Folding of peptide fragments comprising the complete sequence of proteins

J. Mol. Riol. (1992) 226. 795-813

Folding of Peptide Fragments Comprising the Complete Sequence of Proteins Models for Initiation of Protein Folding I. Myohemerythrin H. Jane Dyson?, Gene Merutka, Jonathan P. WalthoT Richard A. Lerner and Peter E. Wright? Department of Xoleculnr Biology The S’cripps Research Institute 10666 North Torrey Pines Road La Jobln. CA 92037. T’.S.=2. (Received 13 December 1991: accepted 17 Nl-lnrch,19.92) an attempt to delineate potential folding initiation &es for different protein structural motifs. we have synt,hesized seriesof peptides that, span the entire length of the polypeptide chain of two proteins, and examined their conformational preferences in aqueous solution using proton nuclear magnetic resonance and circular dichroism spectroscopy. We describe here t,he behavior of peptides derived from a simple four-helix bundle protein. myohemerythrin. The peptides caorrespondto the sequencesof the four long helices (the .I. K. c’ and 11helices), the X- and (‘-terminal loops and the connecting sequencesbetween thrb helices. The pept.ides corresponding to the helicrs of the folded protein all exhibit, preferences for helix-like conformations in solut,ion. The conformational ensemblesof t,hr ,Aand D-helix peptides contain ordered helical forms. as shown by extensive seriesof mediunrange nuclear Overhauser effect’ connectivities, while t,he 1% and C-helix peptides exhibit conformational preferences for nascent helix. All four peptides adopt ordered helicual conformations in mixtures of trifluoroethanol and water. The terminal and interconnecting loop peptides also appear to contain appreciable populations of conformers with backbone 4 a.nd rl/ angles in t.hr g-region and include highly populated hydrophobic clust.er and/or turn conformations in some cases. Trifluoroethanol is unable to drive t’hese peptides towards helical conformations. Overall. the peptide fragments of myohemerythrin have a marked preference towards secondary structure formation in aqueous solution. In contrast, peptide fragments derived from the fl-sandwich protein plastocpanin are relatively devoid of secondary structure in aqueous solution (see accompanying paper). These results suggest that the two different protein st.ructural mot.ifs “a? require different propensities for formaCon of local elements of seconda,ry structure to mltiate folding, and that there is a prepartitioning of conformational space determined by the local amino a.cid sequence that is different for the helical and /?-sandwich structural motifs. In

h’ryzL1ord.s:protein folding initiation; nuclear magnetic resonance: peptide conformation. mpohemerythrin: helix

molecular biology. For most monomeric singledomain proteins,-& vitro folding is a spontaneous The detailed mechanism by which proteins fold event and all of the information required for correct remains one of the central unsolved problems of folding is contained within the amino acid sequence. Recent, technological advances have provided t Authors to whom all correspondence should be important insights into the folding pathways of addressed. small globular proteins, and it is now generally $ Present address: Department of Molecular Biology accepted that folding proceeds via identifiable interand Biotechnology, University of Sheffield, P.O. Box mediates (for reviews, see Kuwajima, 1989: Kim & 594, Sheffield SlO 2UH. UK. 795

1. Introduction

0022-2836/92/1,5079~~23

$03.00/O

0

1992

Academic

Press

Limited

ISiLlti\~ itI. IWO: F’isc*lIrr & Sc*htttid. I!)!IO: .l;rtvii~4ii~. 1991: f’fitsvtr. I!191 ). Nttc~lc~ilr ttl~gtlc~i ic t’~~Ottiltl~‘~’ (tt.trt.r.t) pnlsr li~lx4ing rsIwritnc~ttls willi (,~.tci (~lltY,tnc~ (‘. rilxtnuc~lease. I)arnase and I!-sozymr (Rotl~~r rt (11.. 198X: I!dgaonk;tr & RaIdwin. 198X. l!M): I%\-c-rofi. P/ trl.. 1990; Miranker rt (I/.. IWI) have iclrntifird c~otlll)u(~l kinrtic itttvrtn4iatrh that itt’(’ fi~rtltetl on il millisec~o~td timwC&lr atltl (:ontaiti IttUc~tl of‘ 1 lw secwndar~ htrti(Lt nw of t Iw ii;it ivrz prott~itts. I~ormation of’ nU11vca trrtiar>. sltwc4uw t twrr follows ott att eveIt lonp~t’ titrtescAc. ‘IYIC riIt~,-tlrtt~t’tItitti~l~ tl lP:3cl 10 the‘ first Ol)~f?~‘Vill)l~’ ilrtc~rmediates cwtttainiltg s(‘(‘ott&try st I~uc~ttIIY~. (‘rtltral to il nutntwr of’ l”‘stIIli~t~‘d Iwoirin folding mc~chanisms (f attempts to find foltlcti structures it1 peptjide fragmrnts of proteins were grnrrall~ ttnsuv wssful (ICpantl K- Schrraga. 196X: ‘I’mtut-hi & .Arttinsrn. 1969: Hermans 8r T’uett. 1971) and Ircl to thtb general belief’ that short linear peptidw (lo tlot

t Abbreviations used: n.m.r.. nu&ar tnagnf~tic~ rrsonance: v.d.. c,irc.rtlar dichroism: h.p.l.c,.. high lwrformanw licluict c,hrotna.t.ogral,h?-: TF&\. trifirtoroac~etic, acitl: p.ptn.. parts pvr million: I)TT. dithiothreitol; TSS. trimeth~lsil~lprt,pitllrsrrlfi,rric acicl; 2QF-C’OST. dout)lr quantum filtrrect phase-sensitivr two-dimensional wrrrlated spectrosctopy; TOC’SY. twodimensional total cw4ation spwtroswpy: XOESI’. t v o-dimensional nuclear Orerhausrr effect spwtroswl,-: R.OESY. rotating-f&me NOESY spectroscopy: 21). twoditnrnsional: TFE. -‘.L’,L’-trifluororthaliol: ID. onedimensional: d,,(i. j). dNN(i, j), etc. itItrat~iolecrrlat~ tlistancr betaern the protons C”H and KH. KH and Ml. rtv. on r&dues i and j: XOE, nuclear Overhausrr c~fYwt: p.p.b.. pa.rts per billion: 3JHNa. NH-(‘“H coupling cwtstant

solutions

in *H,O

are unrwrrrctrd S-terminal

(for

loop

solublr philiwd

at pH pptide.

sufficient tnrnts.

The

‘H,O

samples).

pH

-

pvptitlr r..d.

5. upon an insoluble

addition residue

c~wld br attci n.m.r.

rlissolvrd slwratra

at

to test for aggregation. t ttv peptidw derirrrl

rrsiriuw (‘yx!N.

aw \vhirh

ltrrwnt \vvrt

the &helix rlirtttvization

to

varying

hy

factors

from

water-soluble.

in the ittr~luded

rlisulfirlr altprr~sitt~;ttrl,v sralt~i

in

and JWT,BOO temperature (1969). using

prot.ein in the

thv

bonrls. n.ni

sqwnce. peptide

and (TOC’RY:

Has

8

1985)

rf nl.. Bothnrr-H?-

IYi!))

KOESlv 1983).

and ~1..

d

spectrosropv

rotating-frame 1984) were

spwifir. a.ssigntnrnt~ .I Hakun-rvho sequence The mixing

to

usrd for

Sprctra and 32 bpt.wrrn

196’i) phosphate

dtxla\. sprc+r’a. with without

and

300

L’-dimrrtsional r~r~mmrml~

the

t,he

and

iO0

IO pp.ni.

SOESY spwtra \vith a spwtt.al

in

grnerally 1, idth

of

(Rancr ms

in

in

\vater

spvctra. irradiation

hprr+ra

wt’w

Fottrtr~r

I,r~rrntz-to f’uttr.tiotth:

both

time \~as

dimensions.

cwntained 12,.WO Hz

of

the

pla.ced to data were t, noise. points

rlrca~y each wwe

tqutppfvl modified Researr~h). f, employed. anti

fi it It it Sky \-ersiott of

dimensions

atid phase-shiftetl trattsfortnerl spectw

2’048 rra I ltoints itt both dimvnaions. on a (‘ottvex (‘240 computer. or i$‘arrior the

un

array program

sine beil cwntaint‘d

1)atn were prrwessed a Sun3 workstation prorY5sor. FTSMR

using (Hare

Linflar I,asrlint~-r,r,rmc~tion (Dyson ut rtl.. ridge sultprrssioti (Otking et nl.. 1986) A small 1st.orrlrr phase adjustment

usuallg made Temperat)ure

in w, (Dyson cc&icients

ances of each peptide thr plot of r+rnri~~al

were shift

(4 al..

of

the

calculated wr.stt.s

198%). amide from temperatuw.

proton thr

concentrations detertnined

nm

in 5.7 M-puanidinr r~r47i~ient of 1490 wvt’c

buffer

recorded and

(JJH 5).

disulfid~~

.A-

for

hydrochloride. t r*ttl- t

for all for the

pt*ptirlrs 4 lq)tirlrs

formation rvery band Were

lfzast-scjrratw brt\\rett to nm to nttt

a

polynomial 6 anti I5 points. using

on

an

calculaamino case absorb-

in

I tnw drriwri

smoothrtl fit with The

All

various solu-

1wl)tidrs avert

Single

W.5 rum Gdth

~(7311s

with

of

using (Etlelhoc~h.

in

and I)-helix 1 mnl-I)TT

tiotid

by

or. in the of t>-rosittr M

obt~ainerl L)J. taking points constant zlIld using l~xst,linr-r~rtrrt,(~tf,rl spectra

.i (’

rithw

to

protein

Hahn-who

both

I’eptide were

at

using veil pat.hfrngths r~onc~rnt~t~ations \verrl

also intrrwerr

a 4 s timt, of I.5 mtn.

tty using a a window siw rllif)ticity was

\s-ithiti

I ?0

(Johttsoti. u-itttin prsakz

1986) and the \vavelettgth 0.1 ntn using the f.??!). m4.H. of lwnzenr vaprlt’.

I)-lO-~~~~tnI~horsulfonir~

ar.irl ~‘as 260~0

The ser I Uf?rlr’~ of tnyohenwryt hritl ;lnci tllr port ions of’ the sequent-e that r~onstitrttr ear.h peptide are shown in Table 1. to,gether wit,h a tlrwript ion of thts swondary structuw prwcwt in tlw protriri. df~rivrd from the retincrl X-ray crystal structure (Sheriff pf ~11.. 1987). l3ac.h peptitic wxs ehoaw t.o span a region of swondar~~ structure: thew are fitur IOII~ prptides that constitutta the four of’ tnvohemerythrin (A-Mix. R-helix. long heliws (‘-hvlix Nrtr1 lbht~iis). three short itttrt.-helis lr~~p:: (termed the AI<-. I<(‘- anti (‘I)-loops) wttl two longer peptides derived from the S Hntl t’ twmitli of t,hp

1096 cvmplex point’s, in w2 and 5000 Hz in in

rrcrwrlrrl

3. Results and Discussion

solu60 ms

werr recordtd for Spect,ral Lviriths

tt~ansformrd

(;atrs> thr

in

thr the

the water during the

The minimize

to

wr2w

tions rwntaininp the r~r~ntainrrl approximately

at L’!Nb.‘, caIihratvr1 and 266.i

(0,. using \vindon

obtained

the peptides. was sevt=re. was used.

(TFE).

calit,raterl

& Byrd. POESY

(tlmax =

offset

hlwrtra

of peptidt A st,rwk solutions peptidr~. tty measurement

Sprc,tra

rtirtler&ir

for

all of into

lvith 203X complex raarah free indur*tion

t, valurs spectrum.

(%l)j

600

mixing

and the transmitt.er t bra water resonancr. sample sptnning

u vrt\ routittr4y aquirrd or mow sr3an.s for

\vt’t’tl of

Keener

in

spectra spectra

all of by patrd

during

shifts sper+ra overlap spwtra

front tht~ hc~lir~al regions of rn)-ohemerythrin r~ottr,t,tttratiotts of .‘.L’.~-triflnoroethanol

(ROESY: votnplete

all protons was inserted

pulse

analysis &httiix

kl-orrlcr typiwll>.

were used Sdimensional

c..rl.

ancts at 255 an rztittctiort

vt al.. 1983) spertroscop~

(SOESY:

give a tiat, baseline time t Wits 300 to

= 3OOms). For M as suppressed

rrlaxat)ion SOESY rvincirir ar.quirrri

arid the

prepared (1)TT) and

kOESJv t,o obtain

s~v.vtra. IO0 to I.50 ms in ROEST tions and 65 to 100 ms in TOWT and t*max rrsonanr.r

of

2-dimensional

1985: Rance. Phase-sensitive

assignment. Overhausyr effect

Chemical

MM-een 3 and 60 1~ tion of molar rllipticitirs

and

acid

Rance

spin-system ttuclear

sqttencr peptides.

tnvo-

argott.

(2QF-C’OSY: cvrreiation total

Davis.

least-squares.

from the I -dimensional in r~~scs \1-here resonance casr a srries of 2QF-VOS\7

I‘ltr.a\-irtlvt

sprvtromrt~ers. t’alibration of the probe was by the method described tty \‘an(:ret mrthnnol. IXosan. u-ith a r~hrmic~al shift of

sprrtrosr~opy %-dimrnsionai

of

.4$-i\61 1)s r,.tl. sI)t”.t~oI)Olarimeter. of rithrt. 0.2 cm ot’ I.0 cm. Peptide

To avoid of int.er-

\s-ere

under

10

vystrinr

(‘>s35 srquenc~rs

the solutions IO ttt~t~tlithiothrritoi

.r. tutws

of

thr T\\,r,

3.75 p.1t.m. relati\-e to t,ritnethvlsil~lI)ror,anrsulfonir~ (‘MS). was used as an internal standard. Double quantum filkred phase-sensitive rwrrrlatrd

thr lyeHowever.

and 1 )-helix peptides respwtivrl> of thaw pc’pt ides through formation

tnolwular ittr~luding w~rr

c,otir.rntrations

arr

method dirrl$ly ext‘ept which

for t1.m.r. measureof all of thtb pptides

\VPTY rxaminrrl

sequ~ttr~~

in *H,O K-helix and sparingI?-

of water remained

t 0 101) in order Tn p~tieral. Itr~tnerythritt

values

meter readings. The .A-helix. peptides werr appawntlh-

gradient using

a

l98Xa) Were was resonof thr

src4ue11w.

acquired at two concentrations, t.he lower of which was obtained by a I : 10 dilution of the higherconcentration sample. Since the pH can be a critical factor. influencing t,he cshemical shift. wpwially of amide resonances in the vicinit), of carboxyl groups. the pH of the high and low conwntra.tion samples ot each pept’ide were adjusted carefully to within 04% pH units of each other. For the shorter peptides, one dimensional (1 II) n .m .r. sprct)ra were sufficient, t,o resolve all of the prot)on resonances. None of these peptides (the AH-loop. W-loop. (‘D-loop and (‘-krminal loop peptides) showed detectable concent,ration-dependent changes in chemical shift or resonance linewidth for an;\- of the amide prot,on resonances. For the longer peptides, the A-, K- and Ikhelis and K-terminus loop peptides. X’OESY spert,ra were acquired at two concentrations differing by a factor of 10 (approximately 1 my and 0.1 mM); XOEs diapnostic of helix or nascent helix acre present in t.he spectra at both concentrations. The V-helix peptide was not examined in this way, as its structure in solution had previously been shown to be concrntration-independent (Dyson Pt al.. 198Xb). For the A-helix. D-helix and K-terminal loop pept,ides. there was no change in linewidbh ot chemical shift, upon dilut,ion. ?here was a slight difference (approximately 0.01 p.p.m.) in some of the chemical shifk of the B-helix peptide between samples at different concentrations. and the resonance linewidth is slightly deweased at t.he lowrl c*oncent,rat,ion. However. the relative magnitudes of the d,,(i.i-t- 1) and d,,(i,i+ 1) XVOF: wnnectivities for the peptides were independent of peptide cow centration. suggesting that an aggregation phenomenon was neither contributing to t)he KOKs

observed nor infuencing significant’ly the cwnformaGonal preferences of t,he peptide. Since there was no apparent effect. of concentration either. on the NOESJ’ spectrum or the cad. spectrum of thr F-helix peptide. while slight effecats of c*oncerltr,ation were observed upon the linewidths and chemical shifts of the amide proton resonances. V’P cwnclude t,hat thr latter parameters are highly sensitive to the aggregation st’ate of peptides. (b)

’ I/

)I .)I! .I’. stzdb?s

of’ ttt.!~ohc!tttr,r,ythriti

peptides

Complete assignments were made for all peptides using standard %I> n.tn.r. techniques. For the shorter peptides, a 2&F-C’OSY and a NOEKY or ROESY spectrum were usually suficient to allow complete assignments. For t,hr longer peptides. TOCSY speAra were also wnployed. Sample spectra for representat.ive m>-ohrmerythrin peptides are shown in Figures 1 t,o 3. The complete rwonan(~t~ assignments at 278K are shown in Table 2. In addit)ion to their importance for making resow ante assignments. the NOESY spectra can ~~ro\klt~ informatiotl on c~onfof,rnational preferences for turll. nascent helix. This infortnatiotl is helix iItl(l summarized for earh peptide in Figure 4. Several types of NOE are shown, including the mediumrange NOES rharackteristie of preferences for folded conformations. Temperature coefficirnt.s were calculat.ed for thtt amide proton resonances of ail of the myohrm erythrin peptidw. For the shorter peptides. the chemical shift dat,a as a function of temperature could be obtained from 1 I) spectra alone. For the longer peptides, resonance overlap made it nrcessar)- for the amide proton chemical shifts 1.0 he

Folding

of Myohemerythrin

Fragments

V48a-K49N

799 4.0 aa

r56

T51@

T46 ,8 445

-

4.6

F55aTS6N

K49

G ci d c

-I 3.2

-

-2.6 D6:

-

-I 3.4

2.4

2 2 5

- -I

3.6

- -I

3.8

2.8 3.0 8.8

8-6

8.4

8.2

we (rq3.m.)

-

3.2

4.2

Figure 2. Portion of a 506MHz Hahn-echo NOESY spectrum of a solution (54 mM) of the B-helix peptide in *H,O. pH5.1. The probe temperature 90°b ‘H,O/lOq:, was ‘278K and the mixing time 5 was 400 ms. Sequential d,,(i.i+ I) and d,,(i,i+ 1) NOE connectivities are indicated. Peaks of weaker intensity represent intraresidue d,,(i,i) NOES. The d,,(i.,i+ 2) I?oE from Leu47 t.o Lys49 is marked.

4.4 4.6 1

8.8

8.6

F55j

I

4.8

8.4 8.2 a2 (p.p.m.)

Figure 1. Port,ion of a 500 MHz TOCSY spectrum of a solution (54 mrvr) of the B-helix peptide in 90% ‘H,O/lOob 2H20. pH 5.1. The probe temperature was 278K and the spin-lock period was 100 ms using a WALTZ-16 sequence. Vertical lines connect the spin systems of the amino acid residues in the peptide.

obtained from a series of 2&F-COi5Y spectra acquired at different temperatures. The temperature coefficients for the myohemerythrin peptides are included in Table 2. Residues for which the amide proton temperature coetXcient~s are significantly lowered from the mean ( - 7.24 p.p.b./K; standard deviation = 2.11) calculated for all amide protons in all of the peptides are also indicated in Figure 4. (c) Circular dichroism spectra and ejjects of addition of tri$uoroetharrol Circular dichroism spectra were acquired the peptides in water solution. As was

for all of observed

4.6 7 4.8

5 s-

8-2

8.6

8.6

8.4

8.2

w2(p.p.m.1 Figure 3. Poltion of’ a 300 MHz ROESY spec~trum of a mM solution of‘ the AR-IOCJ~J ~Jt~pti& ill !100,, ‘H,O/l00/, ‘H,O, pH 43. The probe temperaturr was 278K and t,he mixing t,ime 5 was 300 ms. using a 4.X kHz spin-locking field. IO

previously for thr C-helix peptidr (I)yson et nl.. IIIXXh). the c.d. spectra of’ t.hr peptides derived f’rom the four helical segments of myohrmerythrin do not show a, double minimum at 208 and 212 nm caharnc>t(Gtic of ordered helix in water solution. Addition of increasing concentrations of TFE t.o these sotutions resutt,s in an increase in the neg:ntivr rllipticit,~~ at 222 nm. to a value indic;Lting maximal helix formation at 40 to 50’!;, TFE. For the peptidrs derived from extra-helical regions of t)hc protein. the cb.d. spectra showed no helix formation in water or in water/TFE mixtures. The c.d. spectra of the A-. IS-. (‘- and D-helix peptides are shown in Figure 5. (d)

Detection,

of helix by n:m.r.

and turn and cd.

strwtmrrs

Helix, reverse turn and nascent helical structures can be detected by n.m.r. and c.d. spectroscopy (for a review, see Dyson & Wright, 1991). Significant)

Folding

of Myohemerythrin

x01

Frcyments

Table 2 Resonance :Imino rrsidur

assignments

and

temperature

coeflicients

of peptide fragments

of myohemerythri?,

arid SHt

(‘“HI

CR3

3~80.3.1 1 4.70

847 827 X.53 8.33 7.98 829 iw X.44 8.45 7.94 i%i

Ml x.32 7.85 8.68 8.69 k9.58 x.45 x.55 8.43 x.37 *31 8% 8~08 X.50 841 7.93 804 8.44 8.30 8.27 8.38 c53 8.14 9.01 n+x) 8.47 829

$14 4.3 1 4.38 449 4.3 1 444 3.99 44x

1.i4.1.84 1%O 2~28.24 1.84 2.21 .l.iH 2~x9275 I.84 :kN

44ti 3.9; $24 *53 412 3.97 4.60 4.35

2.49 1.92.2.02 3.77 3.12.2.94 I.61 1Gi5 3~04.2%~ 3~06.2+~Jl

J.2” 1 4.33 4.26 4.36 4.60 4.11 $10 4.57 123 4.23 4.09 4.61 -t2i 3% 44)7 4.68 -k.58 447 4.13 4.42 440 $15 478 4% 453 4.26

3a3.3.12 1.86.1.97 1.97.2.04 1.65,1,53

113 8.84 8.68

84.5 8.38 8.1 I 8.25 8.26 8.5i 846 fj.55 8.3 1 8.53 8..?8 843 8.26 %69 8.58 t+54 8.46 8.36 84” xY!3

C”Ht

4.67 440

4.66 4.32 4.33 426 $37 4.04 4.34 4.19 4.45 4.30 4.61 4.63 4.72 4.29 4.64 4.29 4% 4.26 4.44 4.5” $54

CYHt

%c2 1.51.1.16 3.68.3.78 2.28 2.0

(“+Ht

Other?

lO.lS(NE’H) 7 15((‘@H)

7%(C6’H) 7.23(PH)

046

0.94 (CYH3)

3.64.3.76 6.93

867 ((:“H)

- A6/AT (pp.b.!K) 7.61(Ce3H) 749(Vc2H)

0.81 .O%l

7.1; 1.36 0.8 1 .O.69

3.04

7.32.7.32 ((‘“H.CiH) 7.22 (N&H)

i.18 7.07

7.32,732 (C”H.CSH) 677 (CH)

7.1%

6.84 (YH)

13.3 %6 11.X

0+3%.@89

2.i5.2.83

1.99.2.03 I .94 3.33.3.16 1.82.1~67

1.67 I.67 030 7.25 I.67

7.29 (CH) 2.96 (CH,) 296 C&H,) 0.79 (CYH; j 7.32,7.30 (C”H.CiH) 2.96 (CH,)

1~03.1~23

077 7.2.i

071 (VHJ 7.32.7.30 (C”H.C‘cH)

1.48.1.20 I%2

0.86

3.17

090 (CHJ 7.21 (N”H)

3.34

7.26 (N’H)

t+:‘,ti

I.i6

1.47 1.47 I~lP.140

3~11.3W 1.X3.1 .i4

1.40

1~77.1.67

I.72 323.2.96 2.62.2.68 2.92 1.88 1.89.1.74

2.60267 2.03 “%1.2%9 2.90.2.97 3.99 I .46 4.01.3.92 1.40 2.29.1%6 2.i7.2.85 I.64 I40 4.19 I~60.1~66 .?m I.79 246 $23 4.16 ‘fin 3a5.3~15 3~00.3~18 4.19 3%?.3.28 1.94.2.06 1.98.210 I .39 2.01 .I‘?.09 1.99.2.13

6.9 t-w 6.5 74 74

2.03

3+%.3+u

1.64

0.93.0.86

1 .%O

I .62 ow,w94 1.43.1.38 0.93.0.96 1.19 1.14

2.99((“H,),7.61(NCH,)

7.6X.7.01 (N6H) 8.5i i.24

7.13 (C&H) 7.33.7.30 (C”H.CcH)

8.62

7.30 (C’H)

I.17 2.40 2.42 2.51.2.59 2.54.2.63

XOP

I/

.J Ih~son rt al. ___-

Table 2 Prptide$

Amino residue

R( ‘-Loop

ASPfi3

(‘-He&x$

(‘II-Ltql

(‘-Terminal

loop

.- A(j, AT ip.p.h li)

acid (“HS

(‘*Hi-

ot11tTt

I .30

I.67 7.11

297 ((“Hz) 6.81 (C”H)

NH?

(‘“Ht

cqBHt

Ala64 Ala65 Lys66 Tyr67 SerSX Glu69 \~a170

8.78 851 %38 843 a.21 8.55 7.86

4.23 4.32 4.21 4.24 4.58 $41 4.72 A3

2~70.2~85 I ,40 1.74 1t 1.70 3.05 2.96 379’ 2.10,1% 2.09

Glu69 Val70 Vnl71 Pro72 His73 Lys7.4 Lys75 Met76 Hisi Lys78 Asp79 PheAO Lea81 OhI82 Lys83 Ile84 Glp8.5 Gl?;86 Lru87

8.86 860 .8.71 8.56 8.64 8.66 &67 8.56 &58 8.28 8.26 8.34 8.50 8.43 8.73 8.38 7.95

4-08 4.14 4.40 4.37 463 4.26 4.27 4.47 4.63 4.25 4.56 4.56 4.29 4.19 $32 4.17 4.0 1.3.94 394 4.19

2.07 2.07 2-02’ 2.04 2.28,1.83 321.3.16 1.78.1.72 1.79.1.74 2.00 3.21.316 1.69.1%9 ?66,2.56 3.12304 1.61 1.52 t 2.02.1.94 1%0.1.75 I ,x5

1.52 2.31.2.27 1.38 1.20.1.49

1.63.1.57

1.57

1.62,1.64 3.84 1.36 232, I .95 2.12 2.67.272

I.64

kk4.5 8.14

3.H4 4.40 $46 4.61 446 $15 4.4 I

4la93 Lys94 ,4SIl95 f’al96 Asp97 TyrQH Cys99 LyslOO GlulOl TIplO

8.73 8.66 8-28 8.39 %20 8.22 8.28 8-34 8.1 I

408 430 4.59 3-99 459 4.38 4?28 $08 414 4.55

1.52 1.75 274,2.64 I.97 2.58.271 2.96303 2.84 I.73 1.98 324.3.32

Leu 103 Vall04 Asn 105 His106 IlelO7 LyxlOH

8.05 7.93 8.21 %14 8.18 8.14

423 392 455 4.5.5 4.12 4.16

1.69 2.06 2-60 2%0,3,10 1.86 1.73

1.58 0.95.088

GlylO9 ThrllO Asp111 Phell2 Lysl13 Twll4 L&31 15 Glyl16 Lysl17 Leull8

%71 858 8.42 826 %21 834 7.77 8.25 8-16

3.87 4.31 4.60 4.51 +lQ 4.46 $24 3.87 4.34 4-20

411 268,258 3.08,3.01 I .63 3.00 1.66.1.81

1.10

C&%6 L&87 k88 Ala89 Pro90 Val9I Asp92

I)- Helix

(rontimed)

CT4 %62 8.46

1.85.1.72 1.60

F33 089.090 >34 2-26 096:090 0~95,090 2.00 1.35 1.39 2-58,2.51 1.35

3~90.3~70 857 - 1.68 - 1.68 %5.i I.67 7.23 0~91.085 - 1.67 086

7.29 - 2.97 -2.97 2.06 7.27 -2.97

(C’“H) (CEHZ) (C”H,) (C”H, J (C’H) (FH*)

7.33.7.28

((‘“H.(‘:H)

- 2.97 (YH L) 0.92 ((“H,)

2+4 0.94.0.9.5

3-64.3.82

1.39

1.62

2.92 ((‘“H,)

tiiti

7.06 ((‘“H)

1.78

2.Q6 ((‘“H,j

lUlG(W’H) 7.06(CC3H) @89.090

i-12((“‘H) ;.lZ(VH)

0% 1.78

0.90 ((‘?H,) 2.95 (CEH,)

1.36

7.3 I 1.61 7.16 1.65

i.33,7.33 ((‘“H,ViH) 2.94 (C”H,) 6.83 (C”H) 296 (CCH,)

1.44 I.60

1.68 o-91,0,86

2.99 (C&H,)

0.76.087

1.47.1.44 230

1.17.1.47 1.45

1.25

t Chemical shifts in p.p.m. fThe proton resonance aassignments for the C-helix peptide have been published previously reproduced for the convenience of the reader. $ All chemical shifts are at 278 K. Solution pH: N-terminal loop: 587; A-helix: 509; A&loop: C-helix: 510; CD-loop: 492; D-helix: 443; C-terminal loop: 518.

(Dyson

7,5l((‘“‘H) 732((‘i’H)

et al., 19886)

4.33; B-helix:

514;

and

are here

BC-loop:

516;

Folding

of Myohemerythrin

-*m-u

d,,@ b;i 1 dm(/,itl)

-*I *I I)

-

I*-*

111

I

A-

d,,,,((itP)

d,,.(/;i)

. . L--

d,&,rt3)

-

d,&itl)

R DNTA

DA~KVSE~

dN,,(r,itI)

dy.(ii) m

d&,i+l)

m*-

d,,,,(i,itI)

m

.

a* --

d,,b,i+3)

--

L

-

--3YF

- -

-

d,,li,it2)

-*-

.

__I*

d,,.(i.i+Z)

d&,it3)

-

*I-

-*I*-.

dg,,(r;rtl)

-

d,,,(Q)

-*-a -.

d&itl)

-- -

d.B(‘;it3)

d~~~~,itl~

803

-*

d,,,,(i,itl) dg,,(i,it

Fragments

-

Kv’:f&L

d,,,i(i) d.,(,,i+l) d,,&itl) dB,,(l;it

-+-* I)

-I

d,,&it2)

-

Figure 4. Schematic diagram showing the intensity of NOE connectivities (filled bars) between the backbone amide and C”H protons observed for all peptides.The diagram includes information from a number of spectra, acquired in some cases under different conditions of pH and temperature. Asterisks in the absence of an NOE connectivity indicate severe resonance overlap. Asterisks and double asterisks associated with pairs of NOES indicate that the particular cross peaks are overlapped with each other and that either or both connectivities may be present. Boxed amino acid residues are those for which the temperature coefficient of the amide proton resonance (Table 2) has a value greater than or equal to 1 standard deviation below the average of all temperature coefficients in this study. Double and triple boxes indicate that t,he values are more than 2 and 3 standard deviations, respectively, below the average. Cross-hatched bar represents connectivities to the CdH protons of a proline residue, which contains no NH.

The temperature coefficients of the B-helix peptide are not significantly lowered from “random coil” values (Table 2), and no medium-range i,i+3 NOES are observed for this peptide. The NOESY spectrum of the B-helix peptide contains &&i,i+ l), d,,(i,i+ 1) and d,,(i,i+ 1) NOES (Fig. 2) and its cd. spectrum in water does not indicate the presence of ordered helix (Fig. 5). As is seen for the C-helix peptide, addition of about 15% (v/v) TFE is necessary to produce a c.d. spectrum that suggests the presence of a significant amount of helix. In addition, there is one unambiguous d&i,i + 2) NOE connectivity in the central portion of the peptide, suggesting that the B-helix peptide forms nascent helix rather than ordered helical conformations in water solution. The n.m.r. spectra provide strong evidence for formation of helical conformations throughout the

cients for some of the A-helix amide protons (Table 2) are significantly lowered, suggesting that intramolecular hydrogen bonding may be occurring. The NOESY spectrum of the A-helix peptide (Fig. 6) shows medium-range d,,(i,i + 3) and d,,(i,i + 3) NOES in several parts of the sequence, as well as extended series of d,,(i,i+ 1) NOES, indicating a threshold population of ordered helical forms. By contrast, the cd. spectrum of the A-helix in H,O gives no indication of ordered helix in water solution. The reasons for such a discrepancy have been discussedelsewhere (Dyson & Wright, 1991; Waltho et al., 1989; Osterhout et aZ., 1989) and are further discussed in the following section. The NOESY spectrum of the D-helix peptide (Fig. 7) contains numerous strong d,,(i,i+ 1) NOE connectivities. The magnitude of the d,,(i,if 1) connectivities is reduced in the C-terminal two

length

thirds

of the A-helix

peptide.

Temperature

coeffi-

of the molecule,

and the temperature

coeffi-

X04

II. J. Dyson

I

200

220

et al

I

I

240

261

I 0

Wavelength

(f) Classijimtion

of helix-like

structures

in solution

The A- and D-helix peptides show behavior similar to that of peptides corresponding to a fragment of the myoglobin H-helix (Waltho et al., 1989, 1990): that is, although the proton n.m.r. results are quite unequivocal in indicating the presence of helix, the c.d. spectrum in water does not indicate a high population of ordered helical conformers. This behavior may reflect a difference in the sensitivity of the two methods to deviations from ideal helical geometry. It has been calculated (Manning et al., 1988) that considerable loss of ellipticity at 222 nm should result from deviations from strict alignment of the chromophores (the backbone carbonyl groups) in a regular helical structure. For a short linear peptide in aqueous solution, it is not at all

I

I

240

260

(nm)

Figure 5. Ultraviolet c.d. spectra of (a) solutions (6.3 PM) of the A-helix peptide (c) solutions (11.0 PM) of the C-helix peptide and (d) solutions (168 concentrations of trifluoroethanol in ‘Hz0 at 278K and pH -5.

cients of several of the amide protons in this region are reduced (Table 2). Medium-range NOE connectivities are present through most of the length of the peptide (Fig. 4), indicating the presence of helical conformations. Like those of the other helix-derived peptides, the c.d. spectrum of the D-helix peptide in water solution does not indicate the presence of ordered helix (Fig. 5), but the ellipticity at 222nm increases in the presence of TFE.

I 220

peptide (b) solutions (3.7 PM) of the B-helix of the D-helix peptide in the indicated y0

PM)

unlikely that deviations from regular helical geometry would occur, perhaps due to participation of bridging hydrogen-bonded water molecules, as seen in molecular dynamics simulations of helix unfolding (DiCapua et al., 1999; Soman et al., 1991; Tirado-Rives & Jorgensen, 1991; Tobias & Hrooks. 1991), or in the X-ray structures of many helical proteins (Sundaralingam & Sekharudu. 1989). In addition, the length of ordered helix required to give the characteristic c.d. signal may well be signihcantly greater than that required for helical conformations to be observable by r1.m.r. Hoth experiments and calculations suggest, that the characteristic c.d. spectrum of a helix is strongly dependent on the length of the helix, and t’hat significant negative ellipticity at 222 nm and 208 nm is observed only when the peptide contains more than 2 to 3 turns of helix (Goodman et cd., 1969: Madison & Schellman, 1972). Thus: either distortions from regular helical geometry or fraying of the ends of a helix in a short linear peptide could make a helix difficult to detect by c.d. spectroscopy. These limitations do not apply to n.m.r., since characteristic NOES will be detectable even for t’ransient helical conformations. When the helix frays, that is, the peptide backbone begins to sample increasing populations of unfolded states, the cd. signal at

Fragments

FoEding of Myohemerythrin

805

G3 la-132N

1 136a-R37N

-f322;F33N - E?s”ST

G31Na

4.2 E’ ci d 4.4

3

H25a-6 H25a-K26N

F29a-6

_

>22a-E2 F29a-K30k -

4.6

F33a-6

-E

“E

8.6

8.4 8-2 w2(p.p.m.l

8-4 E19a$ la)

,&26-i?9

> F29aP

1

-

Y18ap H25ap

Figure 6. Portions of 5OOMHz NOESY spectra of the A-helix peptide. (a) Amide-C”H, amide-amide and aromatic-C”H regions of a Hahn-echo NOESY spectrum in 90% lH,O/lO”/o ‘H,O, pH5*1. Peptide concentration was 2 mM, mixing time 400 ms, 279K. (b) C”H-CBH region of a NOESY spectrum in 99% ‘H,O, pH5.1. Peptide concentration was 2 mM, mixing time 400 ms, 278K.

I

I

I

4.6

4.4

4-2,

w2 (p.p.m.1 (b)

I 4.0

t

KlOOa-

I

4.2

4.6 195Na

Ii d d 5 B.0

8.4

N95N-V96N

8.6

8.4

8.2

-

8-O

WL (t3p.m.) la)

-I

4.6

1

I 3.2

3-o

2.8

I

I

I

9’

I

I

2.6

2.4

2.2

2.0

I.8

I.6

we ( p.p.m. 1 (b)

Figure 7. Portions of a 500 MHz Hahn-echo NOESY spectrum amide) in 90% ‘H,O/lO”/c ‘H,O, pH 4-4. Peptide concentration NH-NH regions. (b) C”H-dH region.

of the D-helix peptide was 5 mM, mixing time

(N-terminal acetyl, C-terminal 400 ms, 278K. (a) NH-C”H and

Folding

of Myohemerythrin

222 nm will be seriously attenuated, but the NOE connectivities characteristic of helix will still be observable, albeit at reduced intensity. For some linear peptides, both the n.m.r. and c.d. methods indicate the presence of ordered helix in water solution (Shoemaker et al., 1987; Osterhout et nl.. 1989: Waltho et al., 1990). This is in marked coontrast to t,he myohemerythrin A- and D-helix peptides which exhibit the n.m.r. characteristics of helix, i.e. (i,i + 3) medium-range NOES, but lack the characteristic c.d. spectrum of helix. As discussed above, this is probably due to the presence of only a small population of helix, which may deviate from ideal geometry and/or may be subject t,o dynamic fraying at the ends. A further increase in conformational flexibility is evident for the B- and C-helix peptides which exhibit t,urn-like conformations in equilibrium with extended forms and that are capable of ordered helix formation only on addition of TFE: we term these peptides truly “nascent helical”. Thus, it appears that peptides may exhibit a continuum of behavior, from totally random conformations, which predominantly populate the B-region of (#,$) space. through nascent helix. frayed helix and ordered helix, with an increasing proportion of regions with significant populations of backbone conformations in the a region of (4,$) space. At the extreme, for some linear peptides in aqueous solution. the majority of the conformers caontain ordered helix (for example, the ribonuclease (I-peptide analogue RN24; Osterhout et al., 1989). (g) (‘onformations

of the

extra-helical

Pragments

X07

13.b

I

‘IOU-s* E6NnI)1 Itx-El2N (W2Na

b

n8ka-

WlONa % 5h7Nn

Q @WZa-E3N

loop peptides d

The extra-helical regions of myohemerythrin fall into two categories: t’he short (3 to 8 residue) segments between the helices (the AH-loop, the BC-loop and the CD-loop) and the two longer regions at the N and C termini. The spectra of the short inter-helical peptides generally show strong and weak to medium-intensity d,,(i,i + 1) d,,(i.i + 1) NOE connectivities throughout. No d,,(i,i +2) NOE connectivities were observed for these loop peptides. and the c.d. spectra are charact,eristic of random structure. We conclude that no highly populated reverse-turn conformations are present in any of these peptides. Nevertheless, the inter-helical peptides apparently have consistent and significant preferences for conformations where the backbone dihedral angles are in the c1 region of ($$) space, as indicated by the d,,(i,i+ 1) NOE eonnectivities. The turn regions of the four-helix bundle protein consistently show a greater propensity for backbone conformations in the a region of (#,$) space than the corresponding regions of the B-sheet prot,ein, plastocyanin (accompanying paper). This is discussed more fully in section (k), below. The n.m.r. and c.d. spectra of the two longer peptides from the N- and C termini contain evidence for a greater population of folded conformations than those of the shorter inter-helical peptides. The NOESY spectrum of the N-terminal loop peptide

8.8

I-

8.6

8.4

8.2

w2bp.m.

8.0

8.8

7-8

1

(a )

Figure 8. Portions of a 500 MHz Hahn-echo NOESY spectrum of the N-terminal loop peptide in 90% ‘H,0/10% ‘H,O, pH59. Peptide concentration was 2 and mM> mixing time 400 ms, 278K. (a) NH-C”H NH-NH regions; (b) C”H-C@H regions.

(Fig. 8) shows a number of d,,(i,i+ 1) and mediumrange NOE eonnectivities in the C-terminal half of the peptide. The spectrum also contains a number of low-intensity cross-peaks in addition t,o those assigned to the peptide ( -5 to lo:/, of the intensity

of the major species). The peptide sample used for n.m.r. was exhaustively purified, and eluted as a single peak using capillary electrophoresis as well as on an isocratic h.p.1.c. run, making it unlikely that these cross-peaks arise from an impurity. Since the peaks appear to occur in regions of the spectrum close to the resonances of the N terminus residues, it appears likely that ci-tram isomerism of one of the proline residues (Pro5 or Pro7) is responsible. NOE connectivities between the C’H protons of both proline residues and the C”H of the preceding residue indicate that the major species has transproline in both positions. The resonances of the minor species are too weak for NOE-based assignments to be made, so that its identity as an isomeric form containing cis-proline cannot be confirmed. We considered the possibility that, some of the crosspeaks assigned to medium-range NOES in the major species of the N-terminal loop peptide arise from the minor species. To ensure that this was not the case, NOESY spectra were acquired at. a number of different pH values and temperatures; the crosspeaks were observed in the positions to be expected for (i,i +2) and (i,i+ 3) NOE connectivities in the major species, even though the resonances themselves had moved. We are therefore confident that these cross-peaks do indeed represent medium-range NOES in the major species. The temperature coefficients for the amide protons of the N-terminal loop peptide are unusual. A series of residues, which appear from the NOE experiment,s to be part of the structured region, have temperature coefficients that. are lowered, in the case of Asp1 1 to a value of - 26 p.p.b./K (Table 2). The Glu3 amide proton also has a significantly lowered temperature coefficient (-2.8 p.p.b./K), which may be due to interaction with its own carboxyl side-chain: at the pH (5.9) used for the experiments with the N-terminal loop peptide. t,his group would be expected to be almost completely deprotonated. Such interactions do not) appear to occur uniformly for all glutamic acid residues: in the same peptide, for example, Glu12 has a temperature coefficient of - 5.2 p.p.b./K, and interaction with carhoxyl groups is unlikely to be the explanation for the lowered temperature coefficients of Phe14 and Argl5. In addition there appear to be a number of unusually high temperature coefficients in the vicinity of the two proline residues. Tt is not known at this time whet,her these values represent an abnormally high degree of solvent exposure. The 3JHNa coupling c0nstant.s in the structured region are not unusually low, indicating that the population of backbone configurations in the a region of (+,$) space is not particularly high. No indication of ordered a-helix or turn conformations is seen in the c.d. spectrum of the N-terminal loop peptide (Fig. 9); t.his spectrum is typical of peptides containing a high proportion of aromatic residues (Manning & Woody, 1989). The conformation of residues 11 to 17 of the intact protein consists of a number of interlinked turns (Sheriff et al., 1987), which is consistent with the conformation observed

220 Wavelength

240 (nm)

Figure 9. Ultraviolet c.d. spectra of solutions (%5 PM) of the N-terminal loop peptide in the indicated y/o concen trations of trifluoroethanol in ‘H,O at 278K and pH -5

in the peptide fragment in aqueous solution. This is discussed more fully in section (i), below. The N-terminal half of the C-terminal loop peptide appears to be largely unfolded in aqueous solution, with backbone conformations in bot’h r and /I regions of (#,$) space (Fig. 4). No mediumrange NOES characteristic of helix are present, and strong d,,(i,i + 1) and weak d,,(i,i + 1) NOE connectivities are observed throughout. There is strong evidence for a significantly populated hydrogen-bonded reverse turn involving residues 113 to 116. The temperature coefficient of the Glyll6 amide proton (Table 2) is exceptionally low: a value of O.Op.p.b./K for an amide proton temperature coefficient in water solution has not to our knowledge been reported previously for an unconstrained linear peptide. The NOE connectivities in t’he vicinity of Gly116, a relatively strong d,,(i&+ 1) between Lysll5 and Glyll6 amide protons and a d,,(i,i + 2) NOE connectrvity between Tyr114 and Gly116 (Fig. IO), are consistent with the formation of a hydrogen-bonded turn involving Lysl13-Tyrll4Lysll5Glyll6. The extent of the protection of tlhe Gly116 amide proton from solvent, may be due not only to the hydrogen bonding associated with the turn conformation, but’, in addition, to the nature of the surrounding residues in thcb sequence. Although lysine is generally classified as a hydrophilic residue, its four methylene groups present a hydrophobic environment near the peptide backbone. Leucine, tyrosine and phenyl alanine are decidedly hydrophobic. Thus, the local environment of the glycine residue at position 116 may account for the extreme extent of solvent protection of its amide proton. Further evidence for a hydrophobic interaction in this region is provided

Folding

of Myohemerythrin

Fragments

Y114EG116a %6

G109a TIlON 09 TllOfi ‘DlllN

K113

K113aY114N K115aGil ~

0.0

6.0

4.0

2.0

K117a-L118N

we ( p.p.m. 1 Figure 10. Portions of a 500 MHz Hahn-echo NOESY spectrum of the C-terminal loop peptide in 900,; ‘H,O/lO% *H,O. pH 5.2. The peptide concentration was 5 mM, mixing time 400 ms, 278K. Inset (a) shows the d,,(i.i+2) NOE connec%tivity. plotted at a lower contour level for clarity. Inset (b) shows a cross-section (column) through the Glyl16 amide proton resonance at 7.8 p.p.m. to indicate the relative intensities of the various cross-peaks in the 2-dimensional spectrum The d,,(i,i+2) peak at 4.48 p.p.m. is indicated by the arrow.

by the observat)ion of an NOE connectivity between C&H (Tyrl14) and C”H (Cly116) (Fig. 10). The cd. spect,rum of the C-terminal loop peptide does not show features typical of a turn conformation. However, since the majority of the residues in the peptide appear to behave as a statistical coil, this is not particularly surprising. (h) Effects of TFE of helical

in the stabilization conformers

The addition of trifluoroethanol to a solution of a peptide may cause it to adopt an ordered helical conformation observable by c.d.. Not all peptides undergo this transition: it appears that the conformations of peptides that populate almost exclusively the p region of (@,$) space, such as the peptides derived from the plastocyanin sequence described in the accompanying paper, are not noticeably affected by the addition of TFE. On the other hand, those peptides that appear from n.m.r. studies to contain nascent or frayed helix in water solut.ion appear to form ordered helix in the presence of TFE. Behavior typical of both helical and non-helical peptides is observed for the myo-

hemerythrin peptides. These studies were carried out exclusively by c.d. spectroscopy. Upon addition of TFE to solutions of t.he AB-loop, BC-loop, CD-loop and C-t’erminal loop peptides, very little change was observed in the c.d. spectrum (data not shown). While the interhelical loop peptides are probably too short for helix to form, it is also probable that they perform the function of helix-breaking sequences, allowing the of the protein to form. tertiary structure Helix-termination behavior has been observed in a pept)ide fragment of myoglobin that contains a fiturn conformation between two helical segments (H. C. Shin, H. J. Dyson & P. E. Wright, unpublished results). The cd. spectrum of the N-terminal loop peptide is dominated by the dichroism associated with its many aromatic residues. An extremely small change (-400 deg.cm2 dmoll’) is seen upon addition of TFE (Fig. 9); this cannot reliably be ascribed to secondary structure formation of any kind. A similar lack of helix formation in the presence of TFE was observed for several of the plastocyanin peptides (accompanying paper). These results suggest that a propensity for helix formation in a peptide is a prerequisite for the induction of

810

H. J. Dyson

ordered helix by TFE. Similar conclusions were reached by Segawa et al. (1991) who found that of the tryptic peptide fragments of lysozyme, only those that correspond to helical segments in the native protein adopt secondary structure in t,he presence of TFE. Each of the four peptides derived from the helices of myohemerythrin shows a marked increase in the negative c.d. minima characteristic of helix upon addition of TFE (Fig. 5). The B- and C-helix peptides, which are shown by the n.m.r. experiments to contain the weakly structured state known as nascent helix, contain very little ordered helix in water, as measured by c.d.. Cpon addition of TFE, however, the ellipticity minima at 208 nm and 222 nm show an increase in intensity, to a maximum value which indicates approximately 50 to 60(& helix in each peptide at TFE concentrations greater than 50%. This behavior has been noted previously for the C-helix peptide (Dyson et al., 19886). for which n.m.r. experiments showed that helix forms only in the C-terminal half of the peptide, where nascent helix is observed in water solution. The n.m.r. data suggest a higher population of helical conformers in the A- and D-helix peptides in water solution, but neither peptide appears to contain ordered helix by c.d. spectroscopy. On addition of small amounts of TFE, a monotonic increase in the negative ellipticity at 222 and 208 nm is observed for both peptides. Interestingly. the final st*ate which is reached at 50 to 70”;, TFE apparently contains less helix than for the B and C helix peptides (40 to 45% helix for the A- and D-helix peptides, compared with 55 to 70% for the R- and C-helix peptides: Fig. 5). Since all four peptide sequences are fully helical in native rnyohemerythrin we presume that extrinsic factors. such as iron co-ordination or long-range tertiary interactions, contribute to stabilize these helices in the native folded protein. (i) Correlation

of peptide

and protein

h-ueture

The conformational preferences observed for the peptide fragments of myohemerythrin can be compared to the secondary structures present in the crystal structure of the intact protein (Sheriff et al., 1987) (Fig. 11). The four helix-derived peptides all show a significant population of backbone dihedral angles in the CI region of (4,1/) space. In some cases, the n.m.r. experiments indicate that small populations of helical structures are formed in aqueous solution, although the c.d. spectra fail to detect the presence of ordered helix. All of the helix-derived peptides are capable of forming 50 to SOyA ordered helix in the presence of TFE. There is thus a good correlation between the helical regions of the myohemerythrin protein and the conformational propensities of the peptides derived from these regions. The X-ray crystal structure of myohemerythrin reveals the presence of a number of turns in the extrahelical loops. The three short interhelical loop

et al.

Figure 11. Regions of the myohemer~thrin molede for whirh the peptide fragments show sigmficant pof,ulwt,ionh of backbone c-onformations in thr c( region of (4.$) +a~. inferred from the relativr> sizes of the d,,(i.i t- I j and d,,(i.i + 1) XOE connetrtivities, mapped onto a rrfjresetltation of the crystal structure of myohemryvthrin (Sheriff cl ~1.. 1987). Red reprrsent,s the rrgions for which thcx d,,(i.i+ I] t,o d,,(i.iS I) rat,io t~xcwtls -lb.-i (tht, d,,(i.i+ I) XOE is relatively strong); yf:llow reprf3flnts thr regions for which the d,,(i.i+ 1) NOE: is present, but thr d,,(i.i+ 1) to d,,(i,i+ I) ratio is less than 0.5. (:rr? rrgions NOES

indicatr parts of the sequrnre where aw observrd in the prptidr fraprnrnts.

no rl,,(

i.i f I )

peptides have a tendency to populate the M region of (4,$) space. but no significantly populat,ed t,urn conformations could be detected. While d,,( i.i + 1) NOE connectivities are present for all of these peptides in addition to the daN(i,i + 1) NOES to be expected for unfolded conformations, no d,,(i,i + 2) connectivities characteristic of reverse turns were observed, and the temueraturc coefficients of the amide protons of these peptides were not indicative of solvent protection by hydrogen bonding. The bturn between residues 88 to 91 in the X-rav crvstal structure of the protein. which forms pa& of the CD-loop peptide, involves a cis-proline residue in a type VI p-turn. Highly populated type VT t,urn I

Y

Folding

of Myohemerythrin

conformations containing cis-proline have been found previously for short peptides (Dyson et al., 1988a). The n.m.r. spectra of the CD-loop peptide show no evidence for cis-proline: &(Ala,Pro) NOE connectivities characteristic of trans-proline are observed in the ROESY spectra of this peptide, and only one set of proton resonances is observed, indicating that no minor &-Pro-containing species is present in solution. As previously mentioned, the N-terminal loop peptide contains a small population of a minor species which probably contains cisproline, but no evidence for the formation of a type VI turn was found. In general, the behavior of the proline residues in the myohemerythrin peptides is consistent with known peptide &s-Pro sequence preferences (Grathwohl & Wiithrich, 1976), exactly as found for the plastocyanin peptides (accompanying paper). The N- and C-terminal loop peptides appear to populate turn conformations in water solution. In the protein crystal structure, the N-terminal loop peptide sequence contains three p-turns, an inverse y-turn and a left-handed helical turn (Sheriff et al.. 1987). The first of the turns in the protein crystal structure is a type VI turn with Pro7 in a cis configuration. As for the CD-loop peptide, the major species in solution for the N-terminal loop peptide is in the trans configuration and there is no significant conformational preference for a type VI turn, although, as noted previously, a very small amount of a minor form of the peptide, possibly containing a cis-proline, is present. The X-ray structure shows a pair of interlocked turns encompassing residues 11 to 15. A number of medium-range (i,i+2) and (i,i+3) NOE connectivities are observed for residues 9 to 15 of the N-terminal loop peptide, which could well correspond to a conformational preference for nascent helix, interlocked turns and/ or different single turn conformers. The lowered t.emperature coefficients observed for residues 11 to 15 confirm the presence of solvent-protected, possibly hydrogen-bonded, amide protons in the same region. Thus, for residues 11 to 15 in the N-terminal loop peptide, it appears that the conformational preferences observed for the peptide closely mirror those of the folded protein, perhaps constituting a folding initiation site whose conformation is retained in the final folded form. A similar close correspondence between the conformational preferences of a peptide fragment of a protein and the intact protein structure has been found for the C-peptide of ribonuclease A and its analogues (Kim & Baldwin, 1984; Osterhout et al.. 1989). The C-terminal region of the native myohemerythrin contains one well-defined type II turn between Tyrl14 and Lysl17. The C-terminal loop peptide apparently contains a turn conformation with a highly protected amide proton in a similar region, but the n.m.r. evidence suggests that the turn conformation lies between Lysl13 and Glyl16. A d,,(i,i + 2) ?JOE connectivity is observed between Tyrl14 and Gly116. The amide proton of Glyl16 has an extremely low temperature coefficient,

Fragments

811

which, as discussed previously, probably indicates solvent protection by local hydrophobic side-chains as well as hydrogen bonding within the turn conformation. The presence of additional conformers containing the Tyrl14 to Lysll7 turn cannot be ruled out, since the d,,(i,i + 2) NOE to be expected for such a turn would not be observable due to the similarity of the C”H chemical shifts of Lysl.15 and Lys117. However, the temperature coefficient for Lysl17 is average for a solvent-exposed amide proton, indicating that the turn conformation, if present at all, is either not hydrogen-bonded or in very low population. Thus, for the C-terminal loop peptide, it appears that the preferred conformation in solution, a reverse turn, is also present in the native protein, but is shifted one residue along the polypeptide chain. A conformation that differs in detail from the one in the final folded state is not unexpected for a putative folding initiation site (Wright et aE., 1988). (j) Hydrophobic

clusters

According to the evidence from the n.m.r. experiments hydrophobic clusters are present in the A-helix and C-terminal loop peptides of myohemerythrin. Both of these examples are associated with glycine residues that show unusual spectroscopic properties. For both of these residues, the temperature coefficient of the amide proton chemical shift is considerably depressed from normal values, and the amide proton resonance itself is shifted upfield by approximately 1 p.p.m. from average chemical shift values observed in many other peptides. In both cases, NOE connectivities are present that indicate the presence of turn conformations: strong d,,(i,i + 1) NOES between the glycine amide proton and that of the preceding residue, and d,,(i,i+2) NOES to the glycine amide proton. The amino acid sequences in the vicinity of the glycine residues are similar: Phe-Lys-Gly-Ile-Phe for the A-helix and Tyr-Lys-Gly-Lys-Leu for the C-terminal loop. For both peptides, NOE connectivities are observed between the glycine C”H and the ring protons of the neighboring aromatic residues (Figs 6 and 10) and the aromatic rings are also apparently in contact with the neighboring lysine, leucine and isoleucine side-chains (NOE data not shown). These observations suggest the presence of hydrophobic clusters in these two peptides in solution. The very low temperature coefficients of the glycine residues can thus be rationalized through solvent protection by a combination of hydrogen bonding in the turn conformations and sequestration from solvent due to the presence of the surrounding hydrophobic residues. An interesting correlation can be made between the chemical shifts of two types of protons and the presence of hydrophobic clusters in the two sets of peptides from plastocyanin and myohemerythrin. There is an apparent correlation between the chemical shift of the CYH, resonance of isoleucine and the presence of NOE connectivities between

812

It. .J. Dyson et al.

d,&S40,

A41) ’ I

0

8.0

6-O w2 (wan

4-o )

2-o

c

IO.0

8-O

we (mm

(a)

IO-0

8-O

6-O

4-o we (P-P m )

6-O

4.0 )

2-o

omo

(b)

2-o

o-0

Cc)

8.0

6-O 4-o w2(p.p m ) Cd)

Figure 12. Representative cross sections (columns) from NOESY or ROESY spectra of comparable peptides from the plastocyanin and myohemerythrin sequences. (a) ROESY spectrum of myohemerythrin AB-loop (Ala41 NH) (see Fig. 3) (b) ROESY spectrum of plastocyanin Pc4 (Phel9 NH) (see Fig. 3 of accompanying paper). (c) NOESY spectrum of myohemerythrin N-terminal loop peptide (Phe14 NH) (see Fig. 8). (d) NOESY spectrum of plastocyanin PC9 (Mu45 NH) (see Fig. 2 of accompanying paper).

this group and an aromatic ring. Thus, the C?‘Hs resonances of Ile28 and Ile32 in the A-helix peptide of myohemerythrin are shifted upfield by about 92 p.p.m. relative to average values obtained from many other peptides, and these methyl groups apparently make contact with the aromatic rings of Phe29 and/or Phe33. Similar observations have been made for plastocyanin peptides Pc6 and Pc7 (accompanying paper). The large upfield shift of the glycine amide protons in the A-helix peptide and the C-terminal loop peptide of myohemerythrin has already been noted. Upfield-shifted resonances in the spectra of thermally denatured lysozyme have also been taken as evidence of the presence of residual structure in the form of hydrophobic clusters (Evans et aZ., 1991).

(k) Comparison of the behavior of plastocyanin and myohemerythrin peptides The genera1 impression given by a comparison of Figure 10 of the accompanying paper and Figure 11 of this paper is that the peptides derived from the two proteins differ in the distribution and frequency

of backbone conformations in the SI region of (4,$) space. Examination of the NOESY and ROESY spectra of the two peptides reveals that this difference is reflected in the NOE intensities as well. One means of evaluating the relative populations of conformers with backbone conformations in the a and /3-regions of ($,II/) space is to compare the relative intensities of the dNN(i,i + 1) and d,,(i,b+ 1) NOE connectivities at a particular amide proton (Wright et al., 1990; Dyson & Wright. 1991; J. P. Waltho, H.J. Dyson & P. E. Wright, unpublished results). This technique is useful in the present case. where NOESY spectra that differ considerably in the absolute magnitude of the cross peaks must, be compared. For all of the peptides derived from myohemerythrin, the ratio of the intensity of the d,,(i,,i+ 1) and daN(i,i+ 1) NOES at a particular residue are uniformly high (> approx. 0.5), indicating that the backbone populates the c1 region of (4,$) space to a significant extent. This is in contrast to the peptides derived from the p-sandwich protein plastocyanin (accompanying paper), where the population of conformers containing dihedral angles in the c( low. This is illusregion of (d,+) sp ace is usually trated in Figure 12, which shows cross sections of

Folding of Myohemerythrin Fragments NOESY and ROESY spectra of peptides able length from the two proteins.

of compar-

(1) Effects of metal co-ordination Myohemerythrin contains a metal prosthetic group consisting of an oxo-bridged two-iron cluster (Stenkamp et al., 1981). The prosthetic group is located between the four helices, at one end of the molecule, with the iron atoms co-ordinated to the side-chains of His73, His77, HislO6, Glu58 and Asp1 11 (Fel) and His%, His.54, Glu58 and Asp1 11 (Fe2) in the azide complex of the protein (Stenkamp et aE., 1981; Sheriff et al., 1987). The conformation of the protein ma.y well be influenced by the presence of the iron atoms. A measure of the extent to which the Fe co-ordination stabilizes the helices can be obtained from a consideration of the conformational preferences in various regions of the helix-derived peptides. One of the bridging carboxyl groups is located in the B-helix (Glu58) and the other in the C-terminal loop peptide (Asp1 11). The other five coordination positions are all occupied by His sidechains, one in the N-terminal end of the A-helix, one in the C-terminal end of the B-helix, two in the N-terminal end of the C-helix and one in the C-terminal end of the D-helix. The regions of the A, B and ‘D helix peptides that contain the co-ordinating residues also contain the helical and nascent helical conformational preferences observed for these peptides. The N-terminal end of the C-helix peptide, which includes the two ligand histidine residues, appears to be without the nascent helical structures which characterize the remainder, and TFE cannot, induce helix in this region (Dyson et aE., 1988b). This behavior, in a peptide which has two co-ordination sites within five residues, strongly suggests that the region including the two histidine residues requires metal co-ordination as a template for helix formation. In addition to the positively charged His side-chains, there are a number of lysine residues in the N-terminal half of the C-helix peptide: as suggested previously (Dyson et al., 1988b), this high concentration of positive charge at the N terminus of t,he peptide would be unfavorable for helix formation, due to interaction with the helix dipole. It is of interest t,o note that this particular sequence takes up a different conformation from that seen either in solution or in the intact protein when bound as a seven-residue peptide fragment to a monoclonal anti-peptide antibody (Stanfield et al., 1990). The peptide is bound as a type 11 p-turn to the antibody, which will also recognize the apoprotein myohemerythrin, but appears to have quite low affinity for the native iron-containing protein (Stanfield et al., 1990). It is not known at this time whether the N-terminal end of the C-helix has a helical structure in the apoprotein, but the crystallographic results imply that it does not, an implication consistent with the results for the C-helix peptide reported herein and previously (Dyson et nz., 1988b).

813

(m) Implications for initiation of protein folding The peptide fragments derived from myohemerythrin are notable for their propensity to form elements of secondary structure in aqueous solution: we observe nascent helix, helix and reverse turn conformations. Some of the structures formed, for example, the turn conformation present in the C-terminal loop peptide, are relatively highly populated, and appear to consist of folded, hydrogenbonded backbone conformations stabilized by hydrophobic interactions between neighbouring side-chains. These observations lend support to hierarchical or “framework” models of protein folding (for reviews, see Kim & Baldwin, 1984, 1990; Goldberg, 1985; Jaenicke, 1987, 1991; Wright et al., 1988; Montelione & Scheraga, 1989; Ptitsyn, 1991), in which there is an initial rapid formation of elements of secondary structure at multiple sites in the polypeptide chain followed by coalescence and collapse to a compact globular state. Coalescence of elements of secondary structure might occur by a process of diffusion-collision (Karplus & Weaver, 1979) or on-site construction (Skolnick & Kolinski, 1989). These processes occur on a sub-millisecond time scale; n.m.r. pulse labeling studies of several proteins reveal formation of compact intermediates containing much of the native secondary structure within a few hundred milliseconds from the start of refolding (Rader et al., 1988; Udgaonkar & Baldwin, 1988, 1990; Bycroft et al., 1990; Miranker et al., 1991). Kinetically detectable slow steps, such as proline isomerization (Brandts et al., 1975) or disulfide bond rearrangement (Creighton, 1978) follow on a much slower time scale. The present work shows decisively that elements of secondary structure can be formed at many sites in an otherwise unfolded polypeptide chain in water solution under non-denaturing conditions. There is clearly a rapid dyna,mic equilibrium between folded and unfolded states in the conformational ensemble: only one set of resonances is observed for each of the myohemerythrin peptides, which means that this process is fast on the (millisecond) chemical shift time-scale. A better indication of the real time-scale of the process comes from measurements of the rate of helix-coil transitions in homopolypeptides, which occur on a N 100 ns time-scale (Lumry et al., 1964; Zana, 1972; Cummings & Eyring, 1975). Further, molecular dynamics simulations of the folding and unfolding of peptide reverse turns in water suggest a nanosecond t’ime-scale for this process (Wright et aZ., 1990; Tobias et al., 1991). Thus, the available evidence supports a model in which the earliest events in protein folding involve format,ion on a nanosecond time-scale of independent elements of secondary structure (or local hydrophobic clusters) in many regions of the polypeptide chain. These transiently folded structures can be studied under equilibrium conditions in isolated peptide fragments. In the case of myohemerythrin, the transient elements of secondary structure detect’ed by n.m.r. in the peptide fragments are similar in nature and

81-C

II.

./.

1Qwn

oc(Lur at, similar positions in the polypeptide chain iti the folded protein. For example, peptides that have a propensity to form helical structures in solution correspond to regions of the sequence that’ are helical in the folded myohemerythrin. An interest,ing observation resulting from the present work is that nascent helical st’ructures are widespread in the peptides derived from the helical segments of myohemeryt,hrin. The term nascent helix (Dyson (4 nl., 1988b) was introduced to describe a dynamic state in which t,he peptide populates a series of turn-like conformations (as shown by a set of sequential d,,(i,i+ 1) NOE connectivties and a number of d,,(i,i+P) NOES) which form and unfold rapidly on the n.m.r. time-scale, t’hus giving rise to a stngle set of resonances. Nascent helical strurtures arp readily stabilized into ordpretl helix by addition of t’rifluoroethanol, which led us to propose a role for turns in t,he initiation of helix (Dyson it ccl.. 1988b). On t,he basis of t,he ohservat,iott t,hat wat,er molecules inserted into hclicses in crystal st’ructures of proteins frrquent’ly result in reverse t,urn-like structures. Sundaralingatn W Sekharudu (1989) also post,ulabed the inrolvement~ of turns in helix formation. Klagdon $ Goodman (1975) earlier postulated helix initiation by rflversf’ turns immediately before t)he N terminus and immediately following the C’ t,erminus of a helix: this differs from our mechanism (Dyson rt al.. 1 SSSh). in whicah the initiating turn sequencers form within the helix. Recent molecular dynamics simulations of helix unfolding (IM’apua rf crl., 1990: Tirado-Rivrs & .Jorgensen, 1991; Soman et al.. 1991) and init’iation (Tobias & Brooks, 1991 ) strongly support the role of turn c~onformations as int,ertncdiate st,at,es in helix-coil transitions. Tn addition. it) appears that t’urns themselves are formed and unfolded in a fairI> facile way in these sitnulations. on a sub-nanosec*ontl t,irne-scale that is quite consist,ent with that c,f prop &in folding initiation. &verse turns are primary candidat,es for init& tion sit,rs for folding. since they limit the conformational space arailablr to the polypeptide chain and may help to direct subsequent folding events (I,ewis ct (II.. 1971: Zimmerman & Scheraga, 1977: Dyson rt ,I,/.. IUX8a: Wright ut frl.. 1988). Several rxperirnrntal and theoretical st,udies have implicated turns in the folding process. .Vonte Carlo simulat’ions of folding pathways for X. fi and mixed r//3 motifs show that relatively stnall (but, not, irrelevant.) local int.rinsic* turn preferences pIa?- an import,ant role in initiation of folding (Skolnick $ Kolinski, 1990. 1991). l‘urns also play an import,ant, role in thr folding of Zhelical hairpins computed using off-latt,ice Krownian dynamics (Rey di Skolnick. 1991). A temperaturrsensit,ive folding defect in a bacteriophage P22 tailspike protein (Yu 8 King, 1988) has been correlated with a reduced propensity for p-turn formation in a short synthetics peptide corresponding to a region of t:he P22 t.ailspike protein containing t.he folding mutation. compared to a peptide with wild-t’ype sequence (Stroup & Gierasch, 1990). Further. these latter studies confirm that> the use of synthetic

et,

al.

[)ept,ide fragments is 8 valid approach tiw f~xtr.trtittttig initiation of protein folding. Finally. it, is interesting to considrr our rt2suit.s Ott folding propensit,ies of myohemrrythrin l)fkl)tidt> fragments in t,he light of recent Monte (‘arIo simula. tions of folding pa,t.hways of four-helix bundles. In the simulations of Skolnick & Kolinski (1991). t,ht earliest folding initiat,iori event,s involve fhrmittiotl of a nat.ivr turn or helix. from M.hi(*h folding proceeds by a process of on-site cwnstruc*tion. Successful folding t,o a unique structure requirf:s att int,rinsic propensit,? for formation of nativt,-like turns, a propensity that is much higher for the fijrtrhelix bundle (5 to XO~o) than for /?-sheet proteins (O..??h). These different ~r)nforrnatic,ttiII propet~sitiw arc’ reflect~ctl in our experimenta,l data.: pf>ptitk f’Filg tnents of the four-helix bundle protein rrt~~ohrttterythrin ha\e pronounced tcntlenc+s to atlol)t i urn. nascent helical and hc>lical structures. \vlwrfas peptide fragments of the /Csandwich protein plastocyanin (acc~otnpanyitip paper) are relatively tif~voitl of elernrnts of secondary struc+nre in il(ltt(‘()lts solution. In summary. the present study has sho\vn that elrment.s of sf~condary striic*ture. in thus form of reverse turns. nascent helix and more ordf!rr~d helical c,orrformat’iorts. as well as lwal hydrophobic (alust’ers. are abundant) in short peptide fragments of a four-helix bundle proi.ein. These trattsicwt ant1 thwmodyr~amira11~ unstable struc’turrs. forttte(l ai rnult,iple sites it1 the polypeptidr chain. are likc*ly to play a fundamental role in t,he initiation of protein folding. \I:e have further shown that two-tlitrrrn. sional r1.tn.r. spevtrowopy. in cwnjunc~tion \vit I-r syti. t hetic peptidtb chetnistSry. is a powerftll tnrt hod f’or identifying potential protein folding itiitiaGott qites iitltl promises to provide new insights into the earliest events in protein fielding. 1%‘~ thank Drs ,JefI’rey Skolnivk and I>aviti C&v for helpful diwussionn, Carry (:ippvrt and Michael Piclue t’or csreativr help with csornputjrr graphics. and 1,itttl;t Tennant. Vicki Fehrr and Steve Svhirbel for ttr,II) \vith peptidr purification. This work was support,rcI t)\, grants CM38794 and C’AZ74RX from thr Nat,ional Jnstit,ut.es of’ Health.

References .Adler.

;\. .J.. (:reenfield. K;. .I. & Fasmarl. (:. 1). (1%:~). ClircLular dichroism and optical rotat’ory dispersion of proteins a.nd polypeptides. :M&od.s En,zyno/. 27. fiif~n.5.

Kax. A. & I)avis, I). (:. (1985). MLECV-17 bast4 twodimensional homonuclear magnetization transfer spect,roscopy. J. Magn. %son. 65, 355-360. Blagdon. 1). E. & Goodman. >I. (1975). Mechanisms of protein and polypr@ide helix initiation. /~iopoZywwrs 14, 241~-245.

Kla~nco. B. .I.. ,Jim&ez, M. A., Rico. M., Santoro. ,I.. Herranz. J. & Nieto, .J. L. (IWI ). Tmdamistat, (12-26) fragment. RiMR chararterization of isolated fl-t’urn foldjng int,ermediatrs. Eur. .I. Jliochem. 200. 44r~R.5

I

Folding

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Bothner-By, A. A., Stephens, R. L., Lee, J. M., Warren, C. D. $ Jeenloz, R. W. (1984). Structure determination of a tetrasaccharide: transient nuclear Overhauser effects in the rotating frame. J. Amer. Chem. Sot. 106, 811-813. Brandts, J. F., Halvorson, H. R. & Brennan, M. (1975). Consideration of the possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues. Biochemistry, 14. 49534963. Brown, J. E. & Klee. W. A. (1971). Helix-coil transition of the isolated amino terminus of ribonuclease. Biochemistry, 10, 470476. Bycroft, M., Matouschek, A., Kellis, J. T.. Serrano, L. & Fersht. A. R. (1990). Detection and characterization of a folding intermediate in barnase by NMR. Nature (London), 346, 488490. Chandrasekhar. K., Profy, A. T. & Dyson. H. J. (1991). Solution conformational preferences of immunogenic peptides derived from the principal neutralizing determinant of the HIV-l envelope glycoprotein gp120. Biochemistry, 30, 9187-9194. Creighton. T. E. (1978). Experimental studies of protein folding and unfolding. Prog. Biophys. Mol. Biol. 33. 231-297. Cummings, A. L. & Eyring, E. M. (1975). Helix-coil transition kinetics in aqueous poly (r-L-glutamic acid). Biopolymers, 14, 2107-2114. DiCapua, F. M.. Swaminathan, S. & Beveridge, D. L. (1990). Theoretical evidence for destabilization of an g-helix by water insertion: molecular dynamics of hydrated decaalaninr. J. Amer. Chem. Sot. 112. 6769-6571, Dyson. H. ,J. & Wright, P. E. (1991). Defining solution conformations of small linear peptides. Annu. Rev. Biophys. Biophys. Chem. 20, 51%538. Dyson. H. J.. Cross. K. J.. Houghten, R. A.. Wilson, I. A., Wright. P. E. & Lerner, R. A. (1985). The immunodominant’ site of a synthetic immunogen has a conformational preference in water for a type-11 reverse turn. ,Vature (London), 318. 48&483. Dyson. H. J.. Rance. M., Houghten, R. A.. Lerner, R. A. Br Wright. P. E. (1988a). Folding of immunogenic peptide fragments of proteins in wat’er solution. I. Sequence requirements for the format,ion of a reverse turn. J. Mol. Biol. 201, 161-200. Dyson. H. J.. Rance, M.. Houghten, R. A., Wright, P. E. & Lerner, R. il. (1988b). Folding of immunogenic peptide fragments of proteins in water solution. II The nascent helix. J. Mol. Biol. 201, 201-218. Dyson. H. J.. Lerner, R. A. & Wright, P. E. (1988~). The physical basis for induction of protein-reactive antipeptide antibodies. Annu. Rec. Biophys. Biophys. (‘hem. 17. 305-324. Dyson. H. J.. Satterthwait. A. C., Lerner, R. A. & Wright. P. E. (1990). Conformational preferences of synthetic peptides derived from the immunodominant site of the circumsporozoite protein of Plasmodium falciparum by ‘H NMR. Biochemistry, 29. 7828-7837. Dyson, H. J., Norrby, E.. Hoey, K., Parks, D. E., Lerner, R. A. & Wright, P. E. (1992~). Immunogenic peptides corresponding to the dominant antigenic region Ala597 to Cys619 in the transmembrane protein of simian immunodeficiency virus have a propensity to fold in aqueous solution. Biochemistry, 31, 1458-1463. Dyson, H. J., Sayre, J. R., Merutka, G., Shin, H.-C., Lerner, R. A. & Wright, P. E. (1992b). Folding of

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Edited by A. R. Fersht