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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).