Synthesis of and Conformational Studies _on Proline-Containing Peptid.es:

I '.-'

. .

^/

..

Synthesis of and Conformational Studies _on Proline-Containing Peptid.es:

"0.:,',<

"" ^.

I

Jv

[on~Binding ~yLinear Tetl'speptides

.• 0 Peter.Hugo Rebse;B.S~1Hons.)

Atbesis.sub;;;itted tothe

Scb.~1

of Gradua.te Studies infia:rti~fulrillmeot of tbe

requirements for thed~gree

or

Master

or

Science

'-,

.oJOep:lttmen~ofBiochemi~try Memdri}ll University of Newround1:md

I?ecember 1985

''.

Newfoundland

.,

\

.~.

/.

I

"'~

. Permission' has be-enc;lrantjld L'"autorisation.aflot!!accordl!e 'to the' National Library of II la Biblioth!que natioode 'Canada- to microfilm thi!L-: du Canada de microfilmer

~g~~~: '~fndth;Oft:;~ ~or-

^{sell :}

~:t~~nJ~:8~e8e\X~emp~~~~:~ ~~

/ . . f'Hm • . '

. The' author (copyright ·owner) '·L'aut.eur (titula"ire du droit

!las' reser"ved' o-ther ;.... d·auteur) se "rl!serve .1;'es p'ublication rights,'~rid :autres draits de publication,

neitOe.r the thesis no[' oi la th~se.01 de lonl/Q

extensive ,extracts from , i t , extraits' de celle-'ci ne.

may be printed'..~['otherwise doivent i!tre 1m'primAs ou reproduced Iwithout .his/her'· autrellient reproauits sans son' wr i t t e n perm!5.Sion. au:torisation ·l!crite. .

ISBN 9-315-))627-7

'.l.

Abstract

The rel~tionshipbetw'teo peptide b3CkbOoc topology arid ion-binding W:L~

investigated through ·proton and c:a.rbon·13~~fR,'CDandInspe<:VoscoPY. '1\

literJ'Lt~r.e~review·

⁰⁰ calciu'm-binding proteins· Ilnd

pe~tides"in4ica:ted

.thb.l"..D structu';e

eba.r:lC~terize..d

^by

two o~erlll.pping

8-turns m:l.Y be,

involv~d.

^{To lest}

t'hi~

hypothesis twopeptides whichco~ldpotel'ltia.llf,r9rrn. thcabovcshucture .were

,y,lh";,,d. >

.~._~~_

^{•.••••}

.The peptide!' N(lt&cPro~AJaAlaNliCH3 ^{lLnd its} glycine' analogue - ...

!'l°t.~cProG[y.~tlNHCH3;,,:,ere ch:tcacterizedi~the un.complex&) 51:1.te.by:\11 or the above mentioned teebniques in' a~variely of solvenls.· Proton NMR temper.ature-dependencc studies in DMSO.d(

and Nt! ^couPJiflg (On5t:1.I~ts,

^along.

with CD "and

m.

spedroscopyiDdic:ated"tbn.t botb peptides in solution were mllde up of

a~Ty_pe_(J'='~turojollowed , .

^by

~n o~e;inppins-'Py-p~turrr.---earbon.I;-~.--.

^. mm,lR aod CDsped~«:,p~jodic'ated that the DAIa-peptideW3S,asexp~ded,' moiest3bl~th3D the Gly-peptide: '

The ,hi'oding of metaliODS

to

the peptides was monitored using CD spectroscopy, Both peptideswe~e,fOuqdto.beio~;~·p~cific.While both·were up"n.ble of binding c:akium ion and in.c3p3bJe of binding sodiuman~lithium toIUlJ'signifieanL exLent, Lhey differed in their ability to bind magnesium. \ While the

N(ttBoc~roO~"aAJ.aNHCH3· p~ptide boun~

^mngnesium

~C3kIY a~

^{n Icyel}

compnr.a~leto its binding to the'~ono';alentions, Lh'e glycine nMlogue bound' magnesiurT\ to a. significant extent, . The Gly-peptide a.lso 'requi.red . a lower

c~ncentration

^of

c~lcium ion~to

reacb binding

s~tur4tion.' Thi~

^WD.Sattributed to the greater

ne~ibi1ty

of glydne a.nd

~ence

^..

ib

enhanced ability to

~.~tje

^metal

~n,

r "

...

;

.

~/, .j

/'

.~.

.-\:..-

, I.: '

. j i

The .~~nrorniatioDalchaDge or the peptides.' upon calcium iob .titfa:tioo .Wll.S.·

[allowed by' proton3n~. e3~bon-~3,~; The changesi~chemical shifts or the carbonyl resonances'andtbe amide proton resona:nces indicated that· aUtOUf

peptide carbonyls (OOrdio3ted to the: ca.lciu'm ion res Itiog in a breaking.or.the

jntramolec~lar

hydroge'o boodsat

th.~

^uocomplexe specieS: Analyses

or

the CD binding-curves. indic:l.l.ed that atI~w cOIl;«n,tra~ioDs0 Icitim(0.pee1ide _a 2:1 p.

e~tide.:ion

CO":,p.

le~' ~as

^torm'ed,

w~ile

^{a 'jg.ber}

c~.

Dceo,ttalions a .1:1

i6~pl~~ was, ....

predominant. The, 2:l. peptide:i.on mpleX~isabletorillall, eight calciu!11 :"c\oordinatio~sites withthe pe e carbOnyls"wliill\th~1:1 complex requires eith'er the perchlorate anion or. water molecule to_{, ~ , . . .}

,

. . .rillthe r.emaining coordination sites.

l'

iii

'Acknowledgements .-

.

~

Special lhftnks to my supervisor;Protess; V.S. i\nanthanaraynnan; the oth"Cf

memb~rs

^{of my'}

supe~vi.~o.ry ~omQlittee,rirs.

^-

Willii~

^S'.'Davidsol) nod

,Br~'l.D

Gregory;. ll.o'd alsoto Dr. Sam.ual K.Att~h.~o~urOt"his _~tient·instruct'ion'iii peptide' ',synthesis

and '_,f't~m

^-

inleip[etati~D:

-'Their" O:Ssist

~ ~,

..o.nd .

rric~dghip pro~~ded

an ideal

environ~en't '£or'tb~

pursuit ot,agrndu'ate'

ret..

^I

, . ' . .,.. ' .. j

I would like .to extend my,ppreciatioD,'t:o Dr. Hooper and QrucC' McDonald· of . the

Atl:Ui.ti~ ~egion. Magn~t'e ~:S0,Dll.ne~.C~n.t're, H~1Wa~~

^{t:Jova;:Scottn, rOt}

ruoo.ing the,mmanalysis and also·to Dr..S~,tr.i,rotpertormining the. Binding . Constant-anal~sis.

. . .

Many thanks to Mr. Lorne Taylor,torhiscritic~lperusal of the, thesis during prqdu~tion.

- - I This rese3reb' was supporledbya grant from the Medictl\ Rcse:l.rch Councilor

Cantlda. ,> •

\.:.

iv

"I)

Tabie of Contents

1.

[~tr~duetion

^,

-U.-Stru~tura)Cbatacteristics~rthe Beta·Turn 1.1.1. DelinitioDs and Nomenclature

1.2.Positional Preterence otArgino Acids in Beta·Turns·

I.~,Peptide Models tor the Beta-Turn 1.3.1.Cyclic "Peptides

; 1.3.2. Linear Peptides 1.4~Functional Role or.Beta~TurDs' ts.Design of Postulated Calcium-Binding Peptides 2. Experimental"

, 2.1. Mnierials, 2.2.-Methods

· 2.2.1. Purity

or

^Peptides

2.2.2. Crystallization

or

Peptides 2.2.3. Melting Point 2.2.4. Elemental Analysis

· 2.2.5.Amino Acid Analysis 2.2.6. HPLe'

~;2.7. Jnrrare~SpeetrosfoPY 2.2.8. Circular Dichroism Spectroscopy 2.2.9, Nuclear Magnetic Resonance 2.2.10. Binding Studies byCircular,Dicbroism 2.2.11. !;IindingStudi~by NMR .2'.3.' Peptide Synthesis

2,3.1. IntroduCtion _2.3.~. N(ltBoeProD~aAl8.NHCH3

2.3.2.1. N(ltBoeProONSU .. 2.3.2.2. N(ltBoeProDAJaOH

2.3.2.3. N(ltBoeProDAJaONSU 2.3.2.4. N(ltBoeProDAlaAlaOH 2.3.2.5. NO'tBoeProDAJaAlaNHCH3

· 2.3.3.N(ltBoePro~lyAlaNHCH3

~ 1 2

,

2

"12 12 13 20 26 2' 29- 29 29 30 30 30 31 31 31 32 - 32 3;l 33 34 34 36-

"36 36 31 -31 38 3'

'J

3. NMR Charaeterb:atlon 3.1. Introduction to NMR 3.2. Catbon-13~m.

3.2.t.lntrodu.ction to CarboQ-13 mm StudieS 3.2:2.Ca~bon.13·

mm

or NQtBocPjoGlyAlaNHCHa

3.2.2.1. Studies UsingDMSO-~6as Solvent 3.2:2..2,.. Studies UsingAcetoDi~rile-d3"asSolvent 3.2.3. 'Carbon-13 NMR

or

NQtBocP.ror;>AJaAlaf'lliCHa

3.2:3.1.

·~.t.ud~es U~~ng DMSO-~6as

^Solvent 3.2.3.2.S\~d~es Us~ng.CHCI3

r

^Solvent

3.2..3:3.St~dlest!SUlg Acelonrd6~.Solvent 3.2.4. Solvent-Dependenc.e orCar~oDyrRes'on:mcts

3.2.4.1.Intr~duction

.J

3.2.4.2.N:'~CPrOGIYAlll.~CH3 .', 3.2.4.3" N l!30cProDAlaAlaNHCHa

a.a.

Proton

1'l1>o.m \ . I

3.3.1. Introduction to Proton NMR Studies 3.3.2..

Proto~

^NMR

~r

NQtBocPrdGliAJaNHCHa

31.2.1. Peak AssIgnments inIOMSO-d

:3

3 2 2 Peak AsSignments iDjCDC13 6 3323 Peak AsSignments10Acetonltflle-da 3':324 Temperature-Depen ence of

N!:!

Protons 333 Proton NMR of NdtBocPrJDAlaAlaNHCH:r

3331Peak Assignments in1DMSO.d6 3332 PeakAsslgnme\~tsin QDCla • 3333 Peak AssIgnmentsIQAcetone-d6 3334 Temperature-Depen ence ofNflProtons 34 Conformational InformatIon Froi Coupling Constanls

341 Introduction

3.4.2. Conformation orNUtBoCP~GJYAlaNHCHa 3.4.3. Conrorm,ation or NatBocPr ,DAlaAlaNHCH;j

-?

Circular Dichroism, Inrrarf!d.and Modf!1 Building 4.1. Circular Dichroism Characterization '

4.1.1: Intrpduction . , 4.1.2.:,t;-I^UtBoCProGlyAlaNHCH3 \

::~:;:~::~:i~s~

^Data

I

4.1.3.NUtBocProDAlaAlaNHCH31

•• ••

_.2

42 43 43

·10

~.,

5,' 0.&

:i.~~

71 72 73· / ' 73 73 75 75 81 80 03 DO DO 103 108 Ill, _~"117 IIi 12.

123 128 128 128."

130 130 13~

137

.\

I

.~.j

",,';dI

,

\ ;

'i

~U.3.L

Spl.'ctf-aI.Data (\ / /

4.2.

Inrra~;~·;;~;::aIYsis

and Campa,rlsoo "..,'.

--!

. 4.3..

Suml'!iar'~

of Ulll::omplexed

Pep~e

Data and M04el Building 3.3.1.N(JtBocProf)AJa.~aNHCH3

, 4.3.2. NO't8{lcProGlyAlaNHCHa 6. [on-Binding

5:1. Introduction , 5.2.NQtBocP~oDAJoAlaNHCH3

5:~U.Circular Dichroism S dies 5.2.1.1. Stu"diesUsin ater as Solvent 5.2.1.2. Studies Using Acetonitrile as Solvent f .

5.2.2. NMR S t u d i e s ' "

I

5.3. NO'tBocPro91yAJaNHCHa .

5~^Circ:ularDichrois~^.Studies 5,3.2. NMR Studies 5.... Ion-Speeilicity Studies

\ 5.5. Ion-Sind.ing Constants - \ 5.6.'Conch:lsioiis andModeqauilding

Reterenees

. '\

131' 14•

142 148.

148 152 1••

155

I.'

15.

IS' 162 • 165

I"

I"

li2 li8 181 182 188

-I

i,.

, !

vji

List of Tables-

Table i-I: ,s.Turn Types:.8s DefiDl1d by Venk3.tach,1lam (1968) Bll,d Lewis dal. (1973)

Table'l-2: <P,'iRnnges or p.Turns asDefi~edby Chandrasek3f3nttal;

(1913). / '

Table 3-1: Carboo-13 NMR Peak,Ass,ignments

or

Na·tBoe~oG1Y~3NHCHIin DMSO.d₆ Table 3.:2: Carbon·13 NMR Peak ¥Ssignments of NQIBoeProGlyAJaOH

jb

_DMSO·d6

Table 3-3:

Oj~/:rrans R3tios~r~atBoeProGIYAlnNHC1-l3

in DMSO.d

6as Deter \Oed by Carbon·13 NMR Table 3-4: Carbon·13 NMR uk AssignmontS of . NatBocProGly 3NHCHain Acetonilrile-da Table 3-6:.Ci,/Trans"R ~osof NQtBocProGlyAlaNHCHa^~n

Ac~lonilrile-3byCarbon-13 NMR • .Table 3-6: Carbon-13" ',mPe3k AssignTl}ents of NQ/BocProOAhOJI

. inDMSO!d6

Table 3-7:

car~o

^lt3

mm.

PeakAssignmentsof . . Nat ,cProDAIa.AJ3NHCH~ⁱⁿD~tSO-d6 Table t\;JV CillTr~n8Ratios of N(llBocProOAlaA1:lI\'HCH" in

IJ.;!SQ-d,

by Cad",.-13 NMR Table

a-g

/

:

^{CarboD-13WtlR}Peak Assignments of N<lItBocPruDAhOIl

in CHCI₃ - " .

T&bi.:e 3· :

qarbon-13

NMR

Peak Assignme!"ts of

, . NO'tBocProDA13AlaNHCH

3in CHCI

3 •

Tabl 3.11: Carbon-13mmPeak Assignments or ,N<lIlBocPropAlo.AlllNHCH3inAcetone-do Ta Ie 3-12: ProtonmmPeak Assignmenlrs of

.' I NO'tBocProClyAJl!oNHCHa in DMSO-do /rabJe3~13: Protonl'{MRPenk Assignments of

j'

NO'tBoeProGlyAJllNHCHa in^COCI~

I

.,/

4.

·Ii

50 ,')2

5;')

58 59.

6~

" r

.1

'0 11

·83

\

. '

viii

Table 3-14: frotoo NMRPeak.Assignments of 88

N(llBocProGlyAlaNHCH3 in Acetoni.trile-da

Table 3-16: Te.!!lperature-D!!pendence ofNHProtons of 04

N

⁴tBocProGlyAJaNHCHa in DMSO-de .

• Table 3-18: Proton NMR PeakAssignments of , 98

NQtBocProDAJaAJaNHCHa in OMSO-de

Table 3-11: Proton NMR Peak Assi&Umcots of . 105 Nat~cPtODAlaA1aNHCH3joCDCla

Table 3-18: . Proton NMR Peak Assignments or" 110

•. N(I'lBocProDAIaAlaNHCHa in.Acetone-:d6

Table 3-Hl: Temperature-Dependence of

N!!

Protons of "115 N~IBocPr?bAlaAJaNHCHi'D' DMSO.de

Table 3-2f?: N⁴tBocProGlyAlaNHCHa .. Angles Derived^r~om'Nl::! 121"

~'esOD:LDCeCouplingConsta~tsⁱⁿD~iso.d6 .

Table 3-21:. ~criBocProDAJaA1aNHCH3~AnglesDerive~fromJ\,!:!.124 Resona\Dce CouplingCoos~aD~sinDMSO-.de

"Table 3-22: NO"IBocProDAlaAJaNHCHa^</JAngles Derived froml\1:! ^J26 Resonance Coupling Constants in CDCI3'

Table 4-01: Maxima and Minima or~TurnCD" Spectral CI:lSses as' 131

DeCined by Woody (IQ74). •

Table4-2: Predicted CD Spectrnl Classes (Woody(Hli4))for .lJ-Turn 132 Types as Defined.-by Lewisdat.(1913)' • Ta.ble .4-3: Circul:u Dichroism Spectra. of N^ClIBocProGlyAbNHCH3 in ,133,

Various Solvents

Table·...: Circul3r~DichroismSpedrl!- ofNCllBocProQAlaA1a~lICH3139

in V:lfio~sSolvl!nts .

Ta:\),le .e-6:

Il!.rrare~

Spectroscopy

~ide

A and Carbonyl Bands of 145

~n~'

^{(a) NCl}tBocProGlyAlaNHCH₃·and (blNCltBocProDAlaAlaNHCH

3in CHCI 3

TableoC~8: ','1'Angles Used in tbe Construction of CPK 'Models of 151

N^CllBocP,ro.I?AJ:LA1aNH

y

"i, .

ix

List of Figures

. \

Figure 1.1:1 Dibwral Angles Figure 1-2: . Geometry of thej.Turn . Figure 1-:l: Consecutive~Turns .

Figur.e

2:1i

Outline of Syntbesis of NIIJBocProDAloAl:aNIICIl_J - 3!;

Figure 3-1:

C~r,b~n-13

NMR Spectrum ofN°tBocp'roGIYAI;I:-.IIIClI:l ^.1-1

in OMSO-dc .

Figure 3'-2: E"xpanded C:l.rbon·13 NMR Spectrumcif

" N°tBocProClyAlaNHCH3

i~MSO.d6

^.•

-:"}igure

3-3:

Carbon-13 NMR Spectrum of NQlDocProGlyA"NlICII_J !)I

joAceto"nitrile-d3 -' . - . -

Figure

3-~:

Carhon-13 NMR Sp;etrum of N^lItBocProDAlaAI:1NIl(;II_J .,50 inDMSO.de

. Figure 3-5: Cllrbon-13mmSpedrum of Nt'OtBocProOAI:1AI:JNHCJl₃- 6.') inCHC1

3 . ' .

Figure 3-8: C~rbon·13NMR Spettrum ofN°tBOtProOt,\l.:lI\la.N~.IGII3 ,0

I in Acetone-de ' . .

Flg~re

3-1: Protol:!

~Nm

Spectrum of NO'lBocProGIfAlnNHCII3 in j6

DMSQ.da ... . ' •

Figure 3-8: Expll.nd.edf'I!HR~&!9D.ofthe Proton NMR Spectrum of_ 78

N°lBoeProClyAl:lNHCH3 in DMSO-de '

Figure' 3..0: Proton

m-m.

'Spectrum of N°lBoeProGlrAI:lNIICJl3 in 82

CDC13 • ' .

F1sure3-10:' E!Cp.o.nded

N!!

Region of the Proton' NMRSp~~trumof 8·1 N.oIBocProClyAlll.NHCH3 in CDC13

Ftg~r'e^3-i'1: Pro~n ~"MRSpectfum of NOfBocProClyAIaNIIC1I3 in 87 A~etonitrile-d3

Figure 3-12: Expanded

N!!

Region of the Proton NMR Spectrum orI00 'NCtjBoeProGlyAlaNHCH; in Acet.o:nitrile-d3 ... . Figure 3--13:" Expanded

.

N!!R~gionof the Proton NMRSpe~trum^' of 01

.-

.'

149 14i 14' 116

lthe

Caldum.Perturb~, NO'I8?cprOGIYAla~CH3

ⁱⁿ

Acetonitrile-d6 .

Frgure 3-14'. Temperllture;Depe;ndetreeofNt!^{Protons of}

II!'

Nll'tBocProGlyAlaNHCH₃

in

_DMSO.d6

'f',~;, Figure 3-16: ProtoD

mm

Spectrum 9f £'lO'tBoeProDAl:1AlaNHCH3iO' gj

.,.~J: I?MSO-dll ' .

FIgnr; 3-16: . Explmd9i

N!!

Regi~~of the ProtoD~MRSpectrum of .100 N/t/Bo~)Sr~DAlaAla['\lHC~a_{in DMSO.d6}

Flzure 3-17: Proton, NMR Spectrum of

N(llB~cProDAIa.AlaNHCH3

^{in 10-1} CDCI,

Flgure...3-.18: _'Ji:~pandedN!!Region ,ofI:'rot~:lD

mOl

Spectrum 'of 10;

'\::Na.J8o:cProD..tlaAlar"ffiCH3:fn CDCla

F1sure,3-1V: ProtoD NMRSpectrumotNO'tBoeProDAlaAt:l.NlIC.H3 in 109",

~. 'Attt"Woe-d!l'-" .' ,." . '

FlguJ'e'~~20iExpnnded

N!:!

Region of the Proton

mm

Spectrum of

Wt.,

~alB~ro~Al~lI.NH.CH3in Acetone-.d6 Figure 3-21': Tcmperature-Depco(lence o(N!!.ProtoDsor

~oIBocProDAlaA1aNHCH3in.DM$O.d6~

F!&!J.re 3-22:,Rel4tions~ip .Betwee~the N!:!·CH Coupling.Const~nt IIlJ

.and tbe~Dihedral Angle '

Figure':J: 'Circuln~Dichroism .Spectra

ot

^NQtBocProGlyAla!'.l-:ICH3 ^13-l ... ' .. in Various Solvenis .

Figure ,....2: Circul:n pichr?is" Spectra or N°tBocProDA1D.Aint'litlCH3 138

in Various Sol'{ents' (, .

Figure~-3: lnlrared~pectrumAmideA Band

pr.

(a)N°lBocPrt;K;lYAla~CH3 ~nd lh) N°lBocProDAla.A!aNHCH;j in. CHCI.3 Figure ..~..: lnriared Sp'llctrum Carbon}'l Reg-ion or

(a) N°lBocProGlyAhNHCH3.and (h)N°lBocProDAJaAlaNHCH3 in CHCI3 .Flgur'.e".-6: SchematicD'i~gramorUncomple~ed

N°tBocProDAlaAJaNHCH3 . • '

Flg':lr'e ....8: CP.K Model

or

Uncomplexed N'"amocProDAliAlaNliCH3 150 , Figure 6-1: Circular Dicbroism Sp!!drl1 o:r N°lBoeProDAJaAlaNHCHi...157

at Varibus'Calcium Chloride to Peptide Rll.tios in. Water and in the Pre!ence pr8MGuHCI and 8M Urea

.:: ')'lgure~2: (81;~^V8. ICa\l;II(~eptidelor N°l60cProDAJaAlaNHCH3'150 ioWater

·.~

xi )

161

185 Iii'

183 18·1 Figure&-3: Circular Dichroism· Spedr:L of. N°IBocProO..\Ja.,\1:lOCH3 160

With aDd Without Calcium100io Water Figure &-4: qircularDic.hrois~.sp~traof N°tBocProDAbAlaOH,

N°lBoePro0A1:LN'fIC,H3aDd N°tBocProDAlaOH in Water; With and \Vithout Calcium Ion ' • Figure &-&: Circular~ichroismSpectra or "N°IBoeProDAI:u\la:'-:IICH₃ 16:1

at Various Calcium to Peptide Ratios in ·Acetonitrile Figure '6-6: 161~;51^VB,1~~2+IIIPeptide]or N"'IBocProDAlaAlIlNIIC'113 16·1

in Acetonitrile '

. ,Figure &-11 Change iD Cliemical Shirl...:i N!:! Reson:lnm _l'81 166 ICiL~+I/IPeptidel Ratio 'of N°tBoeProDAIa.,AIl),NIICII3 in AcettJoe-d

o '

Figure &-8: Change in ChemiC31 Shirt or Carbonyl Rcsona.nce:I va, 16..'1

.JC:i2+I/IP~pti~eJ

Ra.tio or N"'IBoeProDAIa.AlaNIICII3 . in .

Acetoo~de

Figur.,e.&-O:

C?ir~u!ar

iiichroism Spectr:L or N°lOocProCIYAb.to-lICI13 I,D at Various Calcium to Peptide Ratios in Acetonitrile Flg~re6--10:

19l;;'tia,

IC!l~+IIIP.eptidel^ol N°lB(~cProClyAI31\1)ICII3I,-t

in Ace'tonitrile

.Flgu.r~&:<11: Change io Chemic31' Shift -'ofN!!Resonances V8, Ij3 ICa2+I/IPeptide]R:i.ti~of N°lOocProClyAI:LNIIClla in , Aeetonitrife-d

3 '

. Figure &-12: Change'in Chemical Shirt of Carbonyl ResonancC$va, IH (Ca2+I/I~eptidelRatio or N°lBoeProClyN:lNlICUa in

Acetooilrile-d3 '

') Sigure

~13:

• loo-Specificity or N"'tBoeProG)yAlaAJaNlICI13 Figure &-14: Ion-Specificity of N°lBoeProOAlaAI3N1WH3 _....~

Figure &-1&:

lej;;14'~;,

(Mg:!'!'II.IPeptide) or

t'l°tBocP'oCIY~I:1i'l11C:::H3

^180' ioAcetonitrile

Figure &-'18:' Sebem5ticDi3grr.~orK"'IBoerroDAl3AI:~J:mCH3'in 3) Uncomplexed Form aDd bl Calcium-Complex.d Fprm FI&ur~.&-i1: CPK Models

oJ

N°lBocProDAlaA.laNlICII3

in Calcium-Complexed

For~

^{) .}

FII~re

^{&-18: CPK}

~lo~els

^of

N°1l3b~ProDAbAIaNHCH3

^.

in Calcium-Complexed Form Without Calciu'alon

. .

^'..

" )

. . /

,.'

.' ^:'

A"

Aib Ala

A,.

Aip Bu CHCl3 CD COC13 'Cyi DAlto' DCC DCU DMSO DPhe EDTA Glt GuNel Hz 110 ip' HPLC 1R' Leu / ' HCB,

"'eOH 010 NlIR HSU . DEt.

Ph, PiT P"

Sa.

lB••

xii

Abbreviations

.e~t~itril' (I-ami ,oisobut1ric acid.

alani • a.paragiDe ..parttc; acid

'::~~;olo~ .

ClrcularDi,ehroialD deuterated' ,chloroform cy.tdne D:-alanine .:

dic1Cloh,:rylcarbodimidl' dlclclohnyluru di•• thylsulphoxide D-phl111lalanlu

.thrlell..dl&lDl!l.et'~tra.c.ticacid . gl,c1ne

gU&nidi:D.ehJdrochlorl~.

hertz·

isoleucine isoproP11

High Performuc,'LiquidChrOllla~hJ. _ _....

Infrar,d . -,

"/<

;::,:~:

..Calcium-Binding

~arvalbUlDin

^{. \}

IIllth&Jl;o'l llorleucill.l

lI'ucllarUaglilti,c.RUOD&IlCI .1l-h,droxy nccinl'mide

OCH~CH3 phtD~in.

pivot,l pro11nl H-llltbylgl,c1u lertiory"'-But0%7carboIl11

,"'"

_TIIF

. - Ttp T7' UV

xiii t.rifl1l.oroeUluol . t.tr-uydrofllran

tryptophan t.YfOI1I1. • • Ultr&'t'1oh(

I.

-

Chapter 1 Introduction

i\s

3njncr~asi.ngnumber of proteinlhtee--dimeDsio~alstructuresar~ solve~by _

~.ray erys~aJlograpby,

^a.

~g ~Or~ell\ti~n

^between

runcti~n

"and the'bve'rall IOP~10gy

or

the,proteinmolecule is.beco,\ing_{~ViOU!l.Thetopolo~is}depeJ1.deD~

ontb~ r~din'gpatternsof tli,em.o[ecul~and can be clasSified in terms or different .t;peS,orslih·strueture.:d'he

sirnpl~t

struct'ur"alarr"sngements'beyond

th'~

^cOValeD}

interaction of the primary structure (or amino acid sequence) are t.be so-called second3.ry 'structu;es sucb as the O"helix,".struc~lIre^anath~,8-turn. The specific llrrangement orthese structures within a_(unclional domain 01 aprotein (supcrsetondary structure) correbtes very well-with tbe functioul role of tbat domain~For example, the heme bindingdoro~inis characterized by four (t·belices rold~dbi\ck on themselves,

,.

'flie relationship between str\lcture and function (ontinues to mnnifesli~selfat the level of the completely. folded (t~rti3.ri) structure:and .Ihe association of individual protcin subunits in the qunteroary structure (Creighton, 1083).

\

The Hurn is a ;econd:ny strucunl feature pervasive throughou't globular proteins. On-an average;'

abo~t

30 percent' of 'the secondary structure of all globular prot«!ins is made up or tbe ,B-turn.

,.

The .,B-tUrDischaracterized by a '.

reversal or nearlyt8(}"in tbe direction or. peptidecb.i~ ~ndis stabilized by an intramolecular bydrO&en bo;:d betweeo thec~~nyl(C=O)or residueIi)::Lnd tbe amide IN·H) of~eSidueli+3)IZ~ermanDodIcbetag~.IOH; Cr:Lwford~aI.•

l{li3jSmith and Pease, 1'iJ80). By reversing thedireet~oof the polypeptide cha.in, the ,6-turn provides:L.useful device~h.icheO:lbles the more periodic and repetitive secondary structuresto fold intoth~ir^fin:llsuperseco~d':1fY:lnd tertilltY

. I

structures (Sitnnda and Thornton,lOSS). AJthough this effect nn bd Il.ehieved by

.

^{. .}

. a· ·random coil-, the llbilit)' ort~e .8-t~r.nto form a hydrogenbond~ncL"lLSII.

-driving force". The~turnhILS~ee.nimplicated as a.site ornucl~lltionin protein

. .

folding (Ptitsyn, 1081). Sinceitsformal derinitionby·Venkata·fh~I.~in IUG8, ::L co?sider~ble amount of errort has beenexp~n.ded ~. delcrmin~thestrl~ctllral characteristics or the various ,6-turn types in bothprol~iosand pcptidl'S. ·In recent Ye3n,.se~erlL~functioDlllr~les.bo.ve been investigated. I wiU-summnrin below the n;levaot datiL00,6-turnsan~rel:lted areaswbic~would formIIuseful b:lSis for l¥

, . .

stu~

described. in this thest's.

1.1. Structural Characteriatk.

or

~he. Beta~Turn 1.1.1.

D~nnitlona .~d

Nomenelature .

I .

The J-turn, like. oth.ersecgJtd3~ystructures, can be'defined by. et or dihedr.i angles th::Ltdescri~ethecoororm~tionor the peptide bn.ckbone. A dihedr31 angle ,is defined as follows: if. system.~r^{rour atoms}A~B-C-Dis projec d ontoIIplllne oorm!llto borfd B.C, the.angle be.tween the projectililO of A-B a. d the projection ofCDis~~tribed as the dihedral '.ngle. The dihedral • gle is.cO~5idered' . positive or oes:ntive ac'cordiogtowhether,wh~~~e.system viewed..~longtlie

~\

central bond B ... C (or C ... B), the.bon~to the front atom A (or DJisrotated to

. .

the right or to th,e lert. respeetively,·jn order thatit mayeclips~the bondtQt~e

I

^{UfatomD(OfAl (eRe}Handbook of Biochemistry and Molecular Biology, .761. Fig...'-I·d.n.., tho dlh.d..1angl. and

1moD;t'~t"

tho O' and 180' positions of both the4'and the0;angles.which correspond, respectively, to the

~ot3.tions

^{about the'N-Co}

an~ ~on~s.

-<rile dihedral anglew

r~rers

^{to the}

rotlltionnhout the peptid,e(N-C)bond which is given a~3.1ueof180-:'(lrap8 conform3tionl.in .derining the various~iurDtypes although slight deviations from planarity do occur (Ramachandrandal.,1,073).

.

^'

'. : . -

rile JH,urn was originally classified into three main .:rpes-(l,nand ill) andt~e.ir

~irrorimages (1','0' and IlI'1base~.?n.the 41,'1' .:ngles ort~ecorner residues'li+1) and (i+~l (see Table HI (Venkatachalain, lQ68).VE!nkatachalam's. (IQ68)·

cl3SSiri,cation system was dc.rived rrom theoretica.1 calculations. based on a linked three-peptide unit,(Cfto C: and the ability to rorm a 4 -.: I bydrogen bond. The pepiide bond between residues

(i~l)

and (i+2) or Types I andn

diff~r

^by

approximately 180·. The 1J'pe IIIissimilar to the conformation 'of the Type I, . the

rQrme~·

being tbe beginning or a

3

10·belb::. ·The mirror images refered to by the prime (')di~rerTrom their roots~y

s:

sign change. Hence the 41{i+l) or a Type 1:,8-turnis·60· wbile that.or•.Type I' is+60·. A diagram of the P-turq (in the Type I and Type II forms)is· shown in· Figure 1·2

~Iong

^{with the,}

notation9~usei·

for ','"a~glesof the respective amino acid residues.

In' 1073, ChandrllSekarnntl a/.~orrelatedtheqretical stu.dies withex~erimental , data and produced a'range notation (see Table 1·2).

. ^.

^' The experimental data .

•• -.> •

'b

'i~" .

C--;;r--N

<!>'!180'

I

H

H

I · .

⁰

c ~IL i/ _~

C 'l'.(f

l -:

risure1·1: DihedralAbr;les

.

^\

Type

Table 1·11 ~Turn Types as Defined by Veokataehalam (1968) and LewiS dal. (1973)

f'(i+l) ~(i+2)

.,j"'tt-

I -60' -30· ·90· O'

'1' 60' 30' 00· O·

D -50' 120' 80· 0'-

D' 60' ,120' ,80· 0"

m

-60' -30'· ·60' -30·

III' 60· 30' 60,' 30'

rv

exists when two or more or the angles of TypesIthroughill' differby at lellSt 40' ,from any

of

the values listed above.

V · 8 0 · ' 80' ' 80' ·80'

,v· 80'

·80· -80· 80: .

V1 containsa d. proline at position (i+2)

YO a kink in the proteineh~iocteated byf'ti+l)::i:::.180' and 1"'(i+2)1<6O· or1~(i+l)1<60·and~(i+2j:::::1180' . Types

rv

through YO are (rom the expanded definition o( Lewis d

.

^.. al.,1973.

or-

· FIsu"1-2: Gtometry ofthe6-Turo

.. 'i

.~.

" \

\

/ /

Table1~2: _,IiRa~gesof~TurDs as DeCined byCbaDdras~ka.randol. (1073)

B _-(;+1) !J;H -(;H) "'(;+2)

I. I I-'OH-'Or (-.OH-IO), 1-150H70), 10-80·

Ib U (-'0)-(_30)' 80-140· . 20.80' 10-iO·

n. !' 20-80· 10-00· 70-150' -1-'OH-IO) ,

nb U' 30-80' - (-160)-(-70)' . (-SOH-20)' (;70H-IO),

m U (-.0)-1-30)' 70-160' 30-170' (-70,'0'

IV U' 3O-QO^e (-160)-(-70)' (-170)-(-30)' (-80,'0'

A,....- Typea.s·nota~by Chandrasekarlm d at

B

=

Correspondinprype as ,derined byVenka.t~chalam

.(

, /

i:l .

I

"

were based on the crystallographic structures of Iysolyme, chymotrypsi1!, andII.

scri~r:trpeptides, and on the NMR profiles of severnl cyclic peptidl.'s. TIle (ollowing month, in the S3mejournll.l, Le~isel al.(107'3Fprodll"tpdn extensive"ist of·p..W:tls- lXcurring in proteins. They examineJthe crystal

- ~.-,

structure of eight p/~sandidentified'~bend~'as structures )V1\l're11,11' distance between ~he'Cl"'C3rbonsor the ith and (i+3}tQ. fesidul.'S was

'$

i.,t.

Since thederiniti?DS are based upon~stancerather than on thel.'xi~lt'llC·CaLa hydrogen bond, the term "bend" was~sedby these authors rather than "luru", .:xhey diHerel'ltiated the P-bends (r.om

, ,

a-heliees~eqUiting

.

tke a-hC!liclS to h:m four or more consecutiveR_i,{i+3)a-carbon distil ces

or <

8". Usingthe :lbo.ve criteria, they identified 135 .8-bends. FrofQ the above data, and the enera minimization calculations on. a number 'of peptides, thE;Y expanded the d-tUrrl notation of Venkatachalam ,(1968) homSL1to eleven (see Table \-1). The dihedml a.ngies of Types I throughill'as originally proposed by Venbtachalam (lg68) were retained. These "bends" were allowed to have one dihedral angle differing fr0ll\(he ",idenl- value by up

to

50'nnd still maintaini~Sdesignation even if there was no inlramoleeular hydrogen-bond'ing. A Type

rv

bend occ,!Jrred if two or more of the dihedral angles AlScd to define Hurn types I through Ill' differed by more than40'from the ideal. . Type V was represe.nt3tiveat"n,C7

co_n~er

or '1"turn (with an i+2 - j

~r

^{3 -}

11

y drogen bond) as oPPO!l!d to thc C10Hurn which .h3;' ani+3- 0I'or 4- 0I hydrogen bond. The Cx notation refers to the number of atolT}s involved in the ring formed by the hydrogen-

"bonded 'reverse lurn. Type V' was n?l observedbtLewis el al. (lO!31 however, tbey stated tbat it could theoretically exist. Type VI

, ^.

isproduced by aci~proline

in pOsitionfi+2)and Typ:'vnis. effectivelya -kink" in the proteinehai~. In additio,D to the C 7 and CIO~onrorm-er5mentionedabove, Lewis etal.,(19,3).rt~r...

tothe existence of aC-

s

^{conformer(2 -} 1 hydrogen bond). The existence

~

based on the

lit

results of energy minimization calculations

or

•N-3c~tY.l.N'.methyl,AlnAlaAlnAtaAmide. The majorroo9iderationof the work of Lewiselal.(1973)

~nd

C,hnndr:lSek:lrnoetal.(199)istha:t there

~s a

ra.nge of^f,.1f' vnlues for tach of thevari~us/l-turn types.

Inuiis thesi5, .the originalf,t/Iangles andhydrogen-bondingrequirement of

~"katachal,m

(1.68) will b,used. The n;'at!<1n'

,r

L,w;, " 01:

i'.'3)

^and

Chaptlrasek:uan etal. (U)i3lalong withthe

C~

^notationwiil,bereferred 'to on occBSion.

1.2.P~8itionalPreference;or AminoAcidsin Beta·Tt,lrns The ,8-turo, unlike more uniform structures such as a-helices and ,8-sbeels, demonstrates a mar).ed positional preference. of amino acids adding another

~iO~ ~

^lb.

m'~"/b"'k" app~"b U~;

in lb, piedlol:,n01 p,,;ein and . p~ptlde secondary structures. _. . ThIS preference was first recogolzed by Venkatachal3m (UI68) wbopoint~doutth~t.becauseof steric requirements the

"Type I p..turn tends top~ererthe'LL' sequence and tbe Typenprefers the

J.D"

sequence. The notation refers to the particul:u amino acidenantio,"?~rsin positi~Ds(1'+1) and (i+2) of the ,8-turn, respectively. This chinl preference woul\

later.~e used to advantage in tb"esynth~sis'of ,8-tur'nm~ders(seesedioa1.31. \-"

Crawtord d",af.(1973), arter a study of 'the crystal

stF"uc~ure

of 11 proteins, observed that aspartic acid seemedtoQe preferred in the first~ition,proline in

.,..

~htsecond, asparagine in the third,and¹⁰tryptophan inl~e rp~rth. They Sl:ll ...u

tbat the limited dnta pool (125 11l(05) W35 Dot enough

.,

rora-ddinitin'~tlltlr ibey also pointed out what appeared l? be a 'v:lfi:ln('e or positional prr(t·rt'nrt· •.

based on p.turn type.

In 191'7, Chouand

Fasma~mpteted

^nmue-h

m~re

^extensive'stud,· usingthe

ato~jc_

coordinates, as determine.dby

x-ra~ crystallogm~hY,

^or29

p~otcins. ,')1

1 di5covered 460

~turns

as described by Lewis dal. (IQ73). The percent

rr/l~IlCY

ofoccurrence or- eachparticular"type is: Type I, 41.8; "'('ype II, 15',2;T)'j"'ut,18.3.

witb the-other types (]. tbrouf;bytl)occurring with much smalrcr .frequents?' Type I is thusseeDto be by fat the mostcommODro1lowedqyType-Ill'3.0,<1then Type n.The rema.ining p.turc. types are re1:l.titely ra.re. AsdidCrawfordd

af.

(1973). Chou and Fnsmnn(l9i1)..Doti~~davaria.tioo in theposi~ion:J.1prererenc"-.

amino acids based 'on .6-turn type, but ..

~ecause

^{or a.}

Iimit~~

dat:J. pool, t h e y ' "

- • . 4

groupedthe~t.urllStogether.~The most common amino a.cids were; prn;itloni, Asn(17%)1 'rs (l1%); As'p (16%); posi.tion(i+J),Pro (33%). Set11-I%),Lys

tJ ..

(13%); posit'n(i+2),As.n(21%), Asp (20%), Gly (20%); lind at positinn fit3), it: •Trp(19%), Gly (11%), Tyr{1~%1· Sterinonsid~Q,tionsare an importnntf~ctor but it appe:J.rs that there is also dn inyerserel~tionsh1pbl!tween hydrophobic'iti llnd turn·forming potential. Chou and Fa;sman ('l07i) al:!lO 'examined the region! ..."

.~4 residul!5 to eaehside.~ tbe.~turDform,iog It:t:rapeplide ""nd still noled positional pre(erenees. It must ber~memberedthat the aboJit: values are weighted

tow~rds

whilt occurs for

t~

most eommon ".turn type, By looking at the data on no individual p..tUrDbnsis certain sl:!,tementsC:J:Obe made. The mosl cor,n'mon

/

11

li+ 1),(i+2) sequence ort~eP.tufnisProGly. This sequence occurs mosto~tenror Type n rollowed by Type I and t&:oqIbut neverrOtany of the mirror i,:,oges.

This

~s t('l~':-likel)'i,due

totbe

sleri~3.lIy

restrictive

prol~ne.

Glidae witb no side chain could act as either a D or L amino acid and therefore (it the LO

,

.

cQnllg!".atioq J:equitement or the Type ~ ~lUtD !Yenka~achala~, HI68;

Chandrasebran, 1973). Aninteresting pointis'that severalL-nmino.

a~ids

^can also .bc)qund at

~ii+2Jh

position or the Typen,8-tura. 'Although these Type' If JJ-luins ",are probably not

i~etl.l, t;e~'

^LD requirement is bbviously an

.

^{' .}

. . . .

^{~ .} '.

ovetsimpliric:1tion. . The

to'

·'requirement.I was based on the 'so-called alanine -'peptides' (Ramll.chandran

a~d

Snsisekhamn, W6S).

Theposi~ionllJprderoence~ran -amino acid depends' upon .the neighb9uring resit.!u,es-:- This W.as, demonstrated b)'. Ananthanllrayanantl ql,(lgS"') wbo se:uched the sequences of .H globulnr proteins df known crystal sttucture for the

..

\',

lelrapeplide sequence Z-Pro-Y-X,';bichrL'(es prd!ine as the (i+l)th. resid'uf They compared their results with those of Chou and Fasman (10;;) and noted

. , . ' .r'._ ..

, that there were S?me major difrerences. - . Forexam~le, aro~lltieltydrophobic residues su'ehll.S~ryptophnnand,' pbenylalnnine were found.t.o have a relatively high occurrence nt, position i when proline was in . position {i+l.l, . while su.ch hydrophobic residues are generally Dot'preferred int~isposition in the Chou- F35man (lgi7fanal~.sis, Glycine, although one of the top three in, overall oceurre~.ceat 'position

F+2)

in the analysis of Chou and Fil!!mnn (19~7),'hecomes by rar. the

~ost

predominant residue ·ai thiS

po~i.tion

^when

p~oline

^isat position

\ 0 '

12

(i+l). The'eause of maoy of these eHeetsenD be" explnincd.through the results or

: , \

Schimmel and Flot.y (1968) who observed that thepraliDI.' ring restricts the conformational space avnilable toits:immediate N·terminal neigb'bour.(Further e:HI.~inationof_~turns'with more tban one residue rL'\cd becomes difficult because of thelimited pool of protein structural data.

1.3. Peptide Models forthe.Beta~Turn

'Tb'e exnmination of~t~rnsillp~oteinshasitslimitations (see section1.2). The _use of peptidemod~lscnn provide Olu'ch more fundamentalinrormntionsince

not

only' do'one look at a

s.i.m~lcr i~olnted

sys(em but thepolen'tinl

dllt~'

pooi can be increasedoY.er

t~at

^{lLv!-ilaMe'in}

'~r''!t~ins.

A synthetic a.pproach also hIlS .the adped advtLDt:'l.ge or the possible addition or g'roups which 'do not norm:l.lly occur in - "proteins. Amino a.tids such as p..alanine, N-melhylglycini! (Sar), . /

Q.am~~·obut~rY·1

(Ai!>.)' nnd 'the -b.enantiomeric 'amino ncids p,rovide

>..~nique st~ric'

restrictions whIch canbe very userul in the study or peptide conformation.

---

1.3.1. Cyclie Peptldes

Cyclic peplides havebe'e~ wid~ly'usedi~thestud~or~turns. These include

oxyt~cin

(Urry an'd Wnlter, 1(71):

i~

aDnlogue' (Pro3,Gly4J-oxyiocin (Bllll:J.rdiJid al., ~g7S) and wholly synthetic peptides (To.rchia. ~tal., 1072; Blo.h:'l. and '\

Budesinsky,'lg73; Kopp'ledal., tg78; Pease and\yn~n,'IUi8; Nemethy'd?{.,' 1981; Maxfieldt!l

at,

19S1). The major advantage of using cyclic peptidesi~also theirm~jor ~is:1dvantage.Cyc1izationrestric~th"conformational "mobilitr or the peptide. Hence l p..turn would' tend to remain in 'a particular definable conliguration. Because or this.' the cyclic peptide is not the best system~orthe

1

⁰

13 ~ _ study

:'f

^the

re~ative

stabilities of;turns and lh,e effect of

DeigbboUr~

^residues

l?n that stability.. Linear peptides are much more_cODdu~ivetothis type of study.

1.3.2. Linear Pepttdes

The proline-contnining lin(!8.r peptides have aroused the most1nterest in the study of~tutnsfor,tWOte:LSons. ProlineW3Sfoundtobetb~.mos~abundant residue at position(i+1) of the p.turn in proteins (Cho'u and Fa.sman, lQn, Hli8) (see section 1.2) and through conformational e-nergy calculations (Zimmirman and

Sehi!~aga, ~g71;'

^{Zimm:rmnnel01.',}If77) wns shawn to. be 'Iimited to a )nail number of confor;-ations because of the restricted fotation around the N_C- ^ttbond

.

in the"pyr;olidine ring (1

~

^{.GO'} In}lOtO,'Boussl1rd

a

,af.investigated as;ries of proline-conta.ining tripeptides.

lF~e P.tIJr~S wer~ '~,iabili~ed

by hydrogen- bonding between the carbonyl of the N-terminal

pr~tecting

^{group RCO}

and~

^the C-terminal protecting group NHR where R in both cases was either a methyl, isopropyl or a ter/.butyl group. The use of protecting groups to provide the . ,.. carbonyl and NH required for 'hydrogen ~pnd formation and hence p.turn

stahilization is a theme which occurs again and again with synthetic linear peptide models. ·BousSlud

e!

al.{lOjOji'1-vestigoted Pro-X and X-Pro containing sequences using proton NMR and infured methods. They found thnt the Type II .8-turn

~as

^{the most}

f~;ed

conformation with ProDAIa and ProGly

co~;aining

sequences while ProAla contained semi-openedG,.C

6

^{and C}SC7conformers in

.

,

additionto. the expected Type I ".turn. Aubry.el

al.:

(111i7) also found the RcoAla sequence formsIiTypeI.8-turn in solutibli, however, in crystalth~y^found tbnt

N~tBuProAJaNHipr

prefers -ttle"' Type

JJ·.8-~lira

conformation,. Thisis a

"

• n

strl?Il$-reinind~r that the crystal structure does o9t alwllYs reneet the conformation in solution. 10' this 'instance, they blamed the formntion 'of intermolecuiar hydrogen.bonds in crystllltorthe dirrerence. The X-Pro sequencJs s~em'ed

to.

prefer the mirror im"gc .8-turns asderinedbyVenhtllch:l\l\rn(H)l)8) :l.nd were generallylessstabl~

The cUcct of the X residue00the".tUt~Tormingability of the sequence N·

Il.cetytProGly-X.OH WllS investigated by Brahmllchari dat.(IUS:.!). -They tound,

. . ' I .

using Pf.otonNMR" CDand" lR spectroscopytpatthe X residue affected Ihl!

"relative'stability'oC the~tu'rnin ,the order: Leu>Ala>llc'>G1Y>Phe. 'the tripeptide, N-acetylProGlyLeuOH~~d.be,en previously shown. to benea~ly100%.

Type

iI: .8-~uro

in TFE at -40' C,· using vacuum ultnviolet

CD measllremenl~

(Brahmachariel 0/.,19i9). The peptide N-acetylProClyPbcOH wns shown by X·

ray crystallography to form a Type 0 ,6-turn (Brahmncbari elai.,1081). The formation or th_e, 4 .... 1 hydrogen. bond is very imporll1nt in derining. the

,

contormalion or the ,6-tUrD; The cryslal structure of the' dipeptide NQIBocProClyOH,w~ich.canp.ot form a C_IOhydrogen-bonded stru:lureWI\S shown to-,tontain~,"angles for the Pro and Gly residues5i~'lfto those round in n Type l,6-turn (Benedetti, 19i7), This is in

sh:l.r~"contrl1St

to Ute Type lI,6-lurn formed by ProGly sequences in thetriPePtide~"';~^..~tare capable of "?rming n, hydrogen-honded Hurn (see prece"eding paragraph), I~maybe mentioned here' Lhat"although the ProGly sequence is the" most commonly lountl lor 6-lurns in proteins, the conlormational nexibility of the· glycine residuemight~eexpected to lead to other "random"

st~~ctures;

pnrticularily in solution.

1.

~

The erred of the (i+2Ithre.sidueODthe sta.bility of the~turn ~asdemobstr;.ated by Tamburroelat.(19~4).'TbeyiD~estigated,through CD and IR spectroscopy, ,the eercet of the X residue i,n the seq'ucnceNlIlBocPr~X·GlyOEton the stability' . of the HurD. They foundth~tinTFEa~.~~mtemper3.1ure, the molar fraction of ".t'urD conformation(hence

sta~i1ity)

^wasV;I>lIe>!'J1e>Pro,Leu. Ina "Similar study, BOt1SS3rd 311d Marr3ud (19S5) found th.atwaenproline was in position (i+l) or the D-turn there wasaDincrease ib the percentnge .8-tUtn in CH2Clz, going (rom L-nlanine to glycine to D-alanine ,as the (i!t2)th residue. These datll, toge~herwitb the theoretical calculations~r V~kat~eb~la.m(1968).conibi~eto give an interesting,insight into the relative stabilities of Type Land Type IT ,8-turns.

~~cording

^toVenkntachal;l.rn (1068)· the T1P.en,8-turn prefers the -LL- sequcnce whereas the 'TypenIJ-turn prefers/the-LO-sequence. Hence, the

,

^'

'OOAb.

sequence containing peptides would be exp,ected to take on a Typen

•IJ-turn conformation whileexpe<'ted to take00.a Type I IJ-turn, cooformntion. The steric restrictions

t~~

ProAla sequence containing peptides

. ^. w~uld

p~aced^be upon both theL-and the D-alanines are the same; yet a signtriCtllltly gretlter percentage or the pepqde containing theProDAI~sequence forms a ".turn&' This indiC3.t~sa lower stllbility ,of the Type I IJ-turn as comparedtothe Type

n

^8-turll

'when proline is 'Int~e(i+l)th position. This statement is further supported by the observation thnt ProGly containing .8-turns are almost invariably Typeneven th'oughgly~ine,cnn be considered either a0or aLami~oacid (Boussard., 107g;

Brnhmnchari,efal., IgSl, UIS2; see chapter 3). Both crystnland solution studies bn.vesbo~::~'at.8-turn forming peptides with the P!oDAla sequence take on a Typenconformation (Aubry IIItJ~,HI77; Analltbanarayanan and Sbyamasundar,

, ,

^.

/-

)

' /

16.

~9~1;

^Raoet tJl., 1083; Crismll. d al., IOS4). This "bkeo inconjunctionwith the dataofBoussard aDd Marraud (HISS)indicat~that theSUbstit~i~nof thegl~i.ne with D·:llnnlnew~uldstabilize the Type II ,8-turn conformation. Therefore, the inclusionofsteric:llly restrictive

.:m'ino

acids at position (i+2) of the tt-lufn to -lock" the conformation into a limited potentialra~ge^becoml'sattractive.

~'.' _~ber

amino acid residue9,'besides D-lllanine,I

wh~h

do not normntly occur in

\

'

.

peptides, hll.ve been. used to produce conformationnlly restricted.8-turn·rorming

Iinea~

pep tides. These include'otber D

~mino

^acids

sj~

as D·scrineniltl

D"prolin~

^..

(~airIII al.,19jO.;"Boussard andMarralldJ.l~8S)and (l-aminoisohulyric,llcid (Aib)

• (Nagaraj and Balaram,

.~~~,

10SI; Rno e!al.,HJSO; Smithelai.,IUSl; Prtl$nd e!

al.,1082; Jungetaf.,1083; VanRoeyet al.,1083;Bonon.elat.,IUg·l; Crisml1e!

af.,1084). The peptides.containing D-serine and O-proline al. position (i+2jotll.

.8-turn with proline at position(i+t)were fouod to be100%D-lurnns opposed to the00% D-turn achieved with O·alanine in that posilion (Boussardei al., HJ8S.'.

Tbiswoul~be expected since the D-proLine and O·serine side chain. groups arc much bulkier than the methyl groupotO·allloine. Hence they are milchmOle conformatio?ally~estriclh·e. T~eAib group is characterized by lwo mothylson the a-cnrbon as opposed to the one found with, nl:!.nine. The prderpd eon(ormn"lion, whether proline is .itsMlgh~ouron not, appears to be;131O.hc1ix or n~ypeDI(I)JJ-t~rnvnrintion even though PrasndeIai. (HI821 reported:l.Type II .8-turn.with PivProAibNHCHa in etY,stnlnnd solution.Theo!eticnl.confurm:tlion~1

.

^.

...

analysis pertormed by Pr35adeIai. (l08Z1 indicated thnt the Type II p.turIJ:

conformer wasZkeol mol'l more stablethan

t~e

Type HI. They

attribut~d

^the

..

17

3'O·be1ix observedby otbers to long range factors, The Aib..:containing sequence!!

previously studied were';ligopeptides.

The three pointsthat revul themselves arter3Dexamination of the li'terature are that:

• Cyclic {leptides are not required to obt:l.in a stable~turnstructure: A linea.r peptide with im appropriately chosen sequence cO,uld form tbis structure. •

•T~stability Dnd type or".turn formed depends onresi~uesI'through (i+3).

• TheType I p.turn isin~rjDsicallyless stable than the TypeD,1HU~n.

Thenbove_~QDsideralioIiswill become ih.tportant during the design of a double tpturn-r~~mingtetrapeptide. A study. analogous to tbose performed by· Chou and Fasman,pOii, 1978)a~4.Ananth·an3.r3.yan3n ^eIal.(1984) on the occurrence of -

. ";"(~j' .

.8:'turns in pfoteinswu'perform~d

by

Isogili IIIaI.(iQSO). They examined the crystill str,ucures of 23 proteins fOf. multiple bends. A double bend was deCined as II.sequence

~'l

^{whjch two}

~~ccessive

distances b,etween.

C.~

andltC(s'+31

(R~l

^and

C~'+IIilndC~Hl{R3l meet the requirements or Lewis III~l.(lQ73) for il .8-ben,d. _ . [0other words, they, are se.quences with two overlapping4 ...1 b!drogen bonds

which are not, part or .ahelix~. They~ound tha~.4%of ill!resi~uesoccl:!r in mu~tiplebends. or these, thirty eigbtp~rcentha.vea. distance between

Cf

aod C;H

(~

..) or· less than

5.8

A.. These st.ructures are rolde4 more

tightl~'

than o-helices nnd ~hOse doubl~ .6-b~ndswhich arc merelydistort~d helices. The rormatioD or these tightfy wound' structuresisaided by the frequentoccurr~nceor glycine.

18

There ate several peptides which have been proposedtotake onD.double ".tutn st~u.cture.Apart from the Aib containing peptides which. as expected, lake-on a

~3 10·heIL,"~onrormatioll(N3.g:uaj d at., 19;9; Van Roey d at., HJ83j, -there nrc' several peptideswbi~hproducedouble ,8-turns but do not contain Typem

p.turos. Aspointed out in section 1:1, the Type III .8-lurnC30be coo:olidcrcd the

, ,

~eginningor a3l(jhe~ix. Ane~mpleof a <!ou,;"turn whit'h dol'S not contnin a Type mp.turnisPivProProAlal\rICH3which inth~crrstallineSl3t~^exi:;lsas a .. Type D' ,8-turn rallowed by nn

overl~pping Ty~

I 6-turn (Nairet'a1.,lim).

Th~

nb~ve'

stru'cture bas alsg beeJ.1

prop~e.d r~t

^.the,.grt:micidin

S

nnalugllc, di·N-....

.

methYI.ieucin~- gram'ic~~in S~JKu'mn~.

^et

a/~

• .HI,iSl.. Through solution ,speetr cupic

~tudie·s, Venkatachalapa~hi.and-Salaram (1979) ljcscribed PivProProAlaNCH~

as 'an incipient 3 1O-belix althou,gh it is likely that that the'struc~llrcmay actually prove ·to be made up or a polyproline-II-like extended st"ueture r6110wed by a Type II d-turn (AJHlothan:uayanan, personal communication).

With two overlapping p.turns, the (i+2)th residue or the rirst .lJ-llirn is nlso the (i+l)th residuedrth~second. To be sterically ·cbmpntihlc·, their~lIl)wed^4>,lJI angles must be very close. Figure 1·3 plots the4>,1/1angles?fthe (i"·Hlthn~d (i+2)tb residu,es of .8-turn types I, I', OJ U,-aS defined by Venkat:lchnlam(1068)on a Ramnchandran plot (Ramachandran and Sasisekhnf3n,1068). Asseen in Fir;!1re I,.,?,II.Type0.lJ-turn can oBly' be followed by a Typel'or III'. Although not sbown,i'n the rigure, the4>,ofIa~esor the (i+ll^{lh!=S' ueOf,}a Type llI' p.turn

• r

llre identical to that or the Type I'

~tll

(Venkat.:tl: alam,

Ig~8l.

^The

4>~

^angles

corresponding to the (i+l)tb residue or all the other p.turn types areDot.~lqse

\

.

..

^~ 19

Figure 1·3: ;,'1"m.p showing stericalJyaliowed.regions (or both L aDl:! D amino ..

aei~s. ~,

tully allowed tor [,.residu.flj

ffJ,

allowed for

L-alaoine;~.

^tully

allowed tor

ri-residl1ej~,

allowed for D-alanine.

""t~trn

Types I, 1',' nand 0' afe

iDdi~.ted b~

^a.rrowsbegluniog at "tbe 1,'1 angles

~r

residue 'i+l and ending at the , ',' &Dgles orresidueH2.

.. ^...

(

"

20

. enoughtothe

',f

angles of the (i+2)th residue pf the TypenHurD. Also. the

..

'

(i+2)tb residue f,'" angles of types I and I' are quite diUerent hom those of the

,

(i+l)th, residue of (l,oy of the Hurn types. Henceit would be dirricultfota Type lor 'Fype I' tJ-luro<fb be followed by any type of,d-turD.The non.3.o·hclix double 6-Jn would therefore he eitherII.Type

ti

.tHurn rellowN byeitherIt.

Type

I'

~r

m'or aType n' followed by • Type I or m.cHurn. ThellboYe t'onsider3tions become veryim~rtlLDtin designingIIdouble6-1urn-rormingtetupepticlewhich can potentially bind c:sldum ion.(s~esection 1.5).

1.4. Functional Role of Beta-Turns

The high occurrence

of

,lJ-turns,. especitLlly111the surface of proteins (Kllntz, IOi5), ledtothepostul:11ionofa.Dumberorfunttionnl rples in addition to it!

str'!!cturaluse·fuln~S5. These include the /l-turn serving as recognitton sites for en:tymalic phospborylation (Small dal.,IOn), glyc:osyl3tion {Allbut dor.,

10~

and, proline bydroxyla.tion iBrahmlLC}a.ri and Anan'tbaoIHaY3oa.n, IOi8, 10iOI.

~-

.

In1970, Vogt d al. exdmined the loop repoo ofIlseries of homologous calcium·

,

.

binding proteill5 for /l-turo forminr; potentil1l bilScd on thes~onda.rystrudure predidion methods or Chou and Fasm3n (lOiS). They founda.strong cofrel3tion between the ability t9 bind <:a.lcium ion and the

~ition

and ;inu.f density of.

- .Boturn forining residues. A brief description of the struclural data0.0the cnlcium- ' binding regions.~rthese proteins is pre.sented below.

\

The'solution of the X-ray crystallographic structure of the carp. muscle calcium- binding paivalbumin (MCBP) indica-ted the involvement ofDOa-helix-loop-a-helix

... -

.'

21

.~.

slruet~re in calcium-binding aDd suggested that the twelve-.residue long loop

'~ment

^was

r~ponsible

^{f.or the}

~etu31

complexing to the

caJ~ium

^ion

(Kre.tsi~ger

~^{Nockolds,f9i3).} This segmentcontain~^regularlys~cedcarbonyl, carboxr1 and hydroxyl ligands which. could coordi?a.te to the positively cb3.rged calcium ion. There\\!Ctetbr<!c such struduresfoundin parvnlbumin but only two, referred to as1<.:Dbnnd

and

EFband

respectj~ely,

were capable. of binding calcium ion. The .third, referredtoas

As

hand,WlJScharacterized by. aten~

~esi~ue

^loop

segmeDt_instea~

of the twelve residues found in

tb.os~psca~able.ot

bi.oding.

. Or

greater jot,crest was the discovery....oraTypeI~turn

.

at the very beginning of ihe loop segments capable of~iDdrng cal~iumion and the .absence of this structure in the "loop segment which could

no~

bind (Moews·and· Kretsinger:

}975). }'he two deletions in the bUer 'loop segment co'rrespond to positions 6 and

~

of the twelve-residue 'loop segmentS. It

~l\S

^generally

accept~d

^that

~he

^{lack or}

binding was due to the deletions (Kretsinger, iQgOl. These deletions would lIot be expectedtoaffect 'the p...turn structure. A number of ?thercalc~um.b!nding prot~insshowed a good homology in the n-helix-loop-&:-heli.'< region even though the relationshipbet~eenthe entire sequences was often fairly weak., The homologous region!! of prote'ins such as heart troponin C, T4 lysozyme,modul~tor protein, vitamin-O inducedcalciu~-bindingprotein, the alkali-extractable (ALC) nnd dithionitrobenzoate-extradnble (OLC) light chain of lf1uscle myosin,' the EOTA-extrt\ctable light ohain (ELC) from mollusc myosin, and smooth muscle -light chniq. myosin{SLC)were deemedto'bnve. thesam~structure as thilt of parvalbumio" (Kretsinger, 1076, 19801. ,...The solution of the X-ray structures of other ca.lcium-binding proteins such'. as ,bovine intestinal calcium-Mnding protein

. {Stebenyi dat.,1981} a'nd, more recently, troponio C (Henberg and James, 1985;

Sunda.ralingam d 0.1., 1985) and. calmodulin (Ba.bu dol., 1985),sbow~th3t the previous structurlll correlations based on homologywer~. t55e~ti:lllyCOffKt. The lat'k of ca.lcium-bindiol activity exhibited by wme or the homologous~uent'~. . could dot be explained bythe~numberand position of the lig3nds and the number of residues in the loop segmentIW~eds and Mt'Lat'hlip, 10;4; Tu!ty and' Kretsinger, 19i5).

Vogtetat.(19i9)examined the loop segments or the 0t'alc~um-binding·proteins listed above)'· Because or a multitude of homologous regions in. some proteins, they

··:e~e

left with 26 dat::l:telS

~itb

some sequeot'es capable of oinding

'talchim·i·~n

wlfile others were

oOK

They examined all possible tetrapeptidcs ...itbin..ttleI~P segm.eots and calculated tbeir .lJ-tUtnforminfpotenti~.interms· or the probability parameter P_tas derined by Lewis'd 0.1. (19il):

Where f,.. etc. are the frequencies oftesidu~.in~hefour successive positions of tbe .lJ-turn as observed in known protein strudures.. The values used by, Vogt.d al.

(119i91were obtained by. Chou d 0.1.(I07S)from a survey of the X'fay cryst:ll structures of 17 proteins (298~~urlls). Loclli stretches of high P_tvalues indicate a series of overlapping tetupeptides each with a higtl .lJ-turll potentillllLcwis d ai., 11l}7l). In the 26 homologous sequences examined by Vogt d 0.1. (1079) two consecutive peaks with Pto-values great!!r than or

e~unl

to Uie value or 3.0xlO··

were found to occur precisely at the first residue

or

eacb loop region tbat binds

/. f ..

'-

23

calciumiOD. These "-doublets- would be expected fot two overlapping p.turns IVagtct01.,lQj9). The,start of the doubletobtainedfrom the loop :Iequence of the calcium-binding region 'ofMeB?aligned precisely with the start of the MCBP Type I

~turn5.

Very· interestingly.,

tb~re

were no'

suc~

doublets found in tlfe homologous loop sequences which did not bind~aleiumiOD,and neither were C9mpBrab!e doublets found in any other part of the. sequences of calcium-binding proteins. In rabbit skeletal,ttoponin C no

tetrape~e,'

apaFt, from,

th~

in1valved in the doublets, had'ptvaluesgre~ter than or equaltothe value

or

3.0xlo·-t.

Vogt-e.t al.(igj9j also examined the sequences of several non-homologous

';Id"~-b;'d;"

pm',;". Tb,,; ;,d"d,d51,phy''''''"'

"oI",s,.

th"molys;,.•

r

concanavalinAand trypsin. AJtbough doublets do occur at calcium-bindingSitej . they are not

tte

rule as with the homologous proteins. Howev~r,·in the'majority of ClISes one linds at

1~~L

one tetrapeptide with a high P_tvalue

near~

^calcCm-

binding site. ,ThereisthereTorea strong suggestion th;t a p.turn is involved. in

.;-'" .

calcium-bjnding.· It should be noted thattheahove data relied on predictio.n methods based on non-ulcium.bindin.K

r~~gions.

^l-1ence

t~e,8-turn predieti~ns

^refer

. to uncompll!Xed sequences and do nolpertainto the conformation after calcium

.~~ coni.plex~d

^to.tb{ loop segment. Un't'n DOW; the ahov; .obserntioDS

,;~

one of mere

;nter~st.

^{Thelaharator;in which}

th~

results presented in this thesis were obtnin;d bas been Interested In delineati.ng theconrormation~1

. .

' fCeatures orth~,6-turn by spectral methods and elDt!idating its involvement in Cunet}ons such proline-hydrdxylation (An3nthanarnyan3n, lQ83;

-.

ADantbanarayaDan tlal.,'1984; Bu.bmacb.ri and~tballll:D.1antln, 19i5;

Brabmac:hariet al., 19jO, 1981,1082; Chopra and

Aaantbanaray~nl1n,

^1;811..It was tberdore Iogic:al to er.lmine tbe role of ","turns ill metal ton:binding. The final c::atalyst for the.proj~tume from :n examination of cyt'!ic: peptides.

~Iany

c:yelic \ peplidcs, both synthetic and

natur~.

:ate c:ap:ab.le or .selec:ti,·e1;

. c:omplexing with calcium ion or other alkali or alk31i~~:l.fthmetal ions." T,,'l') fea.tures that these ionopbores 'havei~common are ttre~resenc:e

.

.of ,d-tllrnll i'n the.

unc:omplexed_s~:'c:iesand the coordination ofth~ .p'eptid~C:;Lrbonyl groups.totill!' metal iOI) in the C:0':llplexed species.. Both' valinomydn {Degclaentla/.,!Og·lll1nll

. . .

. its I1nalogue cyclo-!AlI1GlyDPhePro)3 (Vishwanl1'th and Ea.sw:nan, l082) were .,' sbown to

eont~iD

^{si..'(4 - I}

hy~rOgeD ~.!\dS. "~he

c:arbonyls wcre

c~dinaltd

^to

c:aldum ion uPon binding aDd this involved the bru.king of 'inlramo!ec:ul:u b~4rogeD ~nds. ~n~Jh~rexample is round iq a paper. by Pease and' Watson.·' (HI78). Tbey designed the penta,peptide cydo-(GlyProDAlaProl witj1 the intent of

. r"

having both a ,.turn and a ..,.tu,n(~-Ihy~rogenbondl·present. Again,4S with

" - tbe valinomycins, tbe hydrogen hoDds were- broken u .tbe c::arbonyls coordiuted c.., to the metal ion. This partic:ular peptide wu subjected to amol~ul:lf m~haniuJ.

study by Lynn aDd Kushick (1084)~ provides a set" of~exc:dlent stereoding1y.ms of tbe

pept~de i~

^l!:oth

i:J uo~mPlex.ed

^and

lithium.comple~ed

form. A similnf set or diagrnms cnD be found in a paper-b,. Duax and Smith

..

(llJ81) wbo were interested in detailing a sequenc:e 'of steps involved in the complexing pf the metal ion to valinomyciD. The dingrnm5 relateIIprogrc1iiive

,

..

~oordinntionor carbonylsto metal ion artl breaking of ;'ntramolecuJaf hydrogen

. .

- ^. . ^.

../

...

~

~\"'; .',

.,:·'1·,

25

f "

bonds. hradditionto the uneomplexed andeomplexed f?tms therear~~aseries 'of

interm~di3,tes.

Several C?ther eyclic-peptides are also

capabl~ or

complexing metal idn's . in:luding

~yc1o-!DPheProGlyDAlaPro)

(Karle•. Itl84). cyclo-(SarSarGIY)2' cyclo-(S:l.f)8 (Sughih3f3:etai., 1976) and cyelo-(P.roSar)ll (0=3,4) (Shimizu and Fujishige, lOgO). The paperby Karle (UI84) c::ompares the crystal structures

ot

the uncompl:;/d

an~, magnesi~m-~omplexed

^species. The carbonyls involved in the

rorm:lti~n

^of

~ntram~lecular'

hydrogen be,sintb: un.complexed species

(.s-

and T"turns) are coordi'[;3Ced to themagnesium lem in the complexed species. The Illtt:r two papers

in~icate'

^{theplesenceoC}'intrJrnolecula;

hYdrO~en

^{bond:' inthe .}

un~ompiexed

^sta'tes

,~

the required c'arbon>:'. coordination to the metdl ion for ..

bindi~g

^{but go...110}

furth~r

^{except to53yth'ere}

~as

,a.p,:ssibilitT o{ multiple.

conformers. Other exa.mples of c.yclic metal ion-binding peptides i.nc1ude , cyc1o-(X.Pro).., :....here· X= Phe, Leu or Lys{Nc.protected)

(~~u:a

a.nd l!flanishi, .

Ig83j lilld theb~Ct,IiF:S,S··Bis-cyc1o-(GlyhemiCysGlyGlyPro) (Sehwyzer et

ar., Igj'Ol~

The la'tt;E!r t"9

pape~o

not talk about the conCotma.lion·of the

pe~tides

1-' '

in the uneomplexed state but do point out tolit the complexed state is achieved :through the coordination of the peptides' carbonyls to. the metal ion. _ The above . ,p,apers 8.re very useful not only in presenting·a·pOssible relationship betwe'en ion-

·'bin.ding O'iliJ'"lhe

~turn

buCalso·for

th~r

technical merit. The methods useJ in the,abov~pa.pers to deter.mine and .foll0j,ion.binding,e~pecially those by Vishwanatb and Easwaran(10S2) and PeiSe

,;d

Watson (ig,S), were used as a.

bUis

·for' the

cal~ium.bindiDg s~udies pr~erited

in this thJ!Sis. These authors characterized the uncomplexed spedes and {ollowed lon~bindlng USlDg both carbon·r3 and proton NMR a;d GO spectroscopy,

~he

peptidesstudied bfpease

f'

26

and Watson (Hlj8) andyishwall:Ltband.Easwaran (1082)-were.like the peptides .studied in this thesis, too sm:sll to be .studied bym:LD)'or thetetb~iqul:'S^((Derally

~ used 'or studying calcium-binding proteins.

The~turn·rormingdoublet p:lttern observtd by Vogtd

at

(lOi9)in the '?Op

'. «

5egm~ntor bOf!lOlogou$ c3.lcium.:bioding proteiD sequences tilong with the resulls

. ,

o.r the eydic,ion·binding peptides ledto l.be belief th3t the,d-turnis:lr:l.vour:l~le . conformationalprereq~isite^rOfiOD.bindi~g. ^{To. testthis"}pos.tu13ted!9,!e,1liot':H peptides,w~icbcould ,mimicthe ov;rlapp!ng.~turns s~n^wit.hth4fmOlogOllS cllldum-bindillg proteins, were synthesized arid theiri~ter:lctionswithcalcium a-nd .othermet31ions werestudiedandco~pllredtosingle "..turno.nd non-p.luro

\. , . '

.

. ,

-

.

forming peptides.

1.S;' Designor,polt.ula~edCaleium-BindingPeptide.

Twoconsi~eralioDshi 'the design 'of peptides u.pahle of torminl two oyer1:l.pping

~~urllS

,and potent!an; bindiog calcium ion have to be made especiallyirone wisbestoeXllimioe the telo.l.iooship betweeo ,true lute and

fu~ction.

^First,th'

peptidecotlfOtmat~DmustIttst3ble in solution and secondly, sinee the hioditl;lof

. .

cllicium. ion probabl; involves a tonform1l.lion:a1 eb:ange. the peptide must

. .,\ .

."

mniotaina'~ertaiD

,

delrte of nexibiliLy. Therefore, the amount of conforrn3tional

.

restriction provjded by the amino acids' incorporated into tbe pcptjde ";ust ,cncet ah,I....

b'lW:~b'

^\wo'

n';~"

^Th';'.plid,

N~IDO'P'~DAI""I'NI1C1l3 ~nd:

^.

. jts poteotinlly mote nCl!ible annloguc N^QIBocProGlYAlaNHCII3 were considercd

~apabltof striking thc balance. Tbe'peptides art tbeor;ticllily clpn.bleofforming' two overln.pping

~tu·rD'.

Thc' first'

~turD

^is

~ilt.biliz.ed

^{by a}

bYd~OICn bo~d