energy d a c e s of trans-PAP7 (A) and cis-PAP7 (B) in

RNase S

PotentiaI energy surfaces of tram-PAP 10 (A) and cis-PAP 10 (B) in

mase S

GRASP surface modek of trans-PAP7-RNase S (A) and cis-PAP7-

RNase S (B)

GRASP surface models of trans-PAP 10-RNase S (A) and cis-PAP 10-

RN= s (BI

CHAPTER 3 AZOBENZENE-REGULLATED

GRAMKIDIM A CHANNELS 3.1 Stereo view of gramicidh A

3 -2 3B2S model for ion transport through a grarnicidin charme1

3 -3 Proposed model for photornodulattion of N-terminal azobenzene- linked

gramicidm A

3 -4 Structure of a p-amùiornethylazobe~l~ene-moaed gramicidin A

channe1

3.5 Structure of phenylazo phenylalanine (PAP), showmg torsion angles

3-6 SmgIe-point energy promes and electrostatic interaction profiles for 81

low energy conformers of cis-PAP 1 -gramicidin A

3 -7 Models of cis-PAP 1 --cidin A (A) and tram-PAP 1 -gramiciciin A 82

(3) showing azo benzene dipoles

3.8 Single-channel curent amplitude histografll~ and representative single- 84

channel events of trans-PAP 1 -gramicidin A (A), and cis-P AP 1 - gramicidin A (B), and native gramicidin A (C)

LlSTOF TABLES

Number Page

CHAPTER 2 AZOBENZELYE-REGULATED RNase S Function o f different amino acids in RNase A catalysis 15-16

Substrate binding subsites in RNase A 17

Chernical shifts o f azobenzene ~g protons in PAP7, PAP7-RNase S, 34

and PAP 1 O-RNAse S

Dissociation Constants for PAP7-RNase S and PAP 2 0-RNase S 55

Collected Values for Observed Constants Va, K, for PAP7-RNase S 60

and PAP 10-RNase S

CaAPTER 3 AZOBENZENE-REGULATED

GRAhlICIDIN A CHANNELS 3.1 Physical properties o f native gramiciclin A and gramicidin A analogues

3 -2 Functional properties o f PAP 1-gramicidi. A channels

CHAPTER 1 GENERAL INTRODUCTION

1.1 PHOTOISOMEWC MOLECULES

GENERAL DESCRIPTION OF PHOTOISOMERLC MOLECULES

Photoisomeric molecules are molecules that undergo a stereochemical

rearrangement between two or more isomeric forms in response to irradiation, The

direction is determined by the wavelength of the incident Iight used to effect

phot oisornerkation. Several classes of pho tochrome exkt : azo benzenes, stilbenes,

thioindigo derivatives (photoisomerization occurs across a double 'bond), spiro pyrans,

mgides (photoisomerization of (4nt2)x-electron systems), and oxiranes, azindines

(photoisomerization of (4n)n-electron systems) (for a recent review , see 1). In each

case, the photoisomerization process is reversible, although fatigue c m set in d e r

repeated isornerization cycIes. Azo'mnzenes are perhaps the best characterkd of these

photochromes, and are descriid in detail in the next section.

Azobeflzenes and their derivatives were heavily studied throughout the latter half

of the 20" century. UV-VIS spectra and rates of isomerization were examined for many

azobenzene-derived dyes in the 1960's 2-5. The rnechanism of isomerization and fàctors

affecthg isomerization were studied during the 1970s 6-8. Since the 19803, much of the

research has focused on incorporatmg azobeflzene into biological and synthetic systems,

and gaugïng the effect of photoisomerization on these systems 9-12-

Azobenzenes undergo two types of isomerization: thermal isomerization, and

photoisomerization. These processes can be understood by examinhg a simple energy

diagram of azobenzene together with the UV-VIS spectra of the trans- and cis-isomers

(figure 1.1)- From the energy diagram, we see that trans-azobenzene is

thermodynamidy more stable than cis-azobenzene, Tram-azobenzene is a conjugated

planar molecule whose n electrons can be delocaiised over the entire molecule. Steric

clashes between the ring protons in cis-azobenzene cause the moIecule to adopt a skewed

conformation

n-

440 nm

Ground State

State

cis trans

Figure 1.1 A) UV-VIS çpectrum of azobenzene (0-7 x 10' M) in isohexane at room temperature:

1, tram; 2, cis 3. B) Azobenzene photoisomerization energy diagram.

and the x electron debcalisation is dismpted. At equiliirium, in the dark, azobenzene

samples are 100% tram isomer. The trans-azobenzene UV-VIS spectrum has peaks at

440 nm (II-ir* transition) and 320 nm (mir* transition) 14. Irradiation of tram-

azobenzene at 320 n m promotes the ground state molecule to a x-n* excited state, wÏth

very little cis-azobenzene being excited. Since the energy barrier for tram-cis

isomerktion is small for excited azobenzene and very little cis-azobenzene is excited,

cis-azobenzene accumulates upon trans-azobenzene excitation_ Thus, when tram-

P d azobemne is irradiated at wavelengths correspondhg to the z-x* transition E + Z

1 conversion ïs facaated- The mechankm by which E + Z ambenzene

% photoisomerization occurs is heavily debated: photoisumerization is poshilated to occur

via either an intenial conversion mechanism, or a rotation mechanisrn (figure 1.2) 15-19.

Since the absorbance spectra of trans- and cis-azobenzene are markedly Merent, the

photoisomerization process is easily followed by UV-VIS spectroscopy. The trans-to-cis

isomerization is revealed by a strong decrease of the band at 320 nm associated with a

n-n* transition, and a coincident increase of the band at 450 nm associated with the n-n*

transition of the azo chromophore 14.

Thermal isomerization always occurs, however, the rate depends on the transition

state barrier height. The energy barrier between trans azobenzene and the thermal

isomerization transition state is greater than the barrïer between ci. azo benzene and the

thenmal isomerization transition state. Therefore, when t h e d isomerization occurs, the

amount of cis-azobenze is reduced and the amount of tram azobenzene increases Two

possible mechanisrns have been put forward for thermal isomerization of azobenzene.

One mechanism involves rotation about the N-N bond, and the other involves inversion

about the N-N double bond (figure 1.2). Although both mechanisms are possible 6-8,

more evidence exists to support the rotation mechanism.

Figure 12 Mechanisms of cidtrans thenna1 isomerization in azobenzene

Several fàçtors affect the rate of thermal isomerization of azobenzene and its

derivatives. The energy banier for cis to tram thermal isomerization rem- constant

when the reaction temperature increases. However, elevating the reaction temperature

provides a larger number of moIecules with sufZcient energy to cross the barrier, which

increases the rate of thermal isomerization 6. Aromatic ring substituents affect the rate of

thermal isomerktion by acting as electron donors and acceptors 20. This ahers the

degree of double bond character, thereby changing the energy barrier to rotation. The

rate of thermal içomerïzation has also been shown to be strongly solvent dependent 6.

Arrhenius activation energy studies show a decreased E, for thermal isomerization

react io ns when they are performed in increasingly po lar so lvent S. Increased so lvent

poIarities favour a rotation mechanism in which the contribution fkom dipolar resonance

structures reduces the nitrogen-nÏtrogen double bond strength and consequently the

torsional energy barrier to rotation. Lastly, pH c m affect the rate of azo benzene thermal

isornerization 1 722. Here, generd acid catalysis of the formation of an azoniurn

t automer intermediate facilitates rotation about the N-N bond and consequently thermal

isomerization,

Azobenzene-pho toregulation is proving to be an extremely attractive means of

switching actMties in biological and non-biological systems- This is primariIy because

photoregulation is reversible, and can be applied in a non-invasive manner (i-e. simply by

shiriing light on the rnoIecule to be regulated). Furthemore, azobenzene chemistry is

weil established, making it accessible to many non-synthetic laboratones- The prjmary

strategy for pho toregulation is to emplo y structural changes, electronic changes, and

volume changes associated with azobenzene photoisomerization in order to effect a

change in the host system, Examples of biological systerns that incorporate an

azobenzene-photoswitch as the prima.ry source of photoregulation include peptides,

enzymes, oligonucleotides, and ion cbannels.

PEPTIDES

Examples of azo-photoregdation in peptides are both nümerous and diverse,

Vollmer et al have created a peptide system composed of two cyclic octapeptides bridged

by an azobenzene moiety 23. Trans-azobenzene peptides exist as hi&-order oligomers

as a resuit of intermolecular hydrogen bonding between cyclic octapeptides. Azobenzene

isomerization causes the conversion fkom interrnolecular peptide assemblies into single

intramolecular1y hydrogen-bonded species. It is anticipated that photoswitchable

molecular self-organization of this sort couid lead to novel photoactive materials for

optical, electronic, and sensor devices. Another f d y of photoregulated peptides, which

has been studied extensively since the 1 9609s, are the azobenzene-containing polyarnino

acids z4,*5- For instance, Pieroni's group have shown that photoisomerizing

azo benzenesulfonyl side chains in po lyw-((phenylazo pheny1)donyl)-L-lyse) gives

rise to reversible helix to coil transitions in the peptide main chah 26. This phenornenon

has k e n defined as a gatedphotoresponse, in the sense that it ody occurs under specifïc

environmental conditions. Polypeptides of this sort can act as ampliners and transducers

of photoisomerization, which could make thern suitable materials for sensors, optical

switches and other photomodulated devices. Photoregdation of peptide secondary

structure has ako been accomplished in short, non-polymeric peptides 27y2*. Kumita et

al. have designed a peptide whose a-hek content can be photoregulated 1 l . In this case,

an intramolecular azobenzene cross-linker is attached vis cysteine residues at position i

and i + 7 in the sequence. The tram cross-Wer destabilizes peptide helicity, whereas

the cis cross-iinker does not. Thus, helix-to-coi1 transitions are reversiby switched in

response to azobenzene photoisomerization,

The chemicd modification of oligonucIeotides with azobenzene derivatives has

only started to attract the attention of those workïng in the field of photoregulated

biosysterns. Two approaches have been taken: to introduce azobenzene as a side chain

on the oligonucleo tide phosphate backbone 9,*9,30, and to introduce azobenzene as a

linker in the main chain of an oligonucleotide 3 1. In the fomîer case, oligonucleotides

have been prepared where the photo-induced cis-tram isomerization of azo benzene exerts

notable effects on both the physicochemical properties of the oligonucleotides and on

their duplex-forming abiüties. In the latter case, oligonucleotides modified with

azobenzene Mers have been prepared that show efficient tram-cis photoconversion

Although no-one has corne forth wah a successful application, researchers hope to

photoregdate conformational changes within DNA duplexes, triplexes, and ribozymes

via azo benzene-linked O ligonucleo tides,

On-off photostimulation of enzyme activities has been achieved for phospholipase

A2 32, a-chymotrypsin 33, papain 34, and RNase S l*,35- a-Chymotrypsin and papain

both hydrolyse peptide bonds, but their photoregulation was accomplished by very

different means- a-Chymotrypsin actnrities were regdated when the enzyme was placed

in an azobenzene-enriched copdymer to which substrate had been added.

Photostimulated a c t ~ t i e s of the polymer-encapsulated enzyme originated 6rom light-

controlled changes in permeabilîties of the substrate across the poIymer matrices. In

contrast, regulated papain activity was achieved by photostimulating azobenzene groups

that had been attached to protein lysine residues. In this case, the isomerization of

multipIe tram-azobenzene groups was believed to be accompanied by structural changes

in the protein backbone that affected the binding properties of the enzyme toward the

substrate. RNase S is a ninuciease that is activated by the non-covalent reassociation of

its S-peptide and S-protein subunits 36. Two groups have recently demonstrated that

photoregdation of RNase S activity can be achieved by the covalent attachent of an

azobenzene unit near the active site- Thus, unllre similar studies with papain, a single

modification with a photochromic moiecule is ticUfficient for photoswitching enzyrnatic

activity-

ION CHANNELS

Azobenzene-mediated photoregulation has been reported for the ion channe1

gramicidin (Stankovic, 1990 #27; Osman, 1998 #28; Lien, 1996 #29). Gramicidin is a 1 5

amiw acid peptide which forms cation-seiective ion channels in lipid membranes when

two ff3 helical monomen self-associate at thek N-termini 37.38. In work originating

fiom the Woolley group, gramkidin was rnodified at the C-terminal end with either p-

aminomethyl azobenzene or m-aminomethyl azobenzene moieties attached 12. The

proxhhy of the positively charged NH3' group to the charinel entrance and exit aEected

the channel conductance. For the tram-azobenzenes, the NH~' group was extended away

fiom the channel entrance or exit, an& it did not affect the charme1 conductance.

Photoisomerization to cis-azobenzene brought the NH3+ group nearer to the channel

entrance and exit, and channel conductance diminished accordingIy. Thus, isomerïzable

azobenzenes on gramicidin served as photogates to ion channel conductance.

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CHAPTER 2 AZOBENZENE-REGULATED

RNase A ACTlVlTY

GENERAI, DESCRIPTION of RNase A and RNase S

RNase A (Cs75Hs07N1710rs2Si2, EC 3.1.27.5) is a small 13686 Da digestive

enzyme comprised o f 124 amino acids (figure 2.1) l . Primarily, RNase A fünctions as a

non-processive endon'bonucIease. It binds the nucleobases and phosphate moieties o f

RNA substrate in several enzymatic subsites 2, after which RNA is transphosporylated

producing a 5'- RNA m e n t and a 2',3 '-cyclic phosphodiester hgment. A second,

mdependent, step involves hydrolysis of the 2',3 '-cylcic phosphodiester intermediate.

Cleavage o f the P-05' bond in an RNA &and occurs on the 3'-side of a pyrimidine

residue. Thus RNase A exhibits pyrimidine specificity 3. RNase A also shows

preference for purine nucleotides in the 5' position following the phosphodiester bond 4.

Figure 2.1 Riiuclease A bound by an ATTA-DNA inhibitor. S-protein is coloured dark blue, S-

peptide is cdoured iight blue, and two catalytic histidine residues are coloured green.

RN- A was first discovered in 1920 5, as the predorninant fom of the enqme

(hence nbonuclease A) in bos Taurus pancreas l . Since its discovery, riinuclease A has

been and continues to be one of the most heavily researched enzymes. FWase A was one

of the first enzymes to be isolated and purined in crystallioe form 6. It was the fïrst

enzyme and t b d protein for which an amino acid sequence was determuied 738. It was

the third enzyme and fourth protein for which a crystal structure was solved 99 10- As

well, RNase A was the first protein to be examuied by NMR spectroscopy 1 l, the first

protein to be unfolded and refolded in the laboratory 12, and the first enzyme for which a

gene was encoded and synthesized 13. Work in this field has led to the awarding of an

unprecedented four Nobel prizes- In 1972, Stanford Moore and Wiam H. Stein 14 were

co-recipients 'flor their contrr-bution to the understanding of the connection between

chemical structure and catalytic activity of the active centre of the ribunucleuse

moleeule" dong with Christian B. Anfinsen for "his work on ribonuclease, especially

concerning the connection between the arnino acid sequence and the biologzkally active

conformation" 12. In 1984 Robert Bruce Merrifield 1 was the sole recipient of the prize

for "his dèvelopment of methodology for chemical synthesis on a solid matrix", and

specifically for his application of this methodology to RNase A.

The mechanism for the reaction catalyzed by RNase A was rnitially fomulated in

1961 16. Although alternate catalytic pathways for RNA degradation by RNase A have

been pposed 17.18, many other groups W 2 2 have helped to substantiate the original

mechanism. The mechanism postdates that two histidines, His12 and His 1 19,

participate in two, independent processes First, a forward ~ h o s p h o r y l a t i o n occurs,

where there is an in-line attack of the vicinal 2'-hydroxyl group on the phosphorous

atom This attack is hcilitated by the imidazde side chah m Hisl2, which acts as a

B-ed base catalyst that abstracts a proton fiom the 2'-hydroxyl, and thus increases its

nucleophilicity. The imidazole side chah of His 1 19 acts cornplementarily as a Bmnsted

acid catalyst that protonates the 5"-oxygen of the l e h g group. Thus, Hisl19 facilitates

ckavage of the phosphorous-5"-O bond. Overall, the transphosphorylation step

generates a 2',3'-cyclic phosphodiester mtermediate RNA b e n t and 5'-RNA m e n t

(figure 2.2). A second reaction, which follows the forward transpho sphorylation,

involves a back-transphosphorylation, or hydrolysis, of the 2'-3 '-cyclic phosphodiester.

The roles of the histidines are reversed here, with His12 acting as a general acid catalyst

and His 1 19 acting as general base catalyst. Together, Hisl2 and Hisll9 mediate attack

of water on the 2'3 '-cyclic phosphodiester intermediate to generate a

3'phosphomonoester RNA fragment.

Figure 2.2 Putative mechanism for the processive cleavage of poly(A) by RNase A, showing B 1, B2

and B3 substrate binding subites I .

It should be noted that RNase A catalyses transphosphorylation and RNA

hydroIysis separately. 2',3 ' -cyclic phosphodiesters are not enzyme-bound intermediates,

but are tnie reaction products that are released hto solution 23. As well, the

transphophorylation reaction occurs fâster in solution than the hydro1ysis reaction 24.

His 12 and His 1 19 are the primary residues w i t h RNase A that are responsïbIe for

enzyme catalysis. Several other amho acids play roles m the catalysis of RNA

degradation by N a s e A. These amino acids, and their roles, are outiined in table 2.1.

Table 2.1 Function of different amino acids in RNase A catalysis

Amino

Acid

Lys 7 bridges with Arg39, Lys41, Lys66, HislI9 via a water netwark and

Arg 10

Gh 21

His 12

Arg 39

Lys 41

Thr 45

Lys 66

Asn 71

Asp 83

anionic phosphates m the PO, PI and P2 subslites, which stabilizes an

unfàvorable configuration of positive groups in the transition state 25.26

Coulombic interactions exkt between the ArglO side chah and the P2

phosphoryl group of bund nucleic acid which aids in substrate binding,

and depresses the microscopie pKa values of His 1 2 and His 1 1 9 thereby

dowing for op- cataiysis 27 25326

orients substrate and prevents it fiom bmdhg m non-productive mode 28

Side chah acts as a base and abstracts a proton fiom 2'-hydroxyl on RNA

substrate, which facilitates 2'-o~ygen attack on phosphorous

bridges with Lys7, Lys41, Lys66, Hisll9 Ma a water network and anionic

phosphates in the PO, P l and P2 subsites, which stabilizes an wnfavorable

configuration of positive groups in the transition state 26

hydrogen bonding stabilizes excess negative charge on non-bridging

phosphoryl oxygens b the transition date 26729

mediates pyrimidine specificity of substrate in the B1 subsite by providing

side chain steric bulk and by hydrogen bonding to a pyrimidine base 3730

bridges with Lys7, Arg39, L@I, -119 via a water network and anionic

phosphates in the PO, P l and P2 subsites, which stabilizes an unfàvorable

configuration of positive groups m the transition state 26

binds and stabilizes RNA in the B2 subsite 3 1

main chah carbonyl forms hydrogen bonds with pyrimidine base in BI

subsite, which also helps to mediate substrate specincity 3932

1 1 1 binds and stabilizes RNA in the B2 and P2 subsites

His 1 19 side chah acts as an acid and protonates 5"-oxygen on RNA substrate,

which facilitates 5"-oxygen displacement fkom phosphorous

Phe 120 main chain amide hydrogen bonds with the reactmg phosphoryl group to

help stabilizes excess negative charge in the transition state 32733

Asp 12 1 positions purme ring in B2 subsite, which in tum aligns His 1 19 via base

Factors other than direct interactions between the various amino acids in RNase A

and RNA also influence the degree of catalysis by RNase A.. RNase A is cationic (pI =

9.3) 34 at physiological pH, since the number of lys (10) and arg (4) residues outnumber

both asp (5) and glu (5) residues. Research by Record etal. 35736 suggests that RNA

biiuding mvolves seven different coulombic mteractions. These long-range coulombic

forces create a cationic environment that not only attracts polyanionic RNA, but aiso

depresses microscopie pKa values of active-site residues Hisl2 and Hisl19 27. Overall,

the effects of coulombic mteractions mcrease substrate bindmg and catalysis.

RNase A and RNase S STRUCTURE

Subtilisin cleaves RNase A between residue 20 and 21 to generate a peptidic

m e n t , ternied the S-peptide, and a protein fragment, termed the S-protem. In aqueous

solution, S-peptide reassociates with S-protein to form competent RNase S 37. There are

very few merences between RNase S and RNase A, since both have the same activity

and saxne structure,

The structures of mase A and RNase S were initially established using x-ray

crystdography methodologies 9,10,38-40. These structures either had poor resolution

(greater than 5.0 A) or, due to the crystallization technique employed, had a phosphate or

sulfate bound m the active site. T w o particuiariy good structure detenninations of RNase

A, which were devoid of bound inorganic molecules, were made m the Iate 1980's.

These structures were solved at resolutions of 1.5 A and 1.26 A 42 respectively. Soon

d e r , the solution structure of RNase A was solved by NMR methods 43-477 a d these

results were found to be nearly identical to those fiom x-ray crystallography.

Taken together, x-ray crystallography and NMR structures of RNase A can be

summarized by the following gross structural féatures. RNase A has an overail kidney

shaped structure (figure 2.1), with the active site residing in the cIeft. Prominent

secondary structure elements of RNase A include a long four-stranded anti-paralle1 beta

sheet, and three short a-helices, There are four proline residues with cis (E) peptide

bonds preceeding two of the prolyl residues. In addition, four disuEde bonds mvolvùig

all eight cysteines are present in these structures.

In addition to the fiee enzyme, crystal and solution siructwes have k e n solved

for inhibitor-bound RNase A s3 y48,49, where inhiïitors such as d(CpA),uridine vanadate,

and 2'-CMP are either subçtrate, product, or transition state analogs. Results fiom these

studies have alI helped to elucidate contact points between the various nucleobase,

phosphate, and ri'bose moieties of RNA substrate and the enzyme at various stages of

IWase action. The different contact pomts, which are important for substrate

recognition, binding and activity, have been categorized into a series of subsites

(including the active site). Table 2.2 1.2 lists the amino acids that make up each subsite.

Table 2.2 Substrate binding subsites in RNase A

Phosphate Nearby amino Nucleobase Nearby amino

binding sites acids bbdiag sites acids

Lys 1

EXPERIRlENTAL OUTLINE: CHAPTER 2

Photoregdation of RN- A activity has been attempted by several goups 5 0 ~ 5 ~ - The ability to affect RNase activÏty photochemicdy provides an alternative method of

enzyme regulation that is reversible and non-invasive towards cek. Enzyme

photoregulation is typically accomplished by insertmg a photochrome at key positions

within an enzyme. Essentidy, RNase activity is dom-regulated when the photochrome

is in one isomeric state, and not af5ected when the photochrome is in the other isomeric

state. In these cases, RNase A was chosen as a test case for enzyme photoregulation

since iîs structure and mechanism are both well documented. Knowledge of structure and

mechaliism dows one to predetermine possible sites within the enzyme where a

photochrome couid be Ïnserted that would not r e d t in the removal of mechanistically

important residues nor cause the disniption of enzyme tertiary structure, but would

effectively interfere with enzyme activity.

Previous attempts to regdate RNase S a c t ~ t y by photochemical means have met

with varied vccess. Liu et al. inserted the photochrome phenylazophenylalanine (PAP)

(figure 2.3) hto a tnincated S-peptide variant m an effort to photoregulate enzyme

activity upon isomerization of the azobenzene unit 50. When the enzyme activity of

Figure 2.3 A diagram of the nm-naturd phenylazophenylalanine (PAP) residue.

trans- and cis-isomers of PAP"-RN~S S, and P A P ~ - R N ~ ~ ~ S were compared, PAP"-

RNase S showed no Merence in Vmax upon photoisomerization and trans-pAP4-RNase

S showed a maximum activïty 25 % Iess than cis-pAP4. Another mutant, PAP'-RN~S~ S,

showed no nbonuclease activity when excess ~ ~ ~ ' - ~ - ~ e ~ t i d e was added to S-protein.

Photoisomerization between the tram- and c i s - ~ ~ ~ ~ - ~ ~ a s e S mutants was reversMe-

Hamachi et al. have also herted PAP at four different points Ïn the native S-peptide sl. When the activities of tram- and cis-isomers were assayed for each RNase S variant, they

fomd that the initial rates of P A P ' ~ - R N ~ S ~ S and P A P ' ~ - R N ~ S ~ S differed upon PAP

photoisomerization. As well, the activity of PAP'~ could be reversibly switched so that

t r a n s - ~ ~ ~ ' ~ - ~ ~ a s e S retained RNase activity and c i s - ~ ~ ~ ' ~ - ~ ~ a s e S lost its RNase S

activity- A detailed kinetic analysis of Hamachi's RNase S mutants is still pending-

Resuïts fiom both Liu's and Hamachi's research show that site-spec5c insertion of PAP

into RNûse S cm potentially lead to reversible photoregulation of enzyme activity. A

follow-up structural mdysis of tram- and cis-PAP-RNase S variations, which was

neglected in each of these studies, would aid in the understanding of enzyme

photoregulation in each case.

The research presented in this dissertation de& with two major areas of study:

creatmg a f d y of photoregdated RNase S prote&; and characterizhg each of the

modified RNase S enzymes using two dimensional NMR analysis and molecular

modehg. Photoregdation of RNase S has been based upon site-specifïc incorporation

of the photochrome phenylazophenyIalanine near the enzyme's active site, where the

chernical synthesis of S-peptide analogues has provided a convenient means for

uitroducing PAP residues to RNase S,

Overall, this research, coupled with kinetic studies of each RNase S analogue,

should provide valuable insight towards creating a detded workmg model for the

photoregulation of RNase S.

SYSNTHESIS AND PURIFICATION OF PEP7XDES

Native S-peptide was synthesized on a 0.2 m o l scde, using a Pd-resin (0.55

mmoYg capacity) and Fmoc amho acids (both purchased fkom Advanced ChemTech).

The couphg step was done using the appropriate Fmoc amino acid, HATU, and DIPEA

(in a 1 : 3: 3: 6 ratio of PAL resin: Fmoc amino acid: HATU: DIPEA) in 15 ML of NMP

solvent (HATU, DIPEA, and NMP were all purchased AIdrich). Fmoc deprotection was

accomplished using a solution of 25 % piperidine in DMF (piperidine was purchased

fiom Aldrich, DMF was purchased fkorn ACP). Couplings and deprotections were

monitored with Kaiser tests s2. Fmal cleavage fiom the PAL resh was performed ushg

a soIution of 87.5% TFA (14 mL) (Aldrich), 5% &O (0.8 d), 5% thioanisole (0.8%)

(Aldrich), and 2.5% EDT (0.4 mL) (Fhika). Cnide S-peptide was dissolved in a 65%

aqueous acetoIlitrile solution and purifïed by HPLC (9.4 mm x 25 cm Zorbax SB-Cl8

column, Perkin-Elmer 250 Binary LC pump, Perkin-Elmer LC290 UV-VIS spectrometric

detector) over 30 minutes, ushg a hear gradient (5% to 65%) acetonitrile / HzO (+ 0.1%

TFA) ehent. The structure of native S-peptide were c o h e d by eIectrospray

ionization MS (observed 143 1.2 Da; caiculated, C&,NzOO&, 2433. I Da).

Phenylazophenylalanme (PAP) was synthesized accordhg to Liu et al 50.

Peptides with PAP at position 7 (PAP7) and position 10 (PAP10) were synthesized by

HSC Biotechnology service centre (Toronto, Ontario). The prirnary peptide structures

for PM7 and PAPl O were codhned by electrospray mass spectroscopy (PAP7:

observed, 2556.4 Da; calculated, C69H9&1019Sl, 1555.1 Da, PAP10: observed, 1528.4

Da; caIcuIated, C&7N19019S~, 1527.1 Da).

S-protein was insoluble m water at elevated (mM) concentrations and denatured

over tirne. Therefore, PAP-RNase S complexes were obtahed by adding deuterated

PAP-S-peptide which had been dissolved in 99.96% &O to lyophilized, deuterated S-

protein (Sigma) m equimolar amounts. Remaining gisohible matter was iiitered, and the

resulting solution, following lyophihation, was used as a stock for NMR and UV-VIS

experiments.

NRlR SPECTROSCOPY

NMR samples were prepared for RNase A (courtesy of Dr. G.A. Woolley), native

S-peptide, S-protein, PAP7, PAP 1 O, PAP7-RNase S, and PAP 1 O-RNase S. NMR

samples of PAP7 and PAP 10 were created by dissolving the peptides in either a 60% ds-

THF: 40% D20, or a 60% d3-TFE: 40% D20 CO-solvent. NMR samples that were made

for RNase A, native S-peptide, S-protein, PAP7-RNase S and PAPl O-RNase S were

dissolved in 99.98% D20 (Sigma Aldrich). A non-D20 exchanged native S-peptide

NMR sample was also made by dissolving the peptide in 90% H20: 10% D20- TSP was

added to solutions of PAP7, PAPIO, PAP7-RNase S, and PAP10-mase S for use as art

interna1 reference. Each sample was prepared by filtering the solution through cotton

wool and then centrifuging the fiItrate for 5 rninutes. Supernatant was collected and

placed in a 5mrn NMR tube, where final pH and volume adjustments could be made. The

pH of each sample was adjusted to 4 - 5 by adding small aliquots of concentrated DCI

andlor NaOD directly into the NMR tube. An of the pH measurements were made

directly in NMR tubes using a Titrator TTT2 pH meter (Radiometer Copenhagen) and a

microCombination pH EIectrode. Direct meter readings m 40 are reported. The final

sample volumes were brought to 0.75 mL uskg the appropriate solvent or CO-solvent.

For NMR spectra that required the peptides and proteins to be deuterated, the samples

were est lyophdkd, and then dissolved m appropriate solvent and dowed to stand at

room temperature for at least 30 minutes. This procedure was repeated 3 times to e n m e

that a l l of the amide protons had been exchanged with d e u t e h The sampIes were

bubbled with either argon, or nitrogen whenever pom'ble in order to mhimize proton

exchange between NMR samples and water vapour. Concentrations were detennined

spectrophotometncally (as discussed in the follownig section: UV- VIS Spectroscopy),

assumïng E = 24000 (337 nm) for PAP 53, and E = 8989.3 (280 nm) for uncomplexed S-

protein s4. The concentrations of PAP7- and PAP10-S-peptides were determined

directly fiom A337. The concentration of S-protein in PAP-RNase S samples was

determined by subtracting A280 of PAP-S-peptide (determined independently by dividing

the A337 PAP-RNase S by the ratio of A337/A280 fiom S-peptide sample) nom of

PAP-R3Nase S and then evduathg the concentration based on the calcuiated AZ8*. D20,

dg-THF, d3-TFE, DCl, and NaOD were al i purchased fiom Sigma Aldrich

HPLC coupled to UV-VIS spectroscopy has been used to ideinte the relative

arnounts of trans- and cis-PAP isomers in dark-adapted samples and Eght adapted

samples 55. Dark-adapted PAP exists in equiliriium at 96 % tram and 4 % cis isomer,

while 337 nm light-adapted PAP exists in equilibnum at 10 % tram h m e r and 90 % cis

isomer. According~y, NMR spectra of PAP -les that were predoniinantly trans-

isomer were recorded d e r the sample had k e n placed in the dark for seven days. Trans-

to cis-azobenzene photoisomerization (for PAP7, PAP 1 O, PAP7-RN= S, and PAP 1 O-

RNase S) was accomplished by irradiating each sample at 337 nm for approximately five

hours, A nitrogen laser was used as the 33 7 nm light source- Photo isomerization

required that each PAP sample be withdrawn fÎom its NMR tube and injected into a

quartz cuvette (1 cm pathlength) that had been enclosed by a rubber septum and filled

with N2. The cuvette was continuously exposed to 337 nm light, and the NMR soiution

was mixed every 30 minutes using a syringe and needle. For the PAP-S-peptide samples,

trans- to cis-PAP photoisomerization was monitored by UV-VIS absorbance at 337 MI

For PAP-RNase S samples, complete conversion corn dark-adapted (96% trans: 4 % cis)-

PAP to iight adapted (90% cis: 10% tram)-PAP was assumed to occur d e r f i e hours,

since UV-VIS spectra h m identical samples also show complete conversion d e r five

hours. Once trans- to cis-PAP photoconversion occurred, the NMR samples were

withdrawn from the quartz cuvettes and injected into a cleau NMR tube that had ken

k h e d with N2 gas. NMR spectra of PAP compounds that were predominantly cis-

isomer were recorded at this point.

AU NMR spectra were recorded at 500 MHz on a Varian spectrometer equipped

with a Varian PFG II probe. AU spectra were obtained at 23 O C (unles otherwise noted).

For D20-exchanged samples, 1D proton NMR spectra were acquired using a basic 90"

pulse sequence with either 256 or 512 transients. For non-DnO exchamged samples

(native S-peptide), the residual water signal was removed using presaturation. Unless

otherwise noted, all DQF-COSY proton NMR spectra were obtained ushg the fonowing

protocol. A 4 0 lock was obtained and then gradient shimming was peflormed by

constructing a shirnrnap and employing an autoshimmmg routine p r o 4 e d by the Varian

software. The 90" pulse width was c a l i t e d , and spectra were obtained with 1024 t2

data points, 16,32, or 64 transients, and 256 t 1 increments. The DQ-COSY pulse

sequence provided in the Varian software with an additional homospoiI-90-homospoil

sequence preceding the d 1 relaxation delay was used for the acquisition of ail DQ-COSY

spectra 56757- AU 1 D-proton spectra were processed using version 2.3 of the Mestre-C

program- 1D spectra were processed in the following rnanner: the FID was fourier

transformed and the resulthg spectnun was phased and basehe corrected using the

polynomial baseline correction algonthm protided in the Mestre-C software. DQ-COSY

spectral processing was accompiished using the procedures outlined in appendix B.

NmrPipe and nmrDraw software were used for aU spectral processing and displays 58.

Spectral work-up was done on an SGI Octane cornputer.

UV-VIS SPECTROSCOPY

AU W-VIS spectra were recorded with a Perkin-Elmer Lambda 2

spectrophotometer. Since the rnolar absorbtivity of azobemne at 337 nrn is known to be

24000 53, the concentrations of PAP-S-peptide and PAP-RNase S solutions were

detemiined based on their absorbante at 337 nm. PAP7 and PAPlO S-peptides were

dissolved in the same solvent(s) as for NMR spectroscopy (60% ds-TH.: 40% D20 or

60% d3-TF'E: 40% D20), and placed in a 1.0 cm quartz cuvette. For samples requiring

deuteration, the protocol is descriid m the next section (as discussed in the previous

section: NUR Spectroscopy). PAP7-RNase S and PAP10-RNase S were monitored at

NMR-level concentrations in a 0.01 cm quartz cuvette (HeIlma), which ensured >95%

(based on Kd values previously determined 54 binding of S-peptide to S-protein. Once

again, these proteins were dissolved in the same solvent(s) as for NMR spectroscopy

(99.96% D20). Photoisornerization fiom trans- to cis-PAP was accomplished by

irradiating the various samples for 40 minutes (PAP-S-peptides), or five hours (PAP-

RNaseS) at 337 nm, using a N2 laser. Photoisomerization was judged cornplete when no

fbrther changes m UV-VIS traces were observed.

MOLECULAR MODELLING

The RNase S structure that was used for all of our modeis was based on an

original crystal structure produced by E.E. Kim et ai. 60. This crystal structure was

solved at 1.6 A resolution, pH 5.5, with an S04 ligand bound in the active site. The

coordinates for this crystal were downloaded directly fiom the Brookhaven protein data

bank (1RNV.pdb) ont0 an SGI Octane. This RNase S structure was then modifïed using

Hyperchem software (Hypercube Inc.) as folIows: The S 0 4 Iigand was removed,

residues one, two, and three from the N-terminus (S-peptide side) were removed, EN on

Hisl2 was deprotonated, Ma4 (now Alal) was acetylated, and the Ctemimal Ser 1 5 (now

Serl2) was changed to an amide. The nnal charge of the S-peptide was +2 and the S-

protein was +7, which represented the ionic state of RNase S residues at pH 7. Lastly,

the entire complex was geometry optmiized, and put through a molecular dynamics

simulation.

John Karanicolas and Professor GA. Woolley had already modelled and

parameterized tram- and cis-phenyla~ophenylalanine~ Both tram-PAP and cis-PAP were

incorporated mto the Hyperchem amho acid iiirary and used in our studies. PAP7-

mase S and PAP 10-RNase S were created by mutating Lys7 or Arg 1 O for tram-PAP

and cis-PAP residues. Thus, four models were produced: one with trans-PAP7, one with

cis-PAP7, one with tram-PAP 10, and the other with cis-PAP 10. Following mutation into

RNase S, the PAP residue was m i n b k d by the steepest descent algonthm (1000

cycles), and then the Polak-Riiihe algorithm (gradient RMS = 0.00 1). These

minimi7ations were performed keepmg all of the other residues in RNase S constrained,

with the PAP side chah positioned so that van der Waals contacts with other atoms of the

enzyme do not exist.

To determine which conformations of PAP were energeticaw allowed, the

following combinatorid approach was takea Three embedded Hyperchem scripts were

created that, in total, calculated the single point potential energies for 13824 dinerent

conformations of PAP. For each RNase S mode4 the potentid energy slnface of PAP

was detennined by rotating ~ 1 , ~ 2 , and ~3 torsion angles of PAP while evaluating Ïts

potenta energy. Figure 2.3 defines the torsion angles ~ 1 , ~ 2 , ~ 3 , and ~ 4 . The fïrst

script rotated ~1 15 degrees and c d e d upon the second script, wbich rotated ~2 15

degrees and called upon the third script, which rotated ~3 15 degrees and calculated the

single point potentid energy of the PAP residue. The third script continued to calculate

PAP single point energies unti l~3 was rotated through 360 degrees. M e r the third script

was completed, Hyperchem reverted to the second script, which rotated f l another 15

degrees and called the third script aga i . The second scnpt continued unt i l~2 was

rotated through 360 degrees, whereupon Hyperchem reverted to the fist script, which

rotated ~1 through another 15 degrees and recded the second script. Overall, the PAP

side chain was rotated through 360' x 360" x 360° for torsion angles XI, ~ 2 , and ~ 3 ,

allowing the potential energy surface of PAP was to be sarnpled in 243 dinerent places-

Hyperchem log files were generated for these cdculationç, where the potential energy,

and matching ~ 1 , ~ 2 , and ~3 torsion angles were included as part of the total information

output. AII of the ~ 1 , ~ 2 , and ~3 coordinates and their conesponding potential energies

were extracted from these log files and written to a table in ascii format using a simple

PEN? script (Appendix A). Three-dimensional potential energy suditces were

generated fiom this data using the program A m . These three dimensionai surfaces were

evaluated to d e t e d e which combination of ~ 1 , ~ 2 , and ~3 angles gave the lowest

energy PAP conforniers. ~3 and ~4 were set to 180" and O0 for trans-PAP7-RNase S and

for trans-PAP 10-RN= S, while ~3 and ~4 were set to 75' and 55' for cis-PAP7-RNase

S and for cis-PAP10-RN= S. At this point, the Hyperchem scripts were re-mu, rotating

only ~1 and ~2 m 4-degree increments. This allowed for a more compIete sarnpling of

the PAP potential energy sadace. Once again, a PERL scnpt was used to extract the

potential energies fiom each log fle and AXUM was used to display the new surfàces.

M e r analysis of these potential energy d a c e s , molecular models of (tram-, and ch-)

PAP7-RNase S and (trans-, and cis-) PAP10-RNase S were created that had the PAP

residue oriented in a low-energy position. These models were developed in Hyperchem

and displayed using GRASP 61.

2.3 RESmTS

S-PEPTIDE

Figure 2.4 Primary amino acid sequence of the modified S-peptide- The numbers below each amino

acid correspond to residues in native S-peptide.

SynihesrS, Punycation and NMR Spectroscopy

A peptide comprising residues 4-15 of the S-peptide (figure 2.4) was synthesized

using standard Fmoc SPPS procedures. Using electrospray mass specirometry, we were

able to CO& that the two major peaks in the HPLC trace of the S-peptide SPPS

product mixture were rnet(0)-S-peptide (MW 1447), and reduced S-peptide (MW 143 1).

ID proton NMR anafysis was &O used to distinguish the different S-peptides. Figure

2.5a shows the 1-D proton spectrum obtained fiom Dfl exchanged S-peptide (peaks

labeled), while figure 2.5 b shows the 1D NMR of met(0)-S-peptide. It should be noted

that reduced S-peptide was used for aU of the subsequent NMR andysis. Once the

met(0)-S-peptide was separated fiom the reduced S-peptide, non-DzO exchange spectra

were recorded for the S-peptide. ID-presaturation, presat-TOCSY and ps-DQCOSY

spectra were obtained (figures 2.6,2.7, and 2.8) and each peak of the S-peptide spectnim

was identified.

F i 2.5 A) ID 'HNMR of deuterated reduced S-peptide (6.2 mM, 99-96% D20, pH 3.2,23 OC),

B) ID 'HNMR of deuterated met(0)-Speptide (17.4 mM, 99.96% Da, pH 3.17,23OC). Areas that have

been asterisked indicate differences W e e n the two spectra.

Figure 2.6 1D 'HNMR of the S-peptide (6.2 m M S-peptide, 90% &O: 10% DzO, pH 3.2, 23"C),

using a presaturation pulse sapence to eliminate the water signal.

PA' S-PEPTIDE ANALOGS

UV-VIS and NMR Spectroscopy Molecular models were created and analyzed to help ident* where PAP should

be inserted in the S-peptide sequence to achieve maximum RNase S photoregdation. It

was evident £iom initial inspection of these models that two possible candidates for

photoregulaion were PAP7 and PAP 10. Both PAP7 and PAP 10 were synthesized at the

HSC Biotechnology seNice centre (Toronto, Ontario) on a NovaSyn Crystal automated

peptide syiithesizer. UV-VIS spectra were acquired for each peptide to determine XPAP

tram-cis photoisomerization could occur in the context of the S-peptide (figure 2.9a,b).

The spectnmi for trans PAP7 (1 1.2 pM, 60% THF: 40% HzO, pH 4.0,23OC) Iias maxima

at 323 nm (z - x* transition) and at 433 nm (n - n* transition). Tram- to cis-azobenzene

photoisornerkation was obtahed by irradiatmg the PAP7 solution at 337 nm for 30

minutes. The UV-VIS trace of the corresponding cis-PAP7 sample does not have a peak

at 323 nm and there is an increased absorbance for the 433 nrn peak. The UV-VIS

spectnim of PAPlO (5.5 pM, 60% THF: 40% HD, pH 6.0,23"C) was similar to PAP7:

x: - z* transitions occurred at 322 nm and n - n* transitions occurred at 422 nm The

maximum at 322 nm (n - x*) dïsappeared and the maximum at 422 nrn (n - n*)

increased folIowing a 40 minute exposure to 337nm light. Recovery oithe tram-

azo benzene spectnim of each S-peptide variant (same conditions) was monitored either at

330 nm (PAP'i), or 329 nm (PAP 1 O). During the nIst ten hours, the rate of recovery for

trans-PAP7 and tram-PAPIO were 0.7 percent / hou. and 1-0 percent / h o u respectively.

Overall, 100 % recovery of tram PAP was observed for both peptides. Chromophore

photobleachg could no t be detected after multiple cycles of pho toisomerization (3 3 7

nm) and relaxation.

Figare 2.9 A) The initial trans-PAW spectrum is colourd r d , the cis-PAP7 spectnim is coloured

blue, and the recovered tram-PAP7 spectmm is coloured green. The maximum percent recovery of trans-

PAP7 was 80.5 %. B) The initial trans-PAP10 spectrum is coloured i . ~ red, the cis-PAPI0 spectrurn is

coloined green, and the recovered b-ans-PAPI0 spectrum is coloured blue.

Photoisomerization of PAP m the S-peptide was examined by both ID and 2D

proton NMR spectroscopy. Study of the PAP 1 O sample was problematic smce Ï t

gradually precipitated at elevated concentrations (mM), as was confirmed by peak

broadening (data not shown) m the correspondhg NMR spectra ID NMR spectra could

only be produced for deuterated trans-PM10 (2.13 mM, 75% d2-TFE: 25% D20, pH 1.1,

23OC), and with poor signal to noise ratios (figure 2.10). The solubility of PAP 10 was

tested in a variety of solvent systems (TFE: water, DMSO: water, etc-), but was insoluble

at mM concentrations (NMR level) in each case. Therefore, S-peptide NMR studies were

only performed on the PAP7 S-peptide variant-

Figure 2.10 1D 'HNMR of deuterated trans-PAP10 (2.13 mM, 75% d2-TFE: 25% Dfl, pH 1.1,

23OC) referenced with TSF,

Initially, these studies were performed on a deuterated PAP7 sample in either a 60% d2-

TFE: 40% D20 or 60% ds-THF: 40% D20 CO-solvent ID-NMR spectra were recorded

for PAP7 (2.0 mM, d&ïEE: D20, pH 5.6,23 O C ) and the azobenzene peaks were

followed tlrmughout the pho toisomerhtion process (figure 2.1 1 a,b). Tram azobenzene

protons yield peaks at 7.66 ppm @&,)y 7.94 pprn Wb), 7.8 1 ppm @), 7.3 5 ppm (EEd), and

7.43 ppm (EL) (table 2.3, figure 2.12). A second group of peaks appear at 6.98 ppm (Ha),

6.78 ppm a), 6-91 pprn (Hc), 7.42 ppm m), and 7.30 ppm (IE) following five hours of

irradiation at 337 nrn (table 2.3). These peaks correspond to the protons of cis-

azobenzene and were present m the spectrum in addition to the original tram-azobenzene

peaks. Recovery of the original tram-PAP7 spectnim was possi'bIe after the peptide was

kept m the dark for three days (data not shown).

Figure 2.11 1D lHNMR of PAW (2.0 mM, pe5.6, 60% drTKf: 40% &O), A) afkr exposure to

suniight for one week B) after king placed in the dark fot one week, The relative intensities of tram-

and cis-azobenzene peaks can be observed to shift for the light- and dark-adapted PAP7 sample.

Men this experiment was repeated, the ci.-azobenzene peak volumes could not be

uicreased, even d e r longer 337 nm exposure times with this light source. Phenylalanhe

protons gave rise to all of the remaining signals m the aromatic region of the NMR

spectrum. 1D NMR spectra fiom PAP7 in a d2-TFE: DrO co-solvent were not

si@cantIy different fiom those m a d8-THF: DZO co-solvent (data not shown).

Table 2.3 Chernical Shi% of PAP in PAP7, PAW-RNase S, and PAP10-RNase S

Sample, Residue Proton 6 + O.Ol(ppm)

PAP7-S-peptide (PAP7) Ha 7.66 (tram), 6.98 (ch)

H b 7.94 (tram), 6.78 (CG)

& 7.81 (tram), 6.91 (cis)

Ha 7.35 (tram), 7.42 (cis)

& 7.43 (tram), 7.30 (cis)

PAP7-RNase S (PAP7) Ha 7.62 (trans), 7.28 (CG)

H b 7.78 (trans), 6.85 (cis)

a 7.96 (trans), 6.90 (cis)

Ri 7.69 (tram), 7.20 (cis)

Hi 7.39 (trans), 7.32 (cis)

PAP 10-RN- S (PAP 1 O) Ha 7.73 (tram), 7.82 (ck)

Hb 7.45 (trans), 7.18 (cis)

& 7.54 (trans), 6.89 (cis)

E3[d 7.75 (trans), 7.26 (cis)

& 7.40 (trans), 7.21 (cis)

PAP7 (2.0 mM, pH 5.6,23 OC), PAl%RNase S (0.64 m M S-peptide, 0.43 rnM S-protein, pH

5.423 OC), PAP 1 O-RNase S (0.34 m M S-peptide, 0.30 mM S-protein, pH 4.1,23OC)

2D DQCOSY HNMR spectra were also recorded for both tram- and cis-PM7

(2.0 mM, 60 % dg-THF: 40 % DzO, pH 5.6,23 OC). When compared to the analogous 1D

HNMR spectra, these DQCOSY spectra reveal tram- and cis- azobenzene peaks that are

eady identined and weU-resolved (figure 2.12). Smce there are t h e cross peaks apiece

for tram- and cis-azobenzene, the ten protons in trans- and cis-azobenzene can be

assigned according to their coupling pattern (table 2.3). Peaks are also observed for the

phenyl ring protons in pheny1ahhe (7.30 ppm - 7.50 ppm), although they are not wefl

resolved.

Figure 2.12 'H-DQCOSY of PAW (2.0 mM-, pH 5.6,60% drTHF: 40% w) following five hours

irradiation at 337 nm. The ring protons (a - e) of tram and cis-azobenzene are observed.

RNase S AND RNase A

UV- MS and NMR Spectroscopy

An analysis of S-protein was attempted in order to make chemicd shift

cornparisons between active site residues of S-protein and PM-RNase S. UnfortunateIy

this approach was not tenable since the protem was not stable in solution at NMR

concentrations. Solutions containhg S-protein (2.45 dl, pH 8.0,99.96 % D20) became

cloudy after tirne, and eventually yielded sipificant insoluble matter. Gentle heating

and/or sonication of the S-protein solutions didn't relieve the problem of insolubility.

W-VIS spectra show increased Rayleigh scattering after time (figure 2-13), which

suggests increased particdate matter in solution due to the formation of insoluble

denatured S-protein. NMR spectra show peak broadening, which can be caused by the

presence of multiple S-protein variants in the NMR tube during the spectnim acquisition

and by decreased turnblùig rates (figure2.14).

220 270 320 370 420 470 520 m Waveleagth (nm)

Figure 2.13 A) S-protein UV-VIS spectnun (pH 8-0, 100 % ho). B) S-protein W-VIS spectnmi

foiiowing filtration. Significa. Rayleigh scatterhg (Le. exponential increase ofthe UV-VIS absorbance at

decreasing wavelengths) is observed in the first spectrum resulting fiom precipitated S-protem in the

cuvette. The Rayleigh scattering c m be reduced after nitration, however it is not eiiminated.

NMR ana.lysis of a deuterium-exchaaged RNase A sample (5.0 mM, 99.96%

m, pH 4.0,23 OC) was also performed. There are six tyrosine residues and three

phenylalanme residues in native RNase A (and RNase S), and protons fkom the phenyl

rings of these residues gave rise to signals in the aromatic region. Cross peaks fiom these

signals were identifiai in the DQCOSY spectrum of RNase A (figure 2.15).

Figure 2.14 1 D 1 HNMR spectnrm of RNase S-protein (2-45 mM, 99.96% D20, pH 8,0,23"C), NMR

conditions. This spectrum is severely broadened.

Figure 2.15 Aromaîic region of the DQCOSY spectrum of RNase A (5.0 mM, 99.96 % &O, pH 4.0,

23OC)-

PAP-RNase S MWTANTS

W - W S and NMR Speciioscapy

W-VIS, and NMR spectroscopy of the RNase S mutants were used to monitor

PAP photoisomerization. Trans-PAP7 RNase S (0.64 mM PAP7,0.43 mM S-protein,

99.96 % D20, pH = 5.4,23 OC) and trans-PAP 10 RNase S (0.34 mM PAP IO peptide,

0.30 mM S-protein, 99.96 % DzO, pH 4.1,23 OC) UV-VIS spectra show maxima at 325

nm and 432 nm, corresponding to x - n* and n - n* transitions in the azobenzene

chromophore, and at 277 nm, corresponding to n - z* transitions in tyrosine and

phenylaIanine side chains (figures 2.16% fig 2.1 7a).

200 300 400 500 600 I Wavelength (nm)

Figure 216 A) UV-VIS spectrum of dark adapted (tram-) PM-RNase S (red trace) and irradiated

PM-RNase S (green trace). PAP7-RNase S sample was prepared with 0.64 m M P M , 0.43 mM S-

protein in a 99.96% M soIvent, and adjusted to pH 5.4. B) Percent recovery of trans PAW-RNase S

fiom cis-mase! S.

Figure 2.17

O 1 O 20 30 40

Time @ours)

A) UV-VIS spectrum of dark adapted (tram-) PAP10-RNase S (green trace), irradiated

PAPBO-RNase S (red trace), and recovered trans-PAPlO-RNase S @lue trace), The PAPIO-RNase S

sample was prepared with 0.34 mM PAPIO, 0.30 mM S-protein in a 99.96% D20 solvenf and adjusted to

pH 4- 1, B) Percent recovery of tram-PAP1 0-RNase S fiom cis-PAP 10-RNase S,

PAP photoisomerization occurs in both RNase S mutants foltowing fke hours of

sample irradiation at 337 nra Photoisomerization of tram PAP to cis-PAP is marked by

the decrease of the peak at 322 nm and the increase in absorbance of the 432 nm peak

(figuses 2.16b, 2.1 7b)). The process is fulty reversibe and the tram-spectrum can be

recovered when the cis-PAP7-RNase S and cis-PAP 10-RNase S samples are placed in the

dark for three days. During the fïrst ten hours, the rate of recovery for trans-PAP7-RNase

S and tram-PAP10-RNase S were 0.7 percent / hour, and 1.5 percent / hour respectively.

As with the S-peptide samples, 100 % recovery of trans PAP was obsewed for both

proteins. Photobleachmg of the azobenzene chromophore was not detected, even after

multiple photoisornerization cycles.

Preliminary ID proton NMR spectra were recorded on deuterated PAP7-RNase S

(0.64 mM PAP7-S-peptide, 0.43 mM S-protem, 99.96% DzO, pH 5.4,23 OC) and

deuterated PAP10-RNase S (0.34 mM PAPIO-S-peptide, 0.30 mM S-protein, 99.96%

DzO, pH 4.1, 23 OC) samples (figure 2.1 8,2.19). AU of these spectra exhibit poor

resolution due to the high number of RNase S protons giving signals. There are,

however, peaks in the aromatic region (6.8 ppm - 8 -0 ppm) whose intensity either

increases, or decreases foliowing azobenzene photoisornerization. It was believed that

these were azobenzene proton peaks, so two-dimensional NMR spectroscopy was used to

resolve the various spectra.

Figure 2.18 A) 1D 'HNMR of tram-PAW-RNase S (dueterated, 0.64 mM PAPFSpeptide, 0.43

mM S-protein, 99.96% &O, pH 5.4). B) 1D 'HNMR of cis-Pm-Nase S (same sample conditions as

tram-PAP7-RNase S). Peaks that have been asterisked indicate major differences between the two spectra

Figure 2.19 A) 1 D 'HNMR oftrans-PAPl 0-RNase S (deuterated, 0.34 m M PAPl O-S-peptide, 0.30

m M S-protein, 99.96% DzO, pH 4.1). B) 1D 'HNMR of cis-PAP10-RNase S (same sample conditions as

tram-PAPIO-RNase S). Peaks that have been asterisked indicate major diGerences between the two

SP-

2D-DQCOSY spectra were recorded using the same PAP-RNase S samples that

were used for the 1D proton NMR analysis. Spectra fiom tram-PAP7-RNase S and

trans-PAP10-Rnase S samples were obtained first (figure 2.20,2.22). The corresponding

cis-PAP7-RNase S and cis-PAP 10-RN- S spectra were acquired foilowing tram-cis

photoisomerization in the azobenzene chromophore (via five hours exposure to 337 MI

light) (figure 2.2 1,2.23). The results of these studies are outlined in table 2.3. AU of the

tram-azobenzene protons codd be assigned in the trans-PAP7-RNase S and trans-

PAP10-RNase S spectra. As well, peaks corresponding to cis-azobenzene protons were

not observeci in any of the tram-PAP-RNase S spectm Cis-PAP7-RN- S and cis-

PAP10-RNase S spectra show peaks that orïginate fiom both cis and trans azobenzene

protons. Consequently, it was easy to dïsîinguish cis-azobenzene peaks fiom the

previously assigned trans-azobenzene-peaks. There is an upfield shat for all of the cis-

azobenzene signais relative to the tram-azobenzene signais- Our RNase S mutants bave 6

Q~osine and 3 phenylaianhe residues apiece, and aU of the ring protons fkom these

residues can be accounted for in each spectrum.

1

c 7z Cd

rn e . . * 2x tyr 7, 3 @ m !' 7.4

Figure 220 The DQCOSY NMR spectrum of deuterated transPAP7-RNase S (0.64 m M PAP7-S-

peptide, 0.43 m M S-protein, 99.96 % D20, pH 5,4,23 OC) following storage in the dark for three days.

Phe x 2 c \ = : b .. a?

Figure 231 The DQCOSY NMR spectrum of deuterated cis-PAP7-RNase S (0.64 m M PAP7-S-

peptide, 0.43 m M S-protein, 99.96% D20, p H 5.4,23 OC) following sample irradiation at 337 mu for five

hours. Peaks are observeci for protons fiom the tram- and cis-azobenzene side chains.

l Tyr x 3

Figure 2.22 The aromatic region of the DQCOSY NMR spectrum of deuîerated trans-PAP10-RNase

S (0.34 m . PAPIO-S-peptide, 0.30 m M S-protein, 99-96% D20, p H 4.1,23 OC). Crosspeaks are observai

for protons from trans-azobemzene (iabeled A through E) and for the six tyrosine and three phenykianine

residues in RNase S.

Figure 2.23 The aromatic region of the DQCOSY NMR spectrum of deuterated cis-PM-RNase S

(0.34 mM PAP10-S-peptide, 0.30 rnM S-protein, 99.96% DzO, pH 4,1,23"C) following sample irradiation

at 337 nm for f i ie hours. Peaks are observed for protons fiom the tram-azoknzene (A through E) and cis-

azobenzene (a through e) side chains. Al1 of the cis-azobenzene cross peaks have been circled m red to aid

in their observation and identification, The tyrosine and phenylalanine cross peaks have not been labeled;

however they retain the same chernical shifts that were observed in the tram-PAP10-RNase DQCOSY

NMRspectrum-

Figure 224 Recovery of deuterated tram-PAPI 0-RNaseS fiom cis-PAPl O-RNaseS as followed by

DQcOSY NMR (0.34 mM PAPlû-S-peptide, 0-30 rnM S-protein, 99.96% D20, pH 4- 1,23*C). A) after O

hours, B) after 24 hours. C) after 48 hours, D) after 72 hours.

There were no additional cross peaks observed in the aromatic regions of these spectra.

The original trans-PAP7-RNase S and trans-PAP 10-RNase S spectra were recovered d e r

the soIutions were allowed to sit in the dark for three days (data not shown).

Recovery of tram- fiom cis-PAP-RNase S was studied in greater depth for

PAP10-RNase. This was dune by monitoring changes m trans- and cis-azobenzene peak

intensities over successive DQCOSY NMR experiments (figure 2.24 a-d). Acquisition of

the fnst spectrum began hediately after photoisomerization was mduced in the

PAP IO-RNase sampk. Peaks eom both cis and tram azobenzene were observed in this

spectrum. Additional spectra were acquired every eight hows, and these show a

contnlued decrease m cis-azobenzene peak intensities coupled with an increase in trans-

azobenzene peak intensities. A cornpIete regeneration of the tram-P AP 1 O DQCOSY

spectrum occurred a e r 72 hours.

Molecular Mo&lIing

Models of the structurai interactions between tram- and cis-PAP and the rest of

the RNase S enzyme cm be used to help determine why our PAP-RNase S mutants have

photoregulated activities. We were able to create a senes potentid energy surfaces for

PAP in RNase S by cdculating single point energies of PAP whiIe simultaneously

rotating the ~ 1 , ~ 2 , and ~3 torsion angles, and keepmg ~4 constant (figure 2.3).

P M potential energies were evaluated at 2 1 924 different combinations of x 1, ~ 2 ,

and ~3 for each trans/cis-PAP-RNase S mutant, which allowed for a thorough sampling

of the entire PAP potential energy surface in both trandcis-PAP7- and tradcis-PAP 10-

RNase S. The different potential energy surfaces that are generated for PAP have

minima that correspond to energetically dowed positions for PAP in RNase S. Simple

single-pomt energy calcdations on the PAP side ch& (see materials and methods) reveal

that the p o t e n a energy surface of trans-PAP7 (in RNase S) has energy minima at the

following combinations of ~ 1 , ~ 2 , and ~3 (figure 2-25a):

-44' < ~1 < - l32', -52' c ~2 < 8g0, ~3 = 0°, 1 80° (when ~4 = 0')

-52' < -124', 140' < ~2 < -88O, x3 = 0°, 180' (when x4 = O")

The potentîal energy surface of cis-PAP7 (m RNase S) exhi'bits energy minima for ody

one combination of ~ 1 , ~ 2 , and ~3 (figure 2.25 b):

-44' < x1< -84', -52' < x2 < 0°, x3 = 5507 -125' (when x4 = 55")

-76" < XI < -132', -45' < x2 < -88O, x3 = 5S0, -125O (when x4 = 557

AU of the different PAP conformers having potential energies beIow O f i 0 1 were

overlaid onto a mode1 of RNase S, and Grasp 61 surfêces were generated representing the

conformational space availabe to the trans- and cis-PAP7 side chah These models

show that trans-PAP7 can occupy two different areas on the RNase S enzyme. In the fkst

area, trans-PAP7 doesn't interfere with the RNase S active site, however, when trans-

PAP7 occupies the second area it sits in the active site cleft between the two catalytic

histidine residues (figure 2.27 a). The cis isomer of PAP7-RNase S is removed fiom the

active site and doesn't bbck it at d (figure 2.27 b). The sarne analysis was performed

for PAP- 10 RNase S. The potential energy d a c e of trans-PAP 1 0 (in RNase S) has

energy minima at

-44O < ~1 < -148'' -180° < ~2 < 180°, ~3 = O", 180" (when ~4 = 557

(figure 2.26a), while the potential energy d a c e of cis-PAP 10 (in RNase S) has energy

minnna at

-80° < ~1 < -128; -40' < ~2 < -144°, ~3 = 5S0, -125O (when ~4 = 554

-88O < xi < -52O, 40' < x2 < 104°, x3 = 5So7 -125' (when x4 = 550)

(figure 2.26b). Once again, models were constructed by overlaying PAP conformers

corresponding to energy minima on the potential energy surfaces ont0 a mode1 of RNase

S. When Grasp surfaces of PAP 10 were generated, we observed that neither trans- nor

cis-PAP 1 O blocked the active site (figure 2.28 ah).

Figure 2.25 The potential energy surfiices of A) trans-PAP7 with both ~3 and ~4 set at O*, and B) cis-PAf7 with both 113 and ~4 set at 55'.

Figure 2.26

at 55'.

The potential energy surfaces of A) trans-PAPlO with both ~3 set at 180' and ~4 set at 0°, and B) cis-PAP10 with both ~3 set at 75' and ~4 set

Figure 2.27 Grasp surfaw mode1 of A) trans-PAP7-RnaseS and B) cis-PAP7-RNase S. RNase S is coloured light blue, the active site residues (His 12,

Hisl19, and Lys 41) are coloured dark blue and PAP7 is coloured aqua. PAP positions were derived fiom minima on the carresponding potential energy surfaces

for PAPiO-mase S.

Figure 2.28 Grasp surface model of A) hans-PAPIO-RNaseS and B) ois-PAP10-RNase S. RNase S is coloured light blue, the active site residues (Hisl2,

Hisll9, and Lys 41) are coloured dark blue and PAPlO is coloured aqua, PAP positions were derived from minima on the corresponding potential encrgy

swhces for PAP1 0-RNase S.

2.4 DISCUSSION

It is well hown that S-peptide binds S-pro tem reversiily to form competent

RNase S 37.62. As well, S-peptide can be modifïed to contaui an azobenzene moieS;

which dows for the incorporation of a photoisornerizable group into RNase S 50.51.

Our a2m was to study the structural properties of PAP-RNase S interactions through

spectroscopic analysis and rnolecular modelling, and to relate these results to activities of

each enzyme.

S-PEPTIDE A preliminary mvestigation of the S-peptide by NMR was necessary m order to

determine suitable techniques and conditions for the fùture study of PAP-S-peptide

analogs. For our studies, we chose to work with a truncated version of the S-peptide (S-

peptide-2), where the fist three and lad five residues are not included as part of the

peptide. This is the shortest sequence that gives fbll activity. The dissociation constant

for S-peptide-2 ( ' = 5.48 f 0.82 x 10" M) 59 is Iarger than the native S-peptide

dissociation constant (& = 7.0 x IO-' M) 63. Atthough removing eight amho acids on

the S-peptide compromises its binding to S-protein, these bmding constants for S-

peptide-2 (and PAP7 and PAP IO) are sW1 withui an acceptable range for activity and

NMR and spectroscopic studies,

S-peptide-2 contains a single methionine residue, which is prone to oxidation

during the solid phase peptide synthesis process. HPLC and ESMS revealed two major

products fiom S-peptide-2 synthesis. These were identified as met(0)-S-peptide02 and

reduced S-peptide-2 based on the mass merence. We also observed subtle clifferences

between the 1D NMR traces for met@)-S-peptide and reduced S-peptide. The largest

differences between the two spectra occur at 1.75 ppm, 2.35 ppm, 3.20 ppm, and 4.05

ppm where peaks can be observeci for reduced S-peptide and not for met(0)-S-peptide.

Any M e r NMR anaiysis would be complicated ifthe two peptides were not separatecl,

smce there wodd be multiple methionine cross peaks to account for in the spectra

Consequently, reduced S-peptide-2 was pudieci by HPLC and used exchrsiveeîy for d of

the remainùig NMR experiments. DQCOSY and TOCSY spectra were obtained for

native S-peptide, and we were able to accolmt for all of the cross peaks in these spectra

(figure 2.7). Therefore we believed that the NMR conditions employed for the native S-

peptide spectra wodd be suitable for the analysis of PAP-S-peptide and PAP-RNase S

mutants.

PAP S-PEPTIDE ANALOGS

Our strategy for photoregulatuig RNase S activity requires the addition of a

photochrome (PAP) hto the S-peptide, whereby the binding of PAP-S-peptide to S-

protein is not compromised. Photoregdation can occur ifthe PAP side chah interferes

with substrate bmding by sterically blocking the enzyme active site. Stenc blocking must

occur for one isomer of PAP onIy, and should be relieved for the second isomer.

Of course, this type of enzyme regulation can only be possible ifeither tram- or

cis-PAP is situated at a position on the S-peptide that brings it m close proxmiity to the

active site. A series of molecular models were constnicted for RNase S witb tram-PAP

substituted at each position in the S-peptide. From these mdels, we obsenred that tram-

PAP is near the active site when it is substmited at the seventh (Lys7) and tenth (ArglO)

position of the S-peptide. PAP substitution at any O ther site m the S-peptide was deemed

unsuitable for a variety of reasons: either the PAP side chsin would not have access to

the active site, or PAP susbstitution would involve removing an amino acid that is cntical

for the chernical step m nbnuclease mechanistic function @Lis12 l, Glnl 1 59, or PAP

substitution wouid signincantly compromise the binding of S-peptide to S-protein (Phe8

50, Met13 64). Table 2.4 shows that mutations at position 7 and 10 of the S-peptide do

not signincantly compromise S-peptide binding to S-protein when cornpared to S-

peptide-2 59. Based on these arbouments, we proceeded with the synthesis of PAP7-S-

peptide and PAPI 0-S-peptide.

Table 2.4 Dissociation Constants for PAP7-RNase S and PAPIO-RNase S 59

S-peptide Analogue Kd in tram (XI 04 M) & m ck (X 1 o - ~ MJ

The tram to cis azobenzene photoisornerization process was examined for PAP7

and PAPlO followÎng their synthesis and purification UV-VIS and NMR spectroscopy

together proved to be excellent tools to follow azobenzene photoisomerization in the two

peptides. Both of the PAP-S-peptides exhi'bited limited solubility in polar soivents.

Instead, solvent mixtures 60% d8-THF: 40% D20 or 60% d2-TFE: 40% D20 were found

to be suitable. Although PAP7 and PAP 10 were fùlly soluble in 60% ds-THF: 40% &O

at pbf (UV-VIS) concentrations, we employed a TFE as a CO-solvent since TFE is known

to the increase a -hek propensity in peptides 65, including the S-peptide 66. Since the S-

peptide adopts an a-belical structure when bound to S-protein 38.60, PAP peptides in a

TFE solution may be more structuralEy similar to theÎr S-protein-bound counterparts.

UV- MS Spectroscopy of P M Peptides

The UV-VIS spectra of trans-PAP7 and tram-PAP 10 are very s i m ï k they both

have mrvrima that are typical of tram-azobenzene compounds 67-69. M e r the samples

are exposed to 337 nm light, their W-VIS traces change to resemble those of other cis-

azobenzene compounds. Cis-PAP-S-peptide W-VIS traces revert to trans spectra afkr

three days in the dark, a phenornenon that is a h common amongst azobenzene

compounds. Lastly, when compared to the PAP amino acid 59, our PAP-S-peptide

spectra exhiiit nearly identical spectral properties: n-n* and n-n* transitions occur at

simïIar wavelengths, there are s d a r relative intensities for these transitions, and the cis-

isomer half-lives are comparable. Based on ail of the results of these W-VIS studies, we

conclude that replacing PAP at positions 7 and 10 in the S-peptide does not hinder the

azobemne photoisornerkition process.

MMR Spectroscopy of PAP Peptides

The NMR spectral properties of the PAF7 and PAPlO peptides were then

investigated in the same solvents as used for TV-VIS spectroscopy (i-e. eithet 60% da-

THF: 48% D20 or 60% d2-TFE: 40% D20 CO-solvents). Sampie deutemtion reduces the

number of signals in the amide and aromatic regions of NMR spectra and, consequently,

azobenzene proton signais are more likely to be well resolved and much easier to analyse.

The sample concentrations tbat are required for NMR spectroscopy (> 1 KIM)

caused the PAPI O peptide to precipitate, and therefore discussion is limited to NMR

spectra nom PAP7. For the trans-PAF7 peptide, the aüphatic region of the 1D proton

spectnim was poorly resohed. This w s expected since there are over 65 protons that are

expected to have NMR signals in this region. The numbers of signais in the aromatic

region were si@cantly fewer, so subsequent NMR analyses were focussed on that

region of the spectnim When trans-PM7 peptide spectra and native S-peptide spectra

were compared, the tram-PM7 peptide spectra had additional peaks that were noticeably

absent in the aromatic region. These extra signals in the P M 7 peptide NMR spectra

were assigned to trans-azobenzene protons. This assignment was vefied by

photoisomerizing the trans-PAP7 peptide sample and acqun-ing the NMR spectnim of

partiaily cis-PAP7. Photoisomerization ahered the chernical shifts of the azobenzene

peaks as expected, since trans- and cis azobenzene protons have different electronic

environrnents-

At room temperature, cis-PAP7 peptide reverts to bans-PAP7 peptide at a rate of

0.7 % per hour during the fïrst ten hours following photoisomerization (based on UV-VIS

resuits, figure 2.9). Since 1D spectroscopy requires Iess than half m hour and 2D

DQCOSY spectroscopy requires only 8 hours, NMR experknents can be conducted over

a period of tmie that allows spectra of cis-PAP peptide sample to be acquired. The tram-

and cis azobenzene protons a, Hb &, & and &) in PAP7 peptide were all identifïed

based on changes in 1D and DQCOSY NMR spectra following photoisomerization.

Afier the trans-PAP7 peptide sample was photoisomerized, trans-azobenzene cross peaks

were l e s mtense, and cis-azobenzpne cross peaks were observed Samples that were

aliowed to thenu@ isornerize h m cis-azobenzene to trans-mbenzene were marked by

a loss of cis-azobenzene peaks and an inmease in intensity of tram-azobenzene peaks.

Individual proton assignments were strajgh~orward. & was the only proton with two

cross peaks (coupled to H, and &). Cross peaks to I-& were halfas intense a s cross peaks

between other azobenzene protons (since & and a', and Hc and were not resolved).

l& was asçigned by process of elimiBation, since it was the only other proton coupled to

&- Ha and Hb peaks were disthguished based on their relative chernical shifts, which

follows the same trend as H, to & (Hb and H, are both ortho to the azo moiety of PAP,

and Ha and fi are both meta to the azo moiety of PAP). For example, in the trans-PAP7

spectnim H, is downfïeld compared to &. Therefore, Hb was assigned to the downfïeld

peak and Ha was assigned to the upfield peak. All of the trans- and cis-azobenzene ring

protons could be assigned (figure 2.12, table 2.3), and azobenzene photoisomerization

was occurring in the PAP7-S-peptide NMR samples. The fact that both cis- and trans-

azobenzene NMR peaks are readiiy observed, in addition to the resdts ikom UV-VIS

studies on PAP7 peptide, lends credence to the idea that PAP photoisomerization isn't

hindered when the PAP is inserted mto the S-peptide.

PAP-RNase S MUTANTS

Andrew James deterrnined apparent dissociation constants for PAP7-mase S and

PAP 10-RNase S (table 2.4). When the amount of S-peptide, S-protein and the

dissociation constant are all known, the quaciratic formula can be used to calculate

percentage binding of S-peptide to S-protein (Appendix D). We found that 99.8 (k 4.5)

% S-protem was bomd and 32.8 (5 3.5) % S-peptide remained fiee for our trans-PAP7-

RNase S sample, and that 97.5 (f 6.0) % S-protein was bund and 14.0 (+ 6.0) % S-

peptide remained fiee for our trans-PAP10-RNase S sample. Photoisomerization fiom

tram- to cis-PAP does not affect the hction of fiee and bound PAP in solution ->

AIthough some S-peptide remains unbound in solution, we or@ observe one set of

azobenzene peaks in the PAP-RNase S NMR spectra. The chemical sbifts of these peaks

are dflerent compared to the PAP7 peptide, so they must arise fiorn fast excbange

between free PAP peptide and PM-S-protein.

UV- W S SpeciLoscopy of Pm-RNie S MMuf fs

The results fkom UV-VTS and NMR studies on PAP-RNase S are similar to the S-

peptide studies, even though the solvents differ between S-peptide studies (60% ds-THF:

40% D20) and RNase S studies 0 2 0 ) . UV-VIS spectra of both dark-adapted trans-

PAP7-RNase S and da&-adapted tram-PAP 10-RNase S gave typical bands for a - x*

(322 MI) and n - x* (432 am) transitions. We observed changes in UV-VIS maxima

foiiowing photoisomerization, where the na* band disappeared, and the n-x* band

becarne more intense. The spectra of these light-adapted samples are indicative of the

generation of cis-PAP7-RNase S and cis-PAP 10-base S. All of the UV-VIS studies

were performed on mM sample concentrations in a 0.01 cm cuvette. At these

concentrations, at least 67% of the PAP7-S-peptide and 87% of the PAPl O S-peptide is

bound to S-protein, aml more than 97 % of the S-protein remains bound by S-peptide m

either sample. Therefore, W-VIS spectroscopy on the PAP-RNase S samples shows that

PAP photoisomerization can occur when PAP-S-peptides are bound to S-protein. NMR

spectra were obtahed for trans- and cis-PM-RNase S to verify that PAP

photoisomerization occurs while PAP is bound to S-protein.

NMR Spectroscopy of Pm-RNase S Mufun&

Initial ID 'HNMR spectra fiom deuterated PAP7-RNase S and deuterated

PAP10-RNase S ali had excellent signal to mise ratios which suggested q 1 e stability,

and meant that the PAP-RNase S mutants would be amenable to M e r examination by

NMR 1D NMR spectra taken before and after sample photoisomerization are different

in the arornatic region. In particular, peaks decreased downfïeld of 7-40 ppm followuig

sarnple exposure to 33 7 nm light. S i d a r NMR studies of PAP7 peptide indicate that

chernical shifi changes of this sort correlate to trans-cis azobenzene photoisomerization in

PAP. To prove that trans-cis azobenzene photoisomerization was ako occurring in the

PAP-RNase mutants, the shifted peaks had to be identifïed. Poor resolution in the ID

spectra forced the use of two-dimensional spectroscopy to foïIow azobenzene

photoisomerization in the RN- mutants,

When the arornatic regions of PAP-RNase S 2D DQCOSY spectra were analyse&

a number of additional cross peaks were observed in cornparison to analogous S-peptide

spectra. Spectra of RNase A were taken to help assign these peaks. In the RNase A

spectra, cross peaks in the arornatic region were assigned to the ring protons in tyrosine

and phenylalanine residues of the enyme (figure 2.15). The additional cross peaks in the

P m - and PAP1 0-RNase S spectra were therefore assigned to tyrosyl and phenylalanyl

ring protons. The remaining peaks were ail subject to change in chernical shift upon

photoisomerization, so they were assigned to the protons fiom either tram- or cis-

azobenzene. The procedure for i d e n m g and assigning these peaks to individual

protons was identical to that used for PAP7-S-peptide studies. UItÎmately, we were able

to assign peaks to all of the trans-azobenzene and cis azobenzene protons (Ha, Hb, &, &

and H, figures 2.20 and 2.23, table 2.3). OveraIl, ID and 2D NMR spectra ofboth

PAP7-RNase S and PAP1 0-RNase S suggest that azobenzene is in fact isomerinng in the

RNase mutants.

Modering and Kinetics of RNme S Mutants

A b e t i c analysisz was carried out for S-peptide-2 RNase S, PAP7-RNase S and

PAP 10-RNase S samples. The procedure for this d y s i s has already k e n descriid 55,

where the initial rate (V,) of substrate cleavage versus S-peptide d o g u e concentration

were made and apparent rate W.) and dissociation constants a) obtained by ntting the

data to equation 1. It should be mted that the apparent rate (Va), and apparent

dissociation constant (&) are usefùI guides for determining the kinetics of RNA

hydrolysis m our PAP-RNase S mutants.

v', = 5- g?1 (1)

IC8 + Pl

The Va of S-peptide2-RNase S was two times greater than PAP IO-RNase S and withm

error for PAP7-RNase S (table 2.5). Trans-PAP7-RNase S had greater a c t ~ t y than cis-

PAP7-RNase S, whüe the opposite was observed for PAP10-RNase S: cis-isomer had

greater maximum activity than trans-isomer. Apparent rate and dissociation constants all

within the same order of magnitude for each RNase S variant.

Performed by D. Andrew James and G.A. Woolley

Table 2 J Collecteci Values for Observed Constants V, K,

S-Peptide Ka (trans) Va (tram) Ka (cis) V, (cis)

Analogue (X 104 M sec-') (X IO-'^ M WC-') (1 106 M sec-') Cx 10-" M -9)

PAP7-RNase S 0.7 + O. 1 64+2 1-7 2 0.2 5 4 k 2

PAP 10-RNase S 1.2 + 0.2 27+1 0.6 I 0.1 31tI

S-peptide-2-RNase S K, = 0.8 t 0.1 x 104 M, and Va = 50 f 2 x 10-L0 M sec-'

The Va values for our PAP 10-RNase S analogue is decreased in cornparison to the

native S-peptide-2-RNase Va value. PAP1 0-RNase has one less positive charge

compared to native S-peptide due to substitution of PAP for the Arg 1 O residue- This

automaticdy reduces the binding interaction between enzyme and substrate since both

Arg is known to contribute to electrostatic stabilization of enzyme-substrate transition

state structure 25. A reduced binding mteraction between RNA and RNase S would

allow for greater substrate flexi'bility in the active site, which could compromise the

enzyme's ability to cleave RNA. Overall, the removal of Arg l O causes a reduction in Va

for RNase S-

From NMR and UV-VIS experiments, we h o w that PAP undergoes

photoisomerization when bound to the S-pro tein Therefore, when the PAP side chab is

converted fiom the tram- to cis- isomer, we are actually measuring effect of

photoisomerization on enzyme activity- Molecular models can offer insight towards

different interactions of trans and cis-PAP in Nase S, which helps to explain the

observed trans- and cis- PAP-RNase S activities.

Molecular modelling was perfomed on each of the two tram- and ch-PAP-

RNase S molecules. We evaluated the energies of PAP with respect to its XI, ~ 2 , and

~ 3 , angles (figure 2.3). When RNase S models that incorporate low energy PAP

conformers are created, we are able to compare the position of PAP in each modeL Thus,

we hoped to identify low energy PAP conformers, and then determine the structure:

activity relationships for each enzyme. Uitimately, we hoped to ident* the cause of

RNase S activity changes upon PAP photoisomerization for each PAP-RNase S mutant.

It is important that the S-peptide structure is not distorted because the proper

folding of RNase S is dependent on S-peptide maintainmg an a-helix structure

Molecular models of both PAP7- and PAP1 0-RNase S show that the PAP residue is

positioned on the outer face of the S-peptide, pointing away fkom RNase S and hto

surroundhg medium, Consequently the presence of a PAP moiety within the S-peptide

should not interfere with iîs binding to S-protein-

When models of low energy PAP7-RNase S conformers were examined, we

found that the trans-PAP side chah can be positioned in two general areas: in the active

site cleft cornprised by Hisl2, His 119, and Lys41, or pointing away fiom the enzyme

(figure 2.29a). The cis-isomer of PAP, by comparison, has its side chain co&ed to a

compact sphere, which protrudes out fiom the S-peptide and away IÎom the enyme

(figure 2.29b). We would expect that smce the trans-PAP side chah enters the active

site, it wodd interfere with RNA substrate binding, causing a reduction in it7s Va versus

the cis-PAP side chain, which should not directly interfixe with substrate binding. In

fact, the opposite is tme and the activity is greater for the trans-borner of PAP7-RNase S

than for cis-borner. A possible expIanation for the observed activities is that more space

is available for tram-PAP to occupy, so that less time is spent in any one position. Since

more space is available to tram-PAP versus cis-PAP, it is possible for the trans-PAP side

chah to occupy positions other than the active site when RNA binds to RNase S, making

it easier for the substrate to access the active site- Essentially, trans-PAP can get out of

the way of substrate binding. It shodd also be noted that since tram-PAP only has a

srnail dipole (Chapter three), and is hydrophobie, there are not likely to be any fàvourable

interactions between trans-PAP7 and the RNase S active site that would permit the side

chah to bind the active site tightly. Therefore, the coulombic and charge interactions

which heb to mediate substrate binding 27 are not lücely to occur for the trans-PAP7

azobeflzene side chah when it is positioned in the active site. Consequently, there is no

reason for the tram-PAP7 side chain to bind m the active site and, in fàct, there is more

reason for it not to bmd the in the active site cleft smce there would be an entropic cost

for doing so. The maximum rate of cis-PAP7-RNase S is reduced slightly in comparison

to the tram-borner, Although the clifference in rates is not great, a possible explmation

for this Merence is that low energy cis-PAP conformers are restricted to a compact

three-dimensional space positioned near the active site yet not Siside of it. Therefore,

during RNA bhding, the ch-PM7 side cham could act to push RNA substrate away

fiom the active site der other RNA binding subsites. Specificdy, the cis-PAP side

chah has a greater chance of making van der Waals contacts with the RNA substrate.

The substrate would then be f~rced to change its position and enter a less-productive

bmdmg mode with the enzyme, so that its arrangement in the active site is not optimal.

This could cause a reduction in Va for cis-PAP7-RNase S.

The trans- and cis-PAP side c h a h of PAPI O-RNase S have the same general

confo~nliitions as for PAP7. The tram-PAP side chah is extended and occupies a large

volume, while the cis-PAP side chah is compact and occupies a small volume (figure

2.3 1 ah). Tram- and cis-PAP IO-RNase S have very similar activities, which suggests

that the PAP residue is too far fiom the substrate binding area for azobenzene

conformation to d e a Herence in substrate binding and therefore enzyme activity-

In general, we have observed only small differences in activities for each PAP-

RNase S enzyme upon PAP photoisomerization Perhaps we would have expected a

greater dserence in activity of RNase S based on simple mode1 examination, however,

our models do no t take molecular motion of residues other than PAP into account. As

weU, for PAP7-RNase S, tram to cis-azobenzene photoisomerization occurred with a 2.5-

fold change in L, and for PAP 10-RNase S, tram- to cis-azobe~lzene photoisomerization

occurred with a 2-fold change m L. Small changes in K, require small changes in fkee

energy (AG = -RTh&), which could be accommodated by minor adjustments in enzyme

conformation to allow substrate fÙU access to enzymatic active site regardess of the

presence of a PAP residue. Thus, the effect on enzyme a c t ~ t y following PAP

photoisomerization is darnpened by these other motions. Overall, it is dangerous to make

too mauy assumptions as to what may cause srnail PAP-RNase S activity changes, since

they can be caused by only subtle changes in enzyme structure, or orientation.

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Chapter 3 AZOBENZENE-REGULATED GRAMICIDIN A CHANNELS

The bear gramicidins are a fâudy of proteins that firnction as cation-selective

ion transport channels in Lipid bilayer membranes 1-6. Gramicidm A is the major

synthetic product of BociZZm brevis at the onset of sporulation 7. The channel protein

supports antispennicidal and anti- human immunodeficiency virus activities 8, and has

been used as an antiotic 9.10.

Gmmicidm A, with the following amino acid sequence 7:

is composed of two penta-decapeptide monorner units that dimerize in a head-to-head

fàshion to f o m an ion channel in a lipid bilayer. In organic solvents it adopts either a

random coi1 monomer structure 1 1, or one of several intertwmed double-stranded dimer

structures 12. The molecular fold of gramicidin A in a lipid bilayer was &st proposed in

the 1971 13, where Urry hypothesized that the alternathg ü- and L- amino acid sequence

induced the backbone to adopt a B-strand structure. Shce this tirne, X-ray crystal

structures 14-16 and high-resolution NMR structures 17718 have been made available.

The results of these studies show that gramicidin A monomer units can self-associate in a

lipid bilayer to form a symmetrical, anti-pardel single stranded dimer (figure 3.1). The

rnonomeric subunits adopt a right-handed P~~ helix secondary structure, and job at theû

amino-termini to yield a 25 Mong cyhdrical pore, with an inner diameter of 4 A The

helnt and chanl~el pore are arranged parallel to the Mayer normal, with the peptide

backbone fo&g the inner wall of the pore and the side-chains projecting away from the

channel lumen Having the backbone lining provides the polar environment necessary

for efficient ion translocation across the lipid bilayer. The dimeric structure of

gramiciclin A is stabilwd by ten intramolecular P-pleated sheet hydrogen bonds as well

as six intermolecular hydrogen bonds at the b e r niterfàce.

Figare 3.1 Stereo view of Gramicidin A l9

MECHANISRI OF ION TRANSPORT

A three-barrier, two-binding site model has k e n widefy adopted for the description of

ion transport through a gramiciclin channel (figure 3.2) 20.21. In this model, there are

thtee maxima and two minima in the potential energy profile of ion passage through the

charnel The largest barrier occurs at the centre of the bilayer. Entry and exit barners

correspond to the stripping of the cation's hydration sphere.

Bindmg constants for various monovalent cations to gramicidin have ken

deterrnined 3.22. Cross et al postulate that ion binding occurs at the channe1 entrance via

mteraçtions with specSc carbonyl oxygens l9. Specifïcally, three leucyl backbone

carbonyls @,eu1 O, Leul2, Leu1 4) coordinate the cation m a stepwise and delocalized

fishion T b mamer of cation bhding helps to overcome the large energy barrier to

Figare 3.2 3B2S (Uuee b-er, two bindmg-site) mode1 for ion transport through a Gramicidin

channei. 00 corresponds to an empty channeI, OX and XO correspond to a moneoccupied charme1 and

XX to a channel with two ions. kl and k2 are the -ation rate constants of the f5st and second ion

respeüively, ki and k2 the rate dissociation constants and 1 is the translocation constant 21.

cation dehydration smce the waters are sequentidy removed fiom the cation hydration

sphere as it moves through the chamel. Structural evidence for specific cation-binding

sites that supports this hypothesis has been obtained ushg Nh4R spectroscopy with and 1 %-labelled gramiciciin A mcorporated in lipid bilayers 23924.

Ion transport occurs m a single file manner in gramiciciin A chatuiels, where water

molecules travel through the pore dong with a cation 25. It has been shown that the

Trp9, Trp 1 1, Trp 1 3, and Trp 15 side chains of gramicidm are important modulators of

channel conductance 26-29- The indole rings of these tryptophans all have dipoles that

are positioned with the positive end projeetmg into bullc water 18. Since the tryptophans

are arranged m a helical M o n in gA, the dipoles act cooperativeiy as one large annula-

dipole 30. The energy barrier in the centre of the channe1 is reduced by the negative end

of the annular dipole, which stabilizes a cation in the centre of a channeL This

s tabht ion can enhance conductance when translocation fiom the entry site to the exit

site is rate limiting. Gramicidin A analogues have been created that demonstrate the

effect of Trp dipoles on charme1 conductance. When one or aU of the tryptophan residues

were substituted with phenyManine residues m gA, conductance of g A channek

decreased 29. When fluorinated tryptophan analogues, which have comparativeiy larger

indole dipoles than native tryptophan, were introduced to gA, the c h e l conductance

was increased 31.

The effect of a dipole at position one of the gA channel on channel conductance

has been studied by a number of groups 32.33- In these shidies a series gA analogues

were constnicted, where val1 was replaced with trinuorovaline, hexduorovaline,

norvaline, norleucine, S-methyIcysteine, methionine, o-fI uoro-phenyIalaniney p- fluoro-

phenylalanine, m- fluoro-phenylalanine, phydroxy-phenyIaIaniney and p-methoxy-

phenylalanine .

Table 3.1 Physical Properties of native gA and gA analogues

Amino Acid at Position 1 Dipole Moment Conductances @S, in 1 .O M NaCI)

Valinea -0.4 12.37 + 0.20

Trifluorovalinea M.8 1.93 I 0.02

Hexafluorovalinea t l . 6 1.42 + 0.04

Norvalinea -0.4 14.69 + O. 15

Norleucine" -0.4 14.74 f 0.14

S-methylcyst einea a . 3 9.90 10.1 1

Methioninea +O. 1 8.28 f 0.13

o- fluoro-phenylaanineb -0.16 9.14 + 0.18

m- flu~ro-~hen~lalanine~ +O .47 6.08 t 0.12

p f lu~ro-~hen~~a lanhe~ M.78 5.86 k 0.17

For dipole moments, positive p values are assigned to dipoles that point towards the channel

lumen. a f?- Russel et al. 32. eom Koeppe II et ai 33

Conductance was found to be modulatecl by the side chains at position 1 (Table 3.1).

Variations in single-channel conductances reflect changes in the energy barriers for ion

and water movement through the c h e L The structure of these modi6ied channek did

not deviate greatly fkom native gramiciciin. rn-fhoro-phenylalanuie andp-fluoro-

phenylalanine have very different electron withdrawing properties (0 = H.06 for p

fluoro-phenylalannie, and o = +0.34 for m-fluoro-phenylalanine), but similar single

channel conductances, so inductive effects did not impact single channel ion

conductance. It has been hypothesized, therefore, that changes in conductance arise fiom

electrostatic interactions between the side chai. and the permeating ion, through ion-

dipole interactions. val'-gramicidin A analogues that have dipoles that point towards the

channe1 lumen have repulsive ion-dipole interactions. This increases the barrier to ion

transIocation causing decreased single channel conductance relative to native gramicidin.

SÙnilarly, val'-~ramicidin A analogues having dipoles that point away fiom the channel

lumen had increased ion conductance relative to native grarnicidùa-

The structure and function of the gramicidin channel are both weu-defïned,

making it an attractive target for photomodulation Optical control over gA channel

conductance wodd be usefid for the development of nanoscale devices such as sensors

34, and, in principle, as a non-invasive tool for studying cell excitabïlity. Several groups

have mitiated studies that utilize azobenzene as the rnodulator of g A conductance *5734-

36- Azobenzene is ideal for manipulating ion charme1 conductance since its

isomerization occurs with si@cant structural and electrostatic changes, which can be

applied towards reversible control over ion transport. The following paragraphs provide

an account of the most recent attempts to photoregdate ion channel conductance using

azobenzene-modi6ed gA analogues-

Stankovic et al. covdently coupIed two gramicidin A monomers at their N-

t e r d ends with a 3,3'-azobis(be~lzeneacetic acid) linker 25. Studies on these

azobenzene linked-gA channels showed two levels of conductance for dark-adapted

tram-azobenzene channels: a main level with longer lifetimes, and a higher level with

shorter Metimes. Tram-to cis-azobenzene pho toisomerization produced another type of

channel with a marked fiickering behaviour, and single-channel current similar to that of

native gA. Stankovic proposed a mode1 that accounts for the observed photomodulation

of N-terminal azobenzene Iinked gk He hypothesized that two molecules of dark-

adapted trans-azobenzene linked gA could associate to form a single pore, or a dual pore

channe1 (figure 3.3). The obsewed main conductance would be nom the single pore,

while the higher-level conductance results fkom dual pore formation Azobeozene

Figure 33 Proposed mode1 for observed photomodulation of N-terminal azobenzene-linked

gramicidin k The diagrams illustrate possible pore structures for the observed charme1 states 25.

photoisornerization to the cis form would disnipt these structures and produce a

unimolecular pore (figure 3.3, right side). Strain at the N-termind-Nterminal interface

could lead to rapid dternation between open and closed states, which would lead to

flickering m conductance.

Attempts have also k e n made to link azobenzene units to the C-tenninus of

gramicidin A to modulate its activity 35936. Three azoberizene derivatives îhat have been

linked to the C-terminus of gramiciciin are p-carboxymethylazobellzene, p-

aminomethylazobenzene, and rn-aminomethylazobenzene.

Para-carboxymethyIazobenzene gA was synthesized by coupling azobenzene

dicarboxyllic acid dire- to the C-terminal ethanolamine residue of gramicidin A 36.

M e r exposure to UV Iight, channel formation of the cis-pcarboxymethylau>benzene gA

was increased relative to native gA and to trans~carboxymethylazobellzene-gk This

phenomenon was f'ully reversible following dark adaptation of the azobenzene-iinked gA

channels. The conductance of cis-p-carbxymethylazobe~lzene, however, was altered by

less than 4 % upon UV-VIS irradiation, Osman et aL have argued that the different

dipole moments of cis and traos-azobenzene may affect the channel fomiing properties of

azobemne-linked g& where the cis-isomer of azobenzene-linked gA is more fàvourably

oriented m the lipid membrane.

Para- and meta-amino-azobenzene have a h been linked to the C-terminal

ethanolamine residue of gA 35. 4,4'-bis(a~omthyI)azobenzene and 3,3'-

bis(amuiometbyl)azobenzene were synthesized and attached to gA via carbamate

Iinkages. Irradiation of these azobenzene-linked gramicidm analogues with 337 nrn Light

caused si@cant changes in thei. single-charnel currents. Dark-adapted trans-

azobenzene-gA channels had small steps in the current, which corresponded to thermal

cis/trans isomerization of the carbarnate Iuikers. Irradiated cis-azobenzene-gA channels

had four current levels which also corresponded thermal cis /tram isomerization of the

carbarnate Mers. The four current Ievels were assigned to trans: trans-, trans: cis

(channel exit)-? cis (channel entrance): trans-, and cis: cis-carbamate isomers of cis-

azobemene-gk The trans: carbamate isomer yidded the highest current, while the

Iowest current was assigned to the cis: cis carbamate isomer.

I 1 Figure 3.4 Structure of paminomethylazobenzene-modifieci gramidin channel, The azobenzene

containhg molecular gates are show in cidcis, and trandtrans arrangements.

Woolley et aL explain these results by hypothesizing that the protonated amino group on

the para, or meta-aminomethyl azobenzene Mer acts as a cation blocker at the channel

entrance (or exit) when azobellzene is in the cis-state (figure 3.4). Channel blocking is

most effective when carbamate linkers are also in the cis-state. When the azobenzene

derivative is tram, however, the amino group is too far removed fkom the chamel

entrance (or exit) to fûnction as a blocker.

EXPEIURlENTAL OUTLINE: CHAPTER 3

In lieu of recent attempts to photomoddate gA channel conductance, a gramicidin

analogue was designed with an N-terminal phenylazophenyldanine (PAP) rnoiety (figure

3.4) 37. Current recordings on dark-adapted (trans) P A P I - g ~ channels, and

photoirradiated (cis) PAP'-~A channels showed cation conductance through cis,cis-

PAP'-~A charnels is lower than trans,trans-PAP'-~A channels and c i s - , t r a n s - ~ ~ ~ ' - ~ ~

conductance is mtermediate. Therefore, ion flux through gA channek is modulated by

azobenzene photoisornerization at position one.

Several groups have demonstrated that side-chai. dipo les mediate conductance in

gA 2630-3373*. Accordingly, we hypothesized that the dipole change associated with

azobenzene isomerization is the principal cause of photomodulated conductance in PAPI-

gA channels. We attempted to validate this hypothesis by modehg trans- and cis-PAPI-

gA channek and, in particular, modeling the electrostatic interactions between the

azobenzene moiety and a cation located in the middle of either a trans- or a C~~-PAP' -~A

chamel. We performed confionnational energy calcuiations to identi@ low energy

conformers for cis-PAP side chahs in gA channeIs. We then calculated electrostatic

interactions between P M conformers and a ~ a + ion Iocated at the centre of the channei.

The calcdated magnitude of electrostatic interactions c m account for conductance

changes observed upon tram-cis PAP photoisomerization of PAP'-~A.

MOLECULAR MODELLING

Al1 modeling was done on a Siücon Graphics Octane system (Imc 6.4) using

Hyperchem (SGI version 4.5, Hypercube Inc.) and Spartan (SGI version 5 -0.3) molecular

modeling software.

Ketchem et al. 18 solved the structure of the gramicidin A channel in a lipid

bilayer. This structure, which was dehed by constramts derived f?om solid-state NMR

measurements of uniformly aligned samples m 1amelIa.r phase lipid bilayers (Brookbaven

protein data bank accession code: IMAG), was used as the starting structure for our

modehg studies. PAP (as modelled by Professor G A Woolley and John Karanicolas;

see Chapter 2.4) was introduced to grarnicidin A (gA) by substituthg trans- or cis-PAP

for val' m both moriomers of the dimenc gramicidin channel. Inspection of cis-PAP

CPK models reveal four major low-energy conformers, where ~3 and ~4 torsion angles

are centred on -57' and -57', +5? and +57', 423O and -123*, and +123" and +123" (see

figure 3 -5 for PAP torsion angle assignments).

Figure 3.5 Phenylazophenylalanine (PAP), showing torsion angles XI, ~ 2 , ~ 3 , and ~ 4 .

Five PAPI-@ modek were therefore bdt7 one for each cis-PAP ~ 3 , ~4 pair, and

one for tram-PAP. A sodium cation was added to the centre of the channe1 lumen in each

case-

The Hyperchem implementation of the Amber force field did not contain

parameters for the ethanolamine group, or for the formyl group of gA. We had to

parameterize both of these groups and enter them into the Hyperchem library since

unknown residues resdted m Hyperchem king unable to perform energy calculations.

Templates that defked a formyl residue and an ethanolamine residue (see Appendix C)

were constructed and added to the chemtpl file. These templates define which atoms are

present in the amino acid, as weII as the comectivities betvveen atoms, and the charges on -

each a t o n The formyl template was based on a pre-exkting template for an acetyl

residue, and the ethanolamine template was based on a pre-e>cisting template for a serine

residue. The carbonyl-bound hydrogen of the formyl group was designated atom type

'HY'- Bending, stretching, non-bonding, and atom type parameters were created and

compiled into the Amber force field parameter files (amberben-PAP-txt,

amberstr-PAP.txt, ambembd-PAP-M, ambertyp-PAP.txt, see Appendk C). The

bending parameters for HY were based on angles determined fiom the solution structure

of gA, while the stretching parameters for HY were based on pre-e>ristmg parameters for

an sp2 hybridized carbon bound to hydrogen The non-bonding parameter for H Y was

made equivalent to the non-bonding parameter for a hydrogen atom bound to an aliphatic

carbon containing one or more electron withdrawing groups. Small errors in these

parameters are not important, since neither the formyl, nor the ethanolamine residues

affect the electrostatic or potential energy calculations m PAP'.

Hyperchem did not recognize the D-amho acids (D-valine, and D-Ieucine) in gA.

To overcome this problem the pdb file for gA was adjusted, where DVA and DLE were

renamed to VAL and LEU respectively and the amide hydrogens, which were improperly

named MN, were renamed to -H-.

It is d i c d that the correct atom-centred partial charges are determhed for cis-

a d trans-PAP since these will directly affect the calculation of how a PAP dipole

interacts with a cation m the chaaneL Partial charges for each of the four low energy

conformers of cis-PAP, and for the trans-PAP conforer ( ~ 3 and ~4 = 1804 were

cdcdated using standard ab initio methods in Spartan. Single-point Hartree-Fock ab

hitio calculations (6-3 1 G* basis set) were perfomed on N-acetyl, N'methyl-

phenylazophenyldanine derîvatives of PAF. The electrostatic fitting algonthm within

Spartan was used to calculate atom-centred partial charges. The same XI and ~2 torsion

angles were used for each calculation. An additional fïfth conformer with dtered ~1 and

~2 torsion angles was checked to ensure that charges on the azobenzene moiety were not

affected by rotations about these torsion angIes. Individual atom charges on the

methylene group (figure 3.5) of PAP were adjusted to maintain a zero charge on PAP.

The Cf3 atom charge was increased by 0.02 units and the charges on the two HP atorns

were each increased by 0.006 units. The adjusted charge sets of the five PAP-gramicidin

conformers were added to their respective pdb files, which were used for electrostatic

energy caIcuIations in Hyperchem.

Potential energy d a c e s and electrostatic surfaces were cdculated for each

PAP'-~A conformer wÏth Hyperchem, using an Amber forcefield (dielectric scale factor

= 5, non-bonded cutofE = none, and a 1-4 scale factor = 0.5). As in chapter 2, a series of

embedded scripts were created that determined the single point energies for 8 1 00

different PAP side chah conformations. The angles ~1 and ~2 were rotated through 360'

m four-degree kcrements, and smgle point energies of PAP' were calculated at each step.

Electrostatic interactions between the PAP dipole and a ~ a + ion at the centre of the

channel were detemimed using the same scripts with Merent PAP'-~A starting

structures. Here, every atom was deleted except ~ a + and those comprishg the PAP side

chain (up to and including C a of PAP). The charges on Ca, Cf3 and HP were set to zero.

M e n the scripts were run, a log £iie was generated containing single point energies, or

electrostatic energies. PERL pro- were developed (ESqarse.pl and SPqarse.pl,

Appendix A) that extracted the potentiai energies and electrostatic energies fiom their

respective log files and tabulated them with respect to ~1 and ~2 PAP' torsion angles.

The output fiom each PERL program was input to where potentid energy

maces and electrostatic energy nufàces were determined for each of the five different

cis-PAP'-& conformers. Molecular models were created for each 10 w-energy PAP-gA

conformer by rotatmg ~1 and ~2 of PAP' to match the low energy positions dictated by

the PAP potential energy surlàces.

Azobenzene dipoles were calculated and superimposed onto models of low

energy cis- and t r a n s - ~ A P ' - ~ ~ conformers as a qualitative representation of the

electrostatic mteractions between the azobenzene moiety in g A and a channel cation,

These dipoles were detennined as foIlows: Ail of the positive atom-centred point charges

on azobenzene were represented by a single ~ a + cation. The charge of ~ a + was

determined by summing the partial charges of positively charged atorns in azobenzene (as

determined fkom Spartan ab initio calculatioas). The location of ~ a + was determined

fiom the average position of all of the positive partial charges on azobenzene m the

PAP*-~A models. AU of the negative atom-centred point charges on azobenzene were

represented by a smgle chloride anion, where the charge and location of Cl- was

determined in the same marner as ~ a f . Five PAP'-~A modek were studied (four low-

energy c i s - ~ A P l - ~ ~ conformers, and one Iow-energy t r a n s - ~ ~ ~ l - ~ ~ conformer), each

having a unique Na+, CI- ion pair. The dipoles of these ion pairs, and thus the dipoles of

azobenzene, could be calculated using GRASP software. GRASP detennined the dipoles

by first calculating the surn and charge weighted average positions of all positive charges

and the similar quantities of all negative charges and then calculating a dipole magnitude

and direction by multipIyÏng the absolute sum of positive and negative charges by the

distance between charge-weighted centres. A dipole vector of a length determined by the

user (in Debye per Angstrorns) was displayed centred at the average position of the

charge-weighted centres. Trans- and C~S-PAP'-~A conforxners, with the PAP' dipoles,

were displayed using GIZASP software (version 1.2) 39.

MOLECULAR MODELLING: POTENTIAL, ENERGY SURFACES

We were Ïnterested in the molecdar origin of photoregulated PAP'-~A channe1

conductance. Trans- and C~S-PAP'-~A models were created and conformational energy

surfaces were generated as a function of ~1 and ~2 torsion angles (for the definition of ~1

and ~2 torsion angles, see figure 3 -5). Four surfaces were generated for cis-~~l?-gA,

one for each Iow energy ~ 3 , ~ 4 pair. These d a c e s (figure 3.6) show that the cis-PAP

side chain is limited to X I = -45' + -180° and x2 = -75" -+ +180° when x3 and x4 are

both -57, or both -123", and that PAP is limited to X I = -4S0 + -180' and ~2 = +75" + O" when ~3 and ~4 are both +5T, or both t123". In addition, we found that the sterïc

component (Es) of the total energy was much larger than the other energetic components

(Le. electrostatic, non-bonding, hydrogen bondmg, bond stretching and bond bending

energies). A sunilar surface was generated for t r a n s - ~ ~ ~ l - ~ ~ (data not shown). From

these d a c e s , it was possible to create models correspondmg to Iow energy conformers

of C~S-PAP'-~A, and of t r a n s - ~ ~ ~ l - ~ ~ (figure 3 -7).

MOLECULAR MODELLING: ELECTROSTATIC SURFACES

Electrostatic maps (figure 3 -6) of each low energy C~S-PAP'-~A ~ 3 , ~4 pair show

that there is a O to +1.5 kcai range for the electrostatic interaction between the

azobenzene side chah and a sodium cation piaced in the center of the gA channel. TO

help visualize these interactions, PAP side cham dipoles were determined for ch-PAPI-

gA (3 -7 D) and for trat ls-~AP'-~~ (0.73 D) (figure 3 -7). It should be noted that the

dipole in C~S-PAP'-~A was equivalent in magnitude for each low energy cis-PAP ~ 3 , x4

conformer. The positive ends of cis-PAP' dipoles were observed to point towards the

charme1 lumen, regardless of ~ 1 , ~ 2 , ~3 and ~4 values There was only a small dipob

associated with the t r a o s - ~ A P ' - ~ ~ (0.73 D). The positive end of this dipole pointed

away fkom the channel lumen dong the N=N axis.

Figure! 3.6 Single point energy profiles (foreground contours) and eiectrostatic interaction profiles

(background contours) for the four low energy conformers of cis-PAP 1 gramicidin. Conformational energy

is lowest ( - O kcaVmo1) for ~1 = -90°, and ~2 = +90° (A, D), or -90' (53, C). The contours begin at O

kcaVmoi (10 kcaVmol per contour) for the singie point energy profiles, The scde for the electrostatic

interaction profiIes is given at the bottom of the figure. A) ~ 3 , ~4 = -56,g0, B) ~ 3 , ~4 = +123,1°, C) ~ 3 ,

x4=+56-gO,D) ~ 3 , ~4=-123.1"-

Figure 3.7 A), B) top view and side view of cis-PAP1 gramicidin, where the PAP residue is a low

energy conformer (XI = 1 80,0°, ~2 = -90.0°, ~3 = 56,g0, ~4 = 56.90)- The azobenzene dipole (3.7 D) is

shown projecting into the channel lumen (0.06 A / D). C), ID) top view and side Mew of tram-PAP'

gramicidm (XI = 180.0°, ~2 = -90.0°, ~3 = O.OO, ~4 = 0-0"). The azobenzene dipole (0.73 D) is shown

parallel to the azo moiety (0.06 A / D).

3.4 DISCUSSION

Gramicidin A (a) was chosen as the test channel for photoregulation because its

structure and function have both been studied in de td 4-6718. The N-terminus of gA

seemed to be an appropriate choice for site-specific modification since previous studies

show that the overail channel structure and stability are not compromised by aromatic

amho acid substitutions at position one 33. We substituted PAP for the N-terminal

valine residue of gA. Photoisomerization of PAP changes its dipole moment 40. The

effects of dipoles on charme1 conductance should tie mawnized at position 1, where the

energy barrier to ion translocation is the highest 20,21?41- Synthesis and purification of

the azobenzene-modXed g A channels has been descnid elsewhere 37.

The azobenzene photoisomerization process was followed by UV-VIS

spectroscopy for PAP'-~A samples. Spectra were recorded that are typical of tram-cis

photoisomerization: following irradiation at 337 MI the n-.n* band at 3 30 nrn decreased

and n-z* band at 430 nm increased. PAP photoisomerization was readily reversible, and

cis-PAP lifetimes were p a t e r than 10 hours. These results uidicate that transkis

photoisomerization was not hindered when P M was substituted at position 1 in

gramiciciin. Consequently, the effect of azobenzene photoisomerization on single

chamel currents could be accurately monitored in PAPI-@ channels.

Current measurements were performed on PAP'-~A channels in diphytanoyl-

phosphat idylcho line/decane membranes with either CsCI or NaCI as the electro lyte.

Dark-adapted t r a n s - ~ A P l - ~ ~ channek yielded only one level of current whose

conductance was 60 % of the conductance of the native-gA channel. Single channel

current recordings show three different types of PAP'-~A channels following irradiation

at 337 nm (figure 3.8). The different PAP'-~A channek, which all had comparable

FQpm 3.8 Single-channe1 current amplitude histograms and representative singIe-channe; events

(inset) of dark-adapted (trans) PAP'-~A (A), extensively photoirrad iated PAP'-~A (B), and native gA (C).

The currents were obtained at +200 mV, 1-0 M NaCl, 5 mM BES, pH 7, DPhPCIdecane membranes;

filtering was at 100 Hz, Several hundred channels of each type were characterized,

l i fehes , corresponded to four types of conducting dimers: trans: trans, trans: cis, cis:

trans, cis: cis. Conductance m the chanwls varied, with the PAP'-~A (trans: trans)

exhî'b'ig the largest conductance, PAP'-~A (cis: cis) exhiibiting the srnafiest

Table 3.2 Functional Proprf3es of PAP L - g ~ Charnels

Channel Type Conductance (1M NaCl) Conductance (1M CsCl)

GA 13 46

PAP'-~A (transltrans) 8 34

PAP'-~A (cis/trans) 6.5 3 1.5

PAP'-~A (cis/cis) 6 29

~ybrid g ~ / PAP'-~A (trans) n.a, 40

~ y b n d g ~ / PAP'-~A (ck) na 38

Single-channel conductance (pS) rneasured at 200 mV

conductance, and PAP'-g~ (trans: cis or cis: trans) having an intermediate conductance

(table 3.2). The dark adapted charme1 corresponded to trans: t r a n s - ~ ~ ~ ' - ~ A , while

predominant irradiated charnel type corresponded to cis: c~~-PAP'-~A.

Based on these results, we see that channel conductance is modulated by PAP

photoisomenzaton. Currentholtage plots for al1 of the PAP'-~A channels gave

superlinear curves. PM' - g ~ (cis: cis) channel gave the most superlinear curves, while

tram: tram PAP 1 -gA channels also produced superlinear I N curves, aIthough

superlinearity was not as pronounced in this case. Superlinear IN curves are indicative

of increased barriers of the rate-lixniting (ion translocation) step for conductance through

PAP'-g~ channels 32. Therefore, trans to cis photoisomerization of PAP'-~A increases

the barrier to allcali cation translocation One explanation for this behavior that cm be

d e d out is that PAP photoisornerization disnipts the locd structure of gA at its dimer

interface. Formation of hybnd gA: PAPI-g~ chamels is strong evidence of structural

equivalence between PAP'-~A and native gA chamel since hybrid channels will only

fom when two Merent monomers are able to adapt at their N-termini and maintain a

P~~ helix structure similar to that of native gA 33742. The fiequency of the hybrid

channek should be related to the Eequencies of the pure channels 42. If the monomers

form helices with identical structures at their joining ends, the fiequency of hybrid

channek, fa should be related to the fiequencies of the pure channels, f, and fb by 42:

When ~A:PAP'-~A(c~s) channels were examined, the ratio was found to be 1.12 (at 200

mV), and when ~ ~ P A P ' - ~ A (trans) channek were examined, the ratio was found to be

1.02. These ratios show that the structure of the gA channel is not disrupted by

substitution of PAP at position 1. Therefore, photocontrolled gA conductance must be

caused by a property intrinsic to the isomerization of PAP, such a dipole moment

switching, and not by distortion of the gA channel.

GRARZLCIDIN MOLECULA. MODELLING

Tram to cis azobenzene isomerization is coincident with a zero to three Debye

change in dipole moment 43. We have hypothesïzed that such dipole switching affects

the translocation of cations through a gA channel (the rate Inlliting step) by either

diminishing, or increasing the barrier to alkali met al passage. For instance, electro st atic

interactions between low-energy cis-PM' conformers and a chaonel cation should

iacrease the translocation barrier enough to cause the reduction in conductance that we

observe for c~-PAP'-@ channels. We therefore decided to examine the conformations

available to PAP'-~A (cis and tram) and to estimate the magnitude and sign of

electrostatic interactions between the PAP tide cbain and a ~ a " ion in the channel.

Conformational searches with the four cis-PAP' conformers revealed that cis-

PAP' single-pomt potential energies were dominated by a steric component, which was

large m cornparison to elecîrostatic, non-bondmg, hydrogen bonding, bond stretching and

binding cornponents. Thw, van der Waal contacts between PAP' and the rest of

gramicidin Iimits low energy &-PA£" conformers to specific regions on the XI, ~2

potentid energy surface.

Dipole position and orientation was examined for these low-energy cis-PAP'

conformations. We found that low-energy PAP' conformek of cîs-azobenzene had a 3.7

Debye dipole whose positive end pointed towards the channel lumen regardless of ~1 and

~ 2 . ~rans-PM', on the other hand, has almost no dipole. Therefore, photoisornerization

fiom tram-PAP to cis-PAP would always be expected to increase the barrier to alkali

cation translocation through a gA channeL A quantitative electrostatic analysis was

necessary to detemime the extent that cis-PAP1 iucreases the translocation barrier.

The electrostatic interaction between azobenzene and ~ a + in the charme1 was

examined for the low-energy cis-PAP' conformations, and was found to be in the range

of 0.2 to 0.9 kcaVmoi, dependmg on the exact values of ~ 1 , ~ 2 , ~3 and X4.1 Assuming

1 Electrostatic d c e s for the azobenzene moiety and ~ a + yielded positive

electrostatic energies over the entire range of XI, ~2 azobenzene torsion angles.

Therefore, the barrier to cation transiocation should be elevated upon tram- to cis-

azobenzene photoisomerization regardless of the orientation of cis-PAPI.

the two cis-PAP' moieties m gA act additively and mdependently, the total electrostatic

contribution of cis-PAP' to the central barrier would be 0.4 - 1-8 kcaI/mol The O bserved

decrease in single channel conductance upon tram- to cis-PA. photoisomerization

corresponds to an increase in translocation bamer height of about 0.2 kcdmol for Naf,

which b the lower range of the calculated electrostatic contribution- Therefore,

pho toswitching of ion dipole interactions can, semiquantitatively, account for the

observeci effects on conductance.

Several aspects of our modelling procedure are worthy of comment. First, we

needed to calculate the atom-centred partial charges on cis-PM in order to detennine the

magnitude and direction of its dipole. Hartree-Fock ab initio calculations were

employed, usmg a 6-3 lG* basis set. This basis set was chosen based on previous results

h m Cotton et aL, which no discernable change in point charges calculated for

tryptophan indole rings when a higher order basis set (6-3 1 1 G**) was used m

cornparison with a 6-3 lG* basis set 38. Second, the magnitude and direction of a cis-

PAP dipole should be the same (with respect to PAP orientation) for each cis-azobenzene

low-energy conformer. Dipotes were calculated and compared for cis-PAP molecules

having identicai x3 and x4 angles, but different ~1 and x2 angles. We calculated an

identical dipole for each cis-PAP conformer (wÏth respect to orientation of azobenzene),

wbich validated the correctness of our ab initio calculations. Third, when the

electrostatic energy of interaction between PAP and ~ a + was calculated, the surrounding

dielectric was approximated at 5. Our choice of dielectric was based on the work of Hu

and Cross, who found s = 5 reproduced weil the effect of tryptophan dipoles in gA

channe1 conductance 26. It should be noted that better descriptions of the dielectric

experienced by the PAP' side chain of gA in a lipid b'ilayer are available 30744- For

instance, Sancho and Martinez treat the dielectric as a two-compownt dielectnc

continuum where the bulk solvent and pore waters are assigned E = 80 and the channel

w d and lipid are assigned e = 2. A more sopbkticated modehg routine wodd be

required to incorporate this description of the dielectric. We chose not to use this routine

since it would be time consuming, and mecessary for the level of modeling we were

performing-

Currently, we have deveIoped a gramiciin analogue that incorporates a PAP side

chah at its N-termnius. Two new channels form upon photoisomerization of tram, tram-

PAPI-~A: trans,cis-PAP l-gA, and c i s , c i s -~AP~-~~. Compared to trans-trans-PM ' -&

the conductances (1 M NaCI) of trans,cis-PAPI-g~ and cis,cis-~A.~' -& are 1 -23 -fo Id

reduced, and 1 -33-fold reduced respective@ (table 3.2)- These merences are caused by

a O to 3.7 D increase m azobenzene dipole moment, which is comcident with trams- to cis-

PAP photoisomerization. Althou& we are able photomodulate channe1 conductance, the

sizes and eEects of photoisomerization are w t large enough to pennit PAP'-~A to be of

use for manipulating ceilular excitability- Therefore, new strategies must be developed

for increasing the effects of photoisomerization on channel conductance.

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APPENDICES

SINGLE POINT POTENTIAC ENERGY EXTRACTION This PERL program searches for and extracts specinc values f?om Hyperchem

single point energy calculation log files and prints them in a tabulated format, with

appropriate column headings. These values include ~ 1 , ~ 2 , and ~3 PAP torsion angles,

the corresponding total potential energy o f PAP, and the energy gradient of the

calcdation,

(Sinfile, %outfile) = @ARGV; #Get the mput and output files fiom the command liw

open(lNFILE,$i.e) II die("can't open ide: $innier');

open(OUTFILE,">$outfïlen) II die("canft open: $outfile");

# Variables

$chil;

$cm;

$cm; $Energy;

$Gradient;

# prints headers to outfile

while(<INFILE>) # read each input line until end of file

iq/(chil = )(Id+)/)

{

$chil = "$2\t";

1 iq/(chi2 = )(Id+)/)

$chi2 = "$2\tw;

1 if(/(chi3 = )(Jd+)/)

$chi3 = "$2\tlT;

1 if(/(Energy=)(- *\d+.\d+)/)

{

$Energy = "$2\tW;

1 iq/(Gradient=)(-+\d+.\d+)/)

$Gradient = "$2\nn;

print OUTFILE " $chil $chi2$chi3 $Energy$Gradient " ;

t t

ELECTROSTATXC ENERGY EXTRACTION This PERL prognim searches for and exhacts values fiom Hyperchem single

point energy caIculation log @es and prints them in a tabulateci fonnat, with appropriate

column headings. These values include XI, ~ 2 , and ~ 3 PAP torsion angles, the

corresponding total potential energy of PAP, the electrostatic component of the total

potentiai energy, and the energy gradient of the calculation.

($Se , boutnie) = @ARGV; #Get the input and output files fkom the command line

open(INRLE,$infïle) II die("can't open me: $$Sinlem);

open(OUTFILE,">$ou~ett) II die("cantt open: $oumiet');

# Variables

$chil ;

$chi2;

$chi3 ;

$Energy;

$Gradient;

$Estatic;

# prints headers to oume

while(dNFEE>) # read each input line until end of file

@/(chi 1 = )(\d+)/)

{

$chil = "$2\tW;

1

nmrPIPE PROCESSING SCHEME FOR DQCOSY SPECTRA

=Pipe -in dbc703d.fid \

1 nmrPipe -fh SOL \

1 d i p e 41 SP -off 0.00 -end 1.0 -pow I -c 0-5 \

1 nmrPipe -fh LP -fb \

1 nmrPipe -fh ZF -auto \

1 nmrPipe -fin FT -auto \

( nmrPipe -fk PS -pO O -pl 0.0 -di \

1 nmrPipe -h TP \

1 nmrPipe -fh SP -off 0.00 -end 1 .O -pow 1 -c 0.5 \

1 nmrPipe -h LP -fb \

1 nmrpipe -fh LP -fb \

1 nrnrPipe -h ZF -auto \

1 nmrPipe -fh ZF -auto \

1 =Pipe -fh FT \

1 nrmPipe -fh PS -pO 0.0 -p 1 180.0 -di \

( ~ i r P Ï p e -h TP \

-out dbc703d5.dat -verb 2 -ov

NmrPipe r ads the script listed above and does the following: reads an fid,

suppresses solvent signal, applies a sinebel window h c t i o n in time domain, Iinear

predicts (mixed forward backward) the data m time domain, zero fills data in time

domain, fourïer transforms data, transposes data so fiequency domah can be processed,

applies a siuebell window fiindon in fiequencydomain, linear predicts (mixed forward

backward) in fiequencydoxnajn, linear predicts(mked forward backward) m

fiequencydomain, zero fils in fiequencydomain, zero fills in fiequency domain, fourier

transforms a, applies ln order phase shift of 180 degrees, transposes, and outputs a data

file.

HYPERCHEM TEMPLATE FILES

Ethanolamine resîdue

; ETHANOLAMINE

HD lm DN:

CA:

(H s -3 s CA s) \

OP^ N -0.5700 imp -3 CA N H \

amber N -0-46.0 imp -3 CA N H \

bio+ NHl -0.3500 ïmp N -3 CA N H \

int C -3 1.335 -2 116.60 -1 180.0

CH 0.2000 imp C S CA N C \

CT 0.0350 \

CHlE 0.1000 imp CA N C CB \

1.449 -3 121.90 -2 180.00

CB: (OG s CA s 1HB s 2HB s)

OP^ C2 0.2650 \

amber CT 0-0180 \

bio+ CH2E 0.2500 \

htCA 1.525 N 111-10 -3 60.00

1HB IDB HE31 HB3: H (CB s)

OP^ none \

amber HC 0.1190 1

bio+ none \

int CB 1,090

(CB s)

OP^ none \

amber HC O.If90 \

bio+ none \

int CB 1.090

(CI3 s HG s)

OP^ OH -0.7000 \

amber OH -0.5500 \

bio+ OH1 -0.6500 \

int CB 1.430 CA 109.47 N 180.00

H (OG s)

OP^ HO 0.4350 \

amber HO 0.3100 \

bio+ H 0.4000 \

int OG 0.960 CB 109.47 CA 180.00

OG:

HG DG HOG:

HYPER-M PARAMETER FILES

Only the parameters pertaining to atom type 'HY" are listed.

ambertype-PM-ad

-

ATOM MASS REFERENCE

HY 1,0080 guess by DCB

O C HY 70,0000 122,9790 mess by GAW

ATOM R STAR EPS REFERENCE

guess for fonnyl group by DCB

B1 B2 K R R E 0 REFERENCE

CALCULATION FOR AMOUNT OF S-PEPTIDE SPROTEIN

COMPLEX

5. Quadratic formula [z] = -b + (b2 - 4ac)-%

For trans-PAP7-RNase S, Kd = 0.38 + 0.01 xlo4 [x<nii] = 0.43 x 1 oe3 M

[YtoM] = 0.64 x 10-~ M

Substituting these nmbers mto the quadratic formula gives 0.429 x 10-~ M RNase S.

Therefore 99.82 % of the S-protein is bound and 0.21 x 105 M f?ee PAP7-S-peptide

remains (32.8 1%).

For cis-PAP7-RNase S, Kd = 0.27 + 0.01 xlo4 M,

= 0.43 x 105 M

[ytOtal] = 0.64 x 1 o - ~ M

Substituting these numbers hto the quadratic formula gives 0.429 x 1 0 ~ ~ M RNase S, and

therefore 99.87 % of the S-protein is bound and 0.21 x lo5 M fiee PAP7-S-peptide

(32.8 1%) remains (assuming 100% conversion fiom trans- to cis-PAP)

Substituting these numbers into the quadratic formula gives 0.292 x 10*~ M RNase S, and

therefore 97.50 % of the S-protein is bound and 4.75 x IO-' M fkee PAP10-S-peptide

remains (13.97 %).

For cis-PAP 10-RNase S, Kd = 0.64 + 0.01 xlo4 M,

[xt,ta1] = 0.30 x 10.~ M

CytOtal] = 0.34 x loJ M

Substitutmg these numbers into the quadratic formula gives 0.296 x IO" M RNase S, and

therefore 98.58 % of the S-protein is bound and 4.43 x 1 o ' ~ M fiee PAPl O-S-peptide

(1 3.03%) remains (8SSUII1II]9 100% conversion fiom trans- to cis-PAP).