J. Mol. BioZ. (1992) 226, 819-835

Folding of Peptide Fragments Comprising the Complete Sequence of Proteins

Models for Initiation of Protein Folding II. Plastocyanin

H. Jane Dyson-f, James R. SayreS, Gene Merutka, Hang-Cheol Shin Richard A. Lerner and Peter E. Wright?

Department of Molecular Biology, The Scripps Research Institute 10666 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.

(Received 13 December 1991; accepted 17 March 1992)

In an attempt to understand the earliest events in the protein folding pathway, the complete sequence of French bean plastocyanin has been synthesized as a series of short peptide fragments, and the conformational preferences of each peptide examined in aqueous solution using proton n.m.r. methods. Plastocyanin consists largely of B-sheet, with reverse turns and loops between the strands of the sheet, and one short helix. The n.m.r. experiments indicate that most of the peptides derived from the plastocyanin sequence have remarkably little propensity to adopt folded conformations in aqueous solution, in marked contrast to the peptides derived from the helical protein, myohemerythrin (accompanying paper). For most plastocyanin peptides, the backbone dihedral angles are predominantly in the b-region of conformational space. Some of the peptides show weak NOE connectivities between adjacent amide protons, indicative of small local populations of backbone conformations in the a region of (#,@) p s ace. A conformational preference for a reverse turn is seen in the sequence Ala65-Pro-Gly-Glu68, where a turn structure is found in the folded protein. Significantly, the peptide sequences that populate the cc-region of (+,+) space are mostly derived from turn and loop regions in the protein. The addition of trifluoroethanol does not drive the peptides into helical conformations. In one region of the sequence, the n.m.r. spectra provide evidence of the formation of a hydrophobic cluster involving aromatic and aliphatic side-chains. These results have significance for understanding the initiation of protein folding. From these studies of the fragments of plastocyanin (this paper) and myohemerythrin (accompanying paper), it appears that there is a pre-partitioning of the conformational space sampled by the polypeptide backbone that is related to the secondary structure in the final folded state.

Keywords: protein folding initiation; nuclear magnetic resonance; peptide conformation; plastocyanin; /l-sheet

1. Introduction

Plastocyanin (Pc$) is a small (99 residues) copper- containing protein involved in photosynthetic elec- tron transfer. Structures of several plastocyanins have been published (Guss & Freeman, 1983; Moore et al., 1988; Collyer et al., 1996) using both X-ray crystallography and n.m.r. methods. The high- resolution structure of French bean (Phaseolus

t Authors to whom all correspondence should be addressed.

$ Present address: Stanford University School of Medicine, Stanford, CA, 94305, U.S.A.

0 Abbreviations used: PC, plastocyanin; n.m.r., nuclear magnetic resonance; h.p.l.c., high performance liquid chromatography; TFA, trifluoroacetic acid; DTT, dithiothreitol; TFE, 2,2,2-trifluoroethanol; p.p.m., parts per million; 2&F-COSY, double quantum filtered phase- sensitive two-dimensional correlated spectroscopy; NOESY, two-dimensional nuclear Overhauser effect spectroscopy; ROESY, rotating-frame NOESY spectroscopy; 2D, two-dimensional; NOE, nuclear Overhauser effect; c.d., circular dichroism spectroscopy; d.n(i,j), d&i,j), etc, intramolecular distance between the protons C”H and NH, NH and NH, etc, on residues i and j; p.p.b., parts per billion.

819 0022-2836/92/150819-17 $03.00/O 0 1992 Academic Press Limited

x20 II. .J. I~yson et al.

1~~1guri.s) plastocyanin has recently been determined in solution from nuclear magnetic: resonance spectroscopy (n.m.r.) distance constraints (Moore et al., 1991). All of these proteins are homologous in primary sequence, and exhibit an identical global fold, that is, a p-sandwich consisting of two inter- linked b-sheets. On the basis of this structural information, we designed a series of peptides based on the sequence of French bean PC (Milne et al., 1974) to probe initiation of folding of plastocyanin.

The mechanism of protein folding is currently the subject of intense interest. Several n.m.r. methods have been used to probe the mechanism of folding of whole proteins, including quenched flow exyeri- ments (Udgaonkar & Baldwin, 1988, 1990; Roder et al., 1988; Bycroft et aE., 1990; Miranker et al., 1991). However, these methods are inadequate for investi- gating the earliest events of protein folding, since these events are invariably rapid, and are complete within the dead time of the flow-quench apparatus. In an effort to elucidate some of bhese early events, we have engaged in the study of peptide fragments of proteins. A number of studies have shown that peptide immunogens that induce anti-peptide anti- bodies that cross-react with the cognate sequence in the intact protein often contain preferences for con- formations other than the random extended-chain conformations normally found for peptides in water solution (Dyson et al., 1985, 1988a.b. 1990, 1992a; Williamson et al.. 1986; Lark et al., 1989; Waltho et al., 1989; Chandrasekhar et al., 1991). This observa- tion has important implications both for the induc- tion of these antibodies (Dyson et al., 1988c) and for the process of initiation of protein folding (Wright et al., 1988).

Calculation of on-lattice folding trajectories by the Monte Carlo method (Skolnick & Kolinski, 1989) implicate the /?-turn in the initiation of folding in mny folding topologies. Using this approach, the complete plastocyanin sequence has been reprodu- cibly and uniquely folded into the correct topology (Skolnick & Kolinski, 1990). It was therefore of interest to determine experimentally the possible sites of initiation of folding of plastocyanin, since this protein consists almost entirely of P-sheet and has several prominent B-turns. Here. we report the results of a study of a series of synthetic peptides corresponding to the sequence of plastocyanin. The peptides span the entire sequence of plastocyanin in an overlapping fashion. Proton n.m.r. was used t’o investigate the peptide conformational preferences in aqueous solution.

2. Materials and Methods

(a) Peptide synthesis and sample preparation

Peptides were synthesized by solid-phase methods and purified by reverse-phase high-performance liquid chromatography (h.p.1.c.) using Vydac C-18 columns and a gradient of water and acetonitrile in 1 o/0 (v/v) trifluoro- acetic acid (TFA). Both the N- and C-termini were unblocked in all peptides. Samples for n.m.r. spectroscopy were routinely prepared from 5 to 10 mg of pure peptide

in BOO:, 1H20/10~;, ‘H,O. So spectra were acquired t’o~ peptides in ‘H,O solution. The sample pH \vas a(ijust,e(l wit,h solutions of NaOH and H(‘1 to between 1.5 ~III(! 5.:; Final peptide concentrations were typically 1 to 5 n1.\;1. Several of the peptides (Prd, Pc6a. PclO. I’cl2. I’(,l,li anti Pc19; see Table 1) were insufficiently soluble for ll.rn.t studies under all conditions of temperature and ptl c~xam ined. Other peptides were sparingly soluble at, ptl .. 5. but were sufficiently soluble at higher pH values f’or r1.m.r measurements to be made, although with c:onsryurnt Iosb of sensitivity for the amide proton resonances dur tt) thud more rapid solvent exchange at the higher pH values. In order to examine the regions of the plastocyanin sequence covered b,v the “insoluble” peptides, alternative. usuall? shorter. peptides were synt,hesized. Thesr UP designated in Table 1 and throughout the text with an additional a OI b. e.g. P&a. One longer peptide was synthesized (PcGa) to investigate the formation of hydrophobic* c~lustrr struts- tures. hut it t’oo was sparingly soluble (Table I j. For peptides containing cysteinta residues. a small amount of dithiothreitol (DTT) was added to t,hr solution and t,he n.m.r. t,uhe was sealed under argon to prrvrnt- the formation of intermolecular disulfide bonds.

For the trifluoroethanol study. a sample of peptidr *as first dissolved in 0.2 ml of ‘H,O and the pH adjust,rd to 5.0. An equal volume of d3-trifluoroethanol (TFE) was added to give 5O”i, [v/v) TFE concentration.

(h) ‘H n.m.~. spwtroscopy

‘H n.m.r. spectra were acquired in the phase-sensitive mode using Bruker AM500 and MST>300 spectrometers. The probe temperature was calibrated using methanol (VanGeet, 1968). Dioxan was used as an internal standard and assigned a chemical shift of 3.75 parts per million @p.m.).

Double quantum filtered phase-sensitive d-dimensional correlated spectroscopy (2&F-COSY) (Rance et al., 1983) was used for spin-system assignment. Sequential assign- ments were made using phase-sensitive S-dimensional nuclear Overhauser effect spectroscopy (NOESY: Jeener et al., 1979) and rotating-frame NOESY (ROESY: Bothner-By et al., 1984) experiments. The mixing time T was 300 to 600 ms in NOESY spectra and 100 to 200 ms in ROESY spectra in water solutions, and 200 ms in water/ TFE mixtures. For all of the spectra, the water resonance was suppressed by gated irradiation during the relaxation delay and during the mixing time of the NOESY experi- ments, and the transmitter offset was placed to coincide with the water resonance. The data were acquired without sample spinning to minimize t, noise. Spectra were routinely acquired with 2048 complex points and 32 or more scans for each free induction decav. Spectral widths were commonly 9 p.p.m. in both dimensions; between 300 and 700 t, values were recorded for ea,ch 2-dimensional (2D) spectrum.

Spectra were Fourier transformed in both dimensions using phase-shifted sine bell window functions; the final matrix contained 2048 real points in both dimensions. Data were processed on a Convex C240 computer, or on a Sun3 workstation equipped with a Sky Warrior arrag processor, using a modified version of the program FTNMR (Hare Research). Linear baseline-correction (Dyson et al., 1988u) and t, ridge suppression (Otting et al., 1986) were employed to enhance the quality of the 2D nuclear Overhauser effect (NOE) spectra. A small lst- order phase adjustment was usually made in o1 (Dyson et al.. 1988a).

Temperature coefficients of the amide proton reson- ances of each peptide were calculated from the gradient of

Folding of Plastocyanin Fragments 821

the plot of chemical shift versus temperature, using the method of least-squares. Chemical shifts were obtained directly from the l-dimensional spectra of the peptides, except in cases where resonance overlap was severe, when a series of 2&F-COSY spectra was used.

(e) C’ircular dichroism spectroscopy

Ultraviolet’ circular dichroism (c.d.) spectra were recorded for selected peptides at 5°C using an Aviv 61DS c.d. spectropolarimeter and 62 or l.Ocm cells, as described in the accompanying paper (Dyson et al., 19923).

3. Results and Discussion

(a) Peptide selection and solubility

The sequences of all the peptides examined are shown in Table 1. The amino acid sequence of French bean plastocyanin (Milne et al., 1974) and the position of each peptide in the sequence is also

indicated, together with the secondary structure present in the folded protein in aqueous solution, derived from n.m.r. studies (Chazin & Wright, 1988; Moore et al., 1991). Peptides were selected according to two primary criteria: firstly, an emphasis was placed upon the turn, loop and helical regions of the intact protein, as being the most likely sites t,o observe a folding initiation site in the peptide, but complete lengths of b-strand were included as a control. Secondly, peptides were selected with a view to optimizing the chances of water solubility, that is, towards the ends of the peptide, hydro- phobic amino acid residues were excluded if possible, while hydrophilic residues were included. The sequences of all the peptides corresponded exactly with the amino acid sequence of French bean plastocyanin (Milne et al., 1974) with one exception: the single cysteine residue (Cys84) was replaced by threonine in three peptides, Pc16, Pc17 and Pc19, to exclude the possibility of dimerization through disulfide formation.

Table 1 Amino acid sequences of French bean plastocyanin showing secondary structure and the sequences of the

synthesized peptides

1 5 10 15 20 25 30 35 40 45 50 LtEVLLGSGDGSLVFVPSEFSVPSGEKIVFKNNAGFPHNVVFDEDEIPA~~V

PC b$bb b b b bbb bbbbb __- bbbbbbbb--_ bbbbbb

# 1 LEVLLGSG 2 levllgsgdgslvf v§ 2a SGDGSL 3 SLVFVPSEFS 4 SEFSV 5 SEFSVPSGEK 6 KIVFKNNA 6a gekivfknnagfphnvvfde 7 KIVFKNNAGFPH 8 KNNAGFPHNV 9 PHNVVFDEDEIP 10 ipagv- 10a EIPAGV

55 60 65 70 75 80 85 90 95 DAVKISMPEEELLNAPGETYVVTLDTKGTYSFYCSPHQGAGMVGKVTVN hhhhhh bb _-bbbbbbbb--pbbbbbbb bbbbbbbb

10 -+ davki sm lob DAVKIS 11 MPEEELL 12 mpeeellnapgetyvvtl 13 ELLNAPGETY 13a NAPGETY 13b APGET 14 getyvvt 1 14a ETYVVT 15 VTLDTKGTY 16 g y y 1. 5 f t 7 16a TYSFYC 17 YTSPHQGAGMV 18 MVGKVTVN 19 gtysfytsphqgagmvgkvtvn

t Amino acid sequence from Mime et al. (1974). 1 Secondary structure derived from the n.m.r. structure of French bean plastocyanin (Moore et al., 1991). 9 Lower-case letters indicate peptides that were not water-soluble. 7 In Pc16, Pc17 and PC19, a threonine residue was used in place of the cysteine residue in the plastocyanin sequence. h. /?-strand; h, helix: _ , position of turn; _ _ _, position of loop.

822 Il. J. Dyson et al.

E43 N’ 4.2 ? E 6. d

5

8.4 8.2 3.2 3.0 2.8 2.6 2.4 2.2 2.0

w2 (p.p.m.1

Figure 1. Portion of a 5OOMHz phase-sensitive ZQF-COSY spectrum of peptide PC9 in 90?; ‘H,O/lOq/, ‘H,O pH 6.5, 278K. The area shown encompasses the so-called “fingerprint region” of the spectrum, containing the cross-peaks between NH and C”H. and the region containing the C”H and C?H cross-peaks. Scalar connertivities for all of the amino

LI

acid residues are indicat,ed.

Several peptides, notably the long peptides for which we had hoped to observe the formation of intramolecular P-hairpins (Pc2, PcBa, PclO, Pc12 and Pc19), as well as Pc14 and Pc16, which each had the sequence of one of the B-strands, were insufficiently water-soluble for n.m.r. studies. To complete the study, by inclusion of the missing sequences, a second set of peptides was designed and synthesized. These included Pc2a. PclOa, PclOb. Pcl4a and PclSa. The latter peptide was synthe- sized with the naturally-occurring cysteine residue included, and precautions were t,a.ken t.o guard against disulfide formation. Since solubility problems were encountered mainly with the longer peptides, the second set of peptides was designed to be shorter. Thus, for example, the sequence from residues 46 to 57 of plastocyanin, which constituted the insoluble peptide PclO, was broken up into two 6-residue sections, PclOa, which included the preceding Glu45, and PclOb, which excluded the hydrophobic Met57 at the C terminus of the peptide.

The possibility of intermolecular association or aggregation must be addressed in studies such as these on small peptides. Most of the longer plasto- cyanin peptides, which had been designed around structural features such as turns and loops in the protein, and which included substantial lengths of protein P-strand sequences at either end, were in- soluble in water. By analogy with the protein struc- ture, these peptides could potentially form P-hairpins in aqueous solution, and their limited water solubility might be an indication that they form strong intermolecular j-structure. These results are consistent with published work that indicates that the P-hairpin structure is difficult to reproduce in a small peptide in water solution. Several attempts have been reported, but either no j&structure was observed (Goodman & Kim, 1990) or extensive intermolecular /?-sheets were formed,

resulting in aggregation and precipitation (Hartman et al., 1974; Osterman & Kaiser, 1985; Muga et al.. 1990). It has even been argued that insolubility of short peptides in water is an indicator of formation of P-sheet structure (Narita et al.. 1988).

A peptide that is apparently water-soluble may also be in an aggregated state. Several observations can be made by n.m.r. to establish the aggregation state of a peptide in solution. The linewidth of the proton resonances is a good indication of the effec- tive molecular mass of the peptide, since it depends upon the correlation time of the molecule. A change in the resonance linewidth with a change of concen- tration can show the presence of monomer-dimer or monomer-oligomer equilibria (e.g. see Waltho et al., 1989). Similarly, a change in the chemical shift. especially of amide proton resonances, with concen- tration provides a highly sensitive indicator of aggregation. In the course of the present work. particular care was taken with those peptides that had shown evidence of limited solubility: in these cases, the spectra were acquired at a pH at least 05 pH units removed from the point of precipita- tion, and the concentration of peptide was also generally lower. A specific concentration-depen- dence of the n.m.r. spectra of t’hree peptides, Pc5, Pc9 and Pc13, was determined by making a 1 : 10 dilution of the original n.m.r. samples and checking for changes in the spectrum. In none of these cases did the spectra show evidence of aggregation.

(b) ‘H n.m.r. studies of plastocyanin peptides

Complete sequence-specific proton resonance assignments were made for all peptides using stan- dard sequential assignment methods (Billeter et aZ., 1982). Sample spectra for representative plasto- cyanin peptides are shown in Figures 1 to 3. The complete resonance assignments of the peptides are summarized in Table 2.

Folding of Plastocyanin Fragments 823

- V39a V4ON

V40 N”

E45a-146N _

E45 Na

I46 N4 -

D42 N’= “-,D42a-E43N

hn D44a-E45N

D44 NOL J

F41aG42N w N38a-V39N

8.6 8-4

w2 ( p.p.m. 1

8.2

4.0

4.4

4.6

Ii ci d

5 8.2

8.6

Figure 2. Portion of a 500 MHz phase-sensitive NOESY spectrum of PC9 in 9076 ‘H,0/100i, *H,O. pH 6.5. 278K. The mixing time t was 400ms. The NH-XH and (‘“H-NH regions are shown, and the sequential connectivities are indicated.

Formation of elements of secondary structure in the peptides can be detected using the nuclear Overhauser effect. For each of the peptides, a NOESY or ROESY spectrum was accumulated for use, firstly, for sequential resonance assignment. and secondly, to detect elements of secondary and other structure in solution. A summary of the d,,(i,i+ 1) NOES observed for the plastocyanin peptides is shown in Figure 4. All of the peptides, without exception, showed a complete series of strong daN(i,i+ 1) NOE connectivities; for brevity, these are omitted from Figure 4.

The temperature coefficients for the plastocyanin peptides are included in Table 2. In addition, Figure 4 also indicates the residues for which the amide proton temperature coefficients were lowered by an

b El8 N’ E18a. F19N

4.2

(b)

8.4

SZON

8.8

8.8 8.6 8.4 8.2 8-O -”

w2 (p.p.m.1

Figure 3. Portion of a 3OOMHz phase-sensitive ROESY spectrum of peptide Pc4 in 900/;, ‘H20/100b ‘H,O, pH 4.3, 278K. The mixing time T was 200 ms. The NH- NH and C”H-NH regions are shown, and the sequential connectivities are indicated. Both positive (diagonal) and negative (cross-peak) contours are plotted without distinction.

amount greater than one standard deviation from the mean value calculated for all the temperature coefficients in Table 2 (mean value = 7.1 p.p.b./K; standard deviation = 1.9 p.p.b./K).

An estimate of the extent to which the peptide adopts local backbone conformations in the u region of conformational space is given by the observation of d,,(i,i+ 1) and other NOE connectivities in NOESY or ROESY spectra, in favorable cases accompanied by a diminution of the intensity of the d,,(i,i + 1) NOE connectivities characteristic of backbone conformations in the fl region of ($I,$) space. Certain medium-range NOE connectivities are characteristic of preferences for folded con- formations in peptides (Wright et al., 1988; Dyson $ Wright, 1991). With one exception (Pc13, see below) no d,,ji,i + 2) or other medium-range NOE connec- tivities were observed for any of the plastocyanin peptides. In a few cases, d&i&+ 1) NOE connecti-

X24 H. J. Dyson et al

PC3 SLVF:SPSEFS B *m

PC4 S~F@h

PC5 SHFBV P S*G& I

Pc6 KIVFENNA *m

PC7 K I V F K N*Nd*G; P H

Pc6 KNNAGFPHNV I*- *I

PC9 PHNV;oF~ED*&I P *-* **w

PclOa E I PAG? *

PclOb D q V K*‘:-.S

Pcll MPEkLL * I

Pc13 ELLNttPGBT? *m I*-

Pcl3a N A P G q T Y I-

Pcl3b A P G E T W*

Pcl4a ETYVVT

Pcl6a 1 Y S*F*Y q

Pc17 Y@P H OG*iteG~V

Pc18 M V G “i V T V N * **

Figure 4. Schematic diagram showing the relative intensity (filled bars) of NOE connectivities between sequential backbone amide protons observed for all peptides. Asterisks indicate that a cross-peak was not observable due to resonance overlap. Boxed amino acid residues are those for which the temperature coefficient (Table 2) is below the average of all temperature coefficients by 1 or more standard deviations.

vities were observed (Fig. 4) and indicate the presence of a small proportion of backbone conformations in the u region of ($,$) space (Wright et al., 1988; Dyson & Wright, 1991). The presence of d,,(i,i + 1) NOE connectivities also varied between different peptides, roughly correlated with the distribution of d,,(i,i + 1) NOE connectivities, but since the presence or absence of this NOE is less informative about conformational preferences than the d&i,i+ 1) NOE, this information has not been

included in Figure 4. For a number of the peptides, a dNN(i,i + 1) NOE connectivity is observed between the residues at the C terminus of the peptide (e.g. seen in Figs 2 and 3). This NOE is frequently observed for short, peptides with unblocked C termini (Dyson et al., 1988a), and probably arises from the influence of the C-terminal carboxyl group rather than from any sequence-dependent struc- turing of the peptide.

When proton resonances, particularly those of

Folding of Plastocyanin Fragments 825

Table 2 Resonance assignments of peptide fragments of French bean plastocyanin

Amino acid NHt residue

Pel 27XK pH 6.00 LWl

ml2 8.87

Va13 8.57 Leu4 861 IAm5 8.56 Gly6 864 Ser7 838 Gly8 8.30

Pr2a 27XK pH 4.01 Ser7 Gly8 89% Asp9 8.63 GIglO 8.67 SW1 1 828 Lru12 8.23

Pc.3 27XK pH 6.14 SW11 Leul2 Val13 832 Phel4 8.62 Vall8 8.23 Pro 16 Serl7 %57 01~18 8.62 PhelS 8.31 Srr20 7.92

Pc4 27&K pH 4.34 Serl7 Glu 18 8.95 Phe19 846 SW20 WY Va121 8.02

PCS 27RK pH If8 Serl7 ClUl8 8% Phet9 8.48 sereo 8.27 Val21 838 Pro22 Ser23 8.66 Gly24 8.60 Glu25 8.33 Lys26 8.19

Pt.6 278K pH 5.04 Lys26 Ile27 !+73 Va128 850 Phe29 8.67 Lys30 845 Asn31 8.58 Asn32 8.55 Ala33 8.02

Pc7 278K pH 4.10 (i) tram Lys26 Ile27 8.72 Val28 8.49 Phe29 8.66 LysYO 8.48 Asn3 1 8.57 Asn32 8.60 Ala33 8.37 Gly34 8.39 Phe35 %13 Pro36 His 37 8.25

C”H? CBHt CYHt (“Ht Othert -A6/AT (p.p.b./K)f.

4.03 $42 $04 $41 4.36 440 450 3.79

1.69,1.74 1.71 1.91,2.04 2.27 2.02 0.91 ,095 1.63 1.59 1.66 1.65

3.86,3+4

420 4.06 $68 4,06,391 4.45 425

400

2.80

388 1.62,1.62

+08 393.3.90 443 1.59 408 1.97 460 2.95307 4.29 1.90 4.26 2.35,1+0 4.36 380,390 418 1.80 4.74 3.28,2.93 4.23 3.86

416 396 426 1.97,1.86 472 3.22,2.97 448 384 407 213

4.14 4.29 4.68 443 4.40 4.42 4,40 3.98.3.98 4.36 4.17

3.94 1.85 317,298 3.78 2.09 2,32,190 3.94,386

1.94 2.11 1.80,1.70 1.40

4.0 1 1.88 4.14 l-73 4.08 1.97 4.60 2.98,308 4.26 1.70 4.61 274,2.84 4.69 %74,2.84 4.09 1.34

402 413 4.08 4.60 4.%4 4.59 465 4.26 3.83 4.88 4.43 4.50

1.88 1.72 1.95 308,2.97 1.69 275J.82 274,2.81 1.38

294,316 2.25,1.93 326,3.15

0.86.0.94 088,0.95

1.62 088,092

1.61 0.88,0.92

0.86,@88 2.00

2.03

(P89,0.93

7.33

3.66

7.27 7.33,7,33 (C”H,CCH)

2.04,2.16 7c25 7.34,7,32 (C’H,CIH)

0.92,095

2.10 7.36 7.27,7.37 (CEH,dH)

096,lfbO 2.02 370.3.91

I +i7 2.98 (CeHz)

1.38 1.14,1.47 087,090

1.34

I.70 085

7.24 1.67

1.39 1.12,1.46 0.90,087

1.34

1.69 084

7.24 1.65

1 .ss 7% 354,:980 857

7.20,7.33 (C”H,6H)

2.99 (COH2) 0.70 (CYH,)

7.33,7.33 (C’H,@H) 2.98 (CeHI)

2.98 (C’H,) @69 (CYH3)

7.31,7.31 (C”H,C’H) 2.95 (CeH2)

7.34,7.34 (C’H,C’H)

55 7.8 9.0 6.1 7.3 6.8 5.9 5.5 Es6

7.28 (C”H) 8.1

6.0 6.9 7.3 5.0 X.0

7.5

93 id.2

8% i.0 6.3 4%

5.2 6.6 4-3 6.5

4.4 6.8 43 R6

X.5 6.5 4.9 7.7

66 82 91 5.8 6.5 64 e5.8

X26 11. J. Dyson et al. - ..____ ___-- ..--- -.---__ -.

Table 2 (continued)

Amino acid NH? residue

(ldHt

-___

Whert as/A? (p.p.h./K): ---

(ii) cis Lys30 8.45 Asn31 8.58 Asn32 8.66 Ala33 8.39 GIy34 8.48 Phe35 8.13 Pro36 His37 8.35

PcX 278K pH 4.73 (i) trans Lys30 Asn31 9.05 Asn32 880 Ala33 8.47 Gly34 8.44 Phe35 818 Pro36 His37 8.75 Asn38 8.69 Va139 7.97

(ii) cis Asn32 18.83 Ala33 8.48 Gly34 8.53 Phe35 8.19 Pro36 His37 8.86 Asn38 18.64 Val39 7.91

Pe9 278K pH 5.10 Pro36 His37 9.09 Asn38 883 Va139 838 Va140 832 Phe4 1 8.48 Asp42 8.44 QIu43 8.46 Asp44 8.48 Glu45 8.20 IIe46 8-39 Pro47

PclOa 278K pH 4.08 Glu45 Ile 46 8.87 Pro47 Ala48 8.61 GIy49 854 Va150 7.81

PclOb 278K pH 5.36 Asp51 Ala52 8.73 Va153 8-40 Lys54 8.63 Ile55 8.55 Ser.56 8.11

Pcll 278K pH 4.07 Met57 Pro58 Glu59 8.73 01~60 860 Glu61 8.55 Leu62 8-45 Leu63 8.22

Pc13 278K pH 498 Glu61 - Leu62 8.86 Leu63 861

426 460 468 4.31 389 4.44 3.83 443

1.69 2,75,2+X) 283 1.41

1.34 1.65 2.95 (C”H,)

2.98 7.19 1.70 350,3.36 3,24,3.10 849

401 472 468 4.26 3.83,3.83 4.87 441 4.75 4.75 4.05

1.91 2.77,283 275,2.84 1.38

1.41 1.71 2.99 (CeH2)

2.92,3.15 2.25,1.86 319,3.30 2.77,2*82 2.11

1.98

088,090

?472 432 392,392 437 3.81 4.71 t4.72 4.04

1.42

2.96,3.04 1.63,1+4 3.153.25

1.46,1.70

437 2.42,2.00 4.73 317,3.23 k68 2.70,2.80 405 2.02 405 1.95 461 2.99,3.12 456 2.59,2.71 423 1.95 460 2.64,2.73 430 1.92,2.01 4.43 1.91 423 2.22,1.88

2.00

082,090 083,088

2.36

2.35 1.18.154 2.00

4.10 4.54 442 432 393,401 412

2.12 1.89 2.33,1.93 1.43

2.40 1.54,1,20 2.07

2.13 0.89,093

422 2.69,2.83 439 1.38 401 2.02 438 1.75,1.82 423 1.92 426 3.84

0.93,097 1.43 1.2OJ.48

4.51 2.14,%21 2.67 4.49 236,1.91 2.04 429 200,2.08 249 433 1.97,2.06 2.47,2.44 4.35 1.97,208 2.43 4.38 1.63,1.68 1.62 4.31 1.64,1.68 1.62

401 SO8 2.36 439 1.60,1@) 1.60 436 1.60,1.62 1.6

7.28 7.33,7.33 (C’H,CcH) 354,3.79 8.60 7.32 (C’H)

335,340 8.56

3.38 8.59 7.27 (C&H) 7.74,7.01 (NdH)

7.24 7.33,7.33 (C”H,@H)

0.87 3.68,3.86

0.99 (CYH3)

6.4 8.2 7.3 8% 46 55 56 3.4 *7

0.89 3.70,3.95

I.00 (CYH3) 7.3

$3 7.1 7.7

1.67 2.98 (C”H,) 0.86 094 (CYH3)

50 97 9.0 9.0 7.7

2.13 (C’H,) 365,3.78

0.87,0.95 087,093

7.4 7.4 7.0 7.7 7.7

089,094 53 086,092 66

6.5 7.7 7.0 6.9 6.2

82 64 7.5

Folding of Phtocyanin Fragments 827

Table 2 (continued)

Amino acid NH? residue

C”Ht C?Ht VHt CdHt Other? -As/AT (p.p.b./K)S

Asn64 859 Ala65 8.41 Pro66 Gly67 8.64 Glu68 825 Thr69 8.32 Tyr70 7.98

Pcl3a 278K pH 4.38 As.1164 Ala65 8.83 Pro66 Gly67 8.71 Glu68 8.11 Thr69 8.34 Tyr70 7.90

Pcl3b 27XK pH 4.09 Ala65 Pro66 -

Gly67 8.81 Glu68 8.25 Thr69 8.14

Pclla 27RK pH 5.“2 * Glu68 Thr69 8.83 Tyr70 8.58 Val7 1 8.28 Va172 8.46 Thr73 7.99

Pc15 ZTRK pH 3.96 Va.172 Thr73 8-82 Leu74 %74 Asp75 8.66 Thr76 8.32 Lys77 8.49 01~78 8.52 Thr79 8.06 TyrHO go0

PclGa 27XK pH 5.40 Thr79 TyrXO 8.91 Ser81 8.36 PheXS 817 Tyr83 8.20 Cys84 7.84

Pc17 27XK pH 4.08 Tyr83 Thr84 8.44 Ser85 8.67 Pro86 His87 X.67 Bln88 8.56 Gly89 %63 Ala90 8.45 Gly91 864 Met92 %27 va193 i.90

PclX 2788 pH 4.35 Met92 Val93 8.77 Gly94 X.72 Lys95 x35 Va196 X51 Thr97 u.53 \?a198 X.48 Asn99 8.33

463 458 440 394 4.36 430 4.42

434 468 439 399,390 443 430 443

4.38 446 402,393 448 4.13

4.03 2.02 431 406 464 2.92,301 406 1.93 411 2.07 414 410

3.88 439 438 4.72 431 4.31 3.97.3.94 430 446

3.81 4.10 4.59 2.92 4.33 366 448 2.93 455 >87,3.09 428 2.88

4.24 4.31 4.64 439 464 433 396 4.33 3.93 451 405

418 416 3~91,400 438 419 4.36 415 451

270,2.74 1.35 2.30,1.93

1.94 416 310,2.86

2.04

2.25 1.14

281,2.96 1.36 2.32,1,95

1.92,1.96 419 3.08,2.89

2.05

2.25

1.51 2.35,1,93

2.04,2.34 422

2.05

2.40 1.18

2-32 1.15

0%8,090 097,099 1.14

2.21 411 159,1.66 2.84,271 430 1.82,1.84

415 2,90,3.10

099,l~OO 1.22 1.61

1.20 1.45

1.11

1.26

3.25,3.35 395 388 2.25,1.80 318,321 1~90,200

1.15

1.99

2.35

1.39

190 2.55,2.50 2.08 0.98,0.99

2.15 2.58 2.08 098

1.72,1.78 2.10 415 2.09 2.80,268

1.40 0.92,0.94 1.19 0.93

7.70,696 (NdH) @2

3,66,3.80

6.80

7.4 48 6.9

7.11 (CFH) 67

7.02, 7.69(NdH) 5u

3.663.84

7.10

7.5 36 7.3

679(C”H) 55

3.62374

7.10 680 (CH)

085.091

1.68

5.10

69 7.7 8-9 7.1

298 (C’H,) 6.3 6.3 5.2

6.80 (C’H) $4

i.06

7.13 7.11

677 (CeH) 7.4 53

7.31,7.32 (C”H,@H) 61 6.82 (C’H) 85

4.2

7.10

3.74387 a.59

6.82 (C”H) 15 X.1

7.28 (C”H) 7.9

7,2

7.9 5.6 6.6

7.0 9.3 7.0 x.4 7.2

7.66,899 (NdH) 6.6 63 7.2 6.9

2.08 (C’H1) 49 63

61 ii.0

I.68 2.98 (CYH,) 6.9 7% 84 X.7 7.7

t Chemical shifts in p.p.m. $ Trmperatore coefficients measured at the same pH as the chemical shifts were measured

N64k - A65N

G67Na

iP66a-G67N

11 E61a-‘l

8.8 8.6 8.4 8.2 8.0 4.6 4.4 4.2 4.0

we (p.p.m.1

Figure 5. Portion of a 500 MHz phase-sensitive NOESY spectrum of Pc13 in go:/,, ‘H,O/lO~/, *H,O. t,ime 600 ms.

amide protons, are overlapped, NOES are less useful in predicting structure. For example, the tempera- ture coefficients of the amide protons of the C- terminal region of PC9 are quite low (Table 2). However, several of the amide and C”H resonances of the residues in question are severely overlapped, precluding observat’ion of the relevant NOES. The pH dependence and exchange behavior of the amide protons of PC9 are also anomalous, which could be an indication of local structure formation which cannot be confirmed by NOE measurements due to resonance overlap.

The case of Pc13 is an interesting one. On the basis of previous work (Dyson et aZ., 1988a; H. J. Dyson, ,J. J. Osterhout & P. E. Wright, unpublished results) we had expected to observe the presence of a /?-turn in the sequence APGE. The ROESY spec- trum of Pc13 shows d,,(i,i+ 1) NOE connectivities. but, only very weak d,,(i,i+ 1) NOES, suggesting a lack of structure, despite the moderately lowered temperature coefficient for Glu68. For peptides of the size of Pc13, ROESY experimenm often give poor results due to rapid rotating-frame relaxation for NH protons. Additional experiments were there- fore performed: a NOESY spectrum of Pc13 was recorded with a long mixing time (6OOms), and two shorter peptides containing the APGE sequence. Pcl3a and PclSb, were synthesized for n.m.r. studies. Roth the ROESY spectra of the shorter peptides and the long mixing-time NOESY spec- trum of Pc13 (Fig. 5) show substantial d,,(i,i+ 1) NOES. The c&(i,i+Z) NOE between Pro66 and Glu68, which would provide confirmatory evidence for a b-turn involving residues Ala65-Pro66-GlyBT- (~1~68, is ambiguous due to overlap of the C”H resonances of Pro66 and Glu68 for all three peptides at pH - 5, but. can be discerned in the NOESY spectrum of Pcl3 at pH 4.1, where the C”H reson-

8.0

8.2

pH 4.1: mixing

ante of Glu68 is shifted upfield to an extent. that the d,,(i,i+2) NOE can now be observed (Fig. 5). ROESY spectra of Pcl3a and Pcl3b were not recorded at the lower pH. The chemical shifts of the

corresponding resonances of residues 66 to 68 for each of the three peptides are virt,ually identical at a given pH, suggesting that each peptide has the same conformational preference. c.d. spectra show a posi- tive ellipticity at 210 nm, which is suggestive of a turn, although the spectrum is complicated by the dichroism associated with the tyrosine side-cahain.

(d) Hydrophobic clusters

For two of the plastocyanin peptides, NOES are present in ROESY spectra which indicate the close approach of non-sequential side-chain protons. Roth Pc6 and Pc7 contain the sequence l,ys26-fle27- Va128-Phe29, and for bot.h of these peptides, NOE connectivities are observed between the aromatic ring protons of Phe29 and the C?‘H3 methyl group of Ile27. These are shown for PCS in Figure 6. In addition, it is noticeable that the resonance of the C’H, of He27 is shifted slightly upfield in Pc6 and Pc7, with chemical shifts of 069 and 0.70 p.p.m..

respectively, compared to the more usual chemical shifts of 0.94 to 1.00 p.p.m. for the U’H, resonances of He46 and He55 in peptides Pc9, PclOn and PclOb. This is consistent with the influence of a ring-current such as could be provided by the aro- matic ring of Phe29, and suggests that these hydro- phobic groups come into close contact in aqueous solution. Literature reports of such interactions are not plentiful, although they are expected between side-chains spaced three and four residues apart when peptides form helical structures (Lim, 1974). In the present case, the hydrophobic interaction apparently occurs in the absence of any preference

Folding of Plastocyanin Fragments 829

I27 Ns CD

V28 NY 0

El ’ 0 V28yF29N

F29 Np

8.8 8.6 7.4 7.2

-127 CyH3

0

0

Q3 0

F29 aromatic-p

0.8

3.2

w2 (p.p.m.1

Figure 6. Portion of a 300 MHz phase-sensitive ROESY spectrum of Pc6 in 90% ‘H,O/lO% ‘H,O. pH 5.0, mixing time 206 ms, showing NOE connectivities between the CYHJ of He27 and the aromatic protons of Phe29.

for secondary structure. Similar hydrophobic inter- actions have been reported (Bundi et al., 1978; Braun et al., 1983) for peptides derived from para- thyroid hormone and glucagon, and have recently been detected in a denatured protein (Evans et al., 1991).

It is interesting to note that the CYH, resonance of Ile27 is also substantially upfield-shifted in native French bean plastocyanin (Chazin & Wright, 1988). From the structure (Moore et al., 1991) it appears that the side-chains of Ile27 and Phe29 are packed on the same side of the sheet, and are in contact (within 3 A). Thus, we may be seeing in the peptide traces of an interaction that also occurs in t’he folded protein.

(e) Role of TFE in helix stabilization

The effect of the addition of TFE to three of the plastocyanin peptides (Pc5, PC9 and Pc18) was examined. The peptides chosen are those that are the most likely to contain helical segments, according to the secondary structure prediction of Chou & Fasman (1978). The solubility of the peptides in water/TFE mixtures was low: neither PC9 nor Pc18 was sufficiently soluble to attempt

n.m.r. spectroscopy. An n.m.r. spectrum was obtained for a sample of Pc5 in a 50% TFE/water solution and a portion of the NOESY spectrum obtained under these conditions is shown in Figure

7. A summary of the results of these experiments is shown in Figure 8 and the proton resonance assign- ment,s for Pc5 in 50% TFE are summarized in Table 3.

Organic solvents such as TFE are well known to promote helical conformations in proteins and peptides (Timasheff, 1970). Nozaki & Tanford (1971) suggested that this is due to the highly hydrophilic nature of the backbone peptide unit. TFE has been found to stabilize helix in regions that already have a high propensity for helix forma- tion (Lu et al., 1984; Leist & Thomas, 1984; Dyson et al., 1988b, 1992b; Gooley & MacKenzie, 1988; Nelson & Kallenbach, 1989). In the present series of peptides, there is an opportunity to examine the effects of TFE on peptides that have no detectable helical propensity.

The c.d. spectra of the Pc5, PC9 and Pc18 peptides were recorded in water, 50”,:, TFE and 90 qb TFE (data not shown). None of the three peptides showed a significant helical content, as measured by c.d. in water, and there was no observ- able increase in helicity upon addition of TFE. This is consistent with the notion that TFE only causes helix formation where an intrinsic propensity exists in the sequence. Similar conclusions are reached by n.m.r.; addition of TFE to aqueous solutions of Pc5 leads to no new NOE connectivities, as would be expected if helical conformations were formed (Figs 7 and 8). The relative intensities of the rE,,(i,i+ 1)

4.0 S23 No,@

4.2

~24 NO@ ‘*G24a-E25N 4-o

m17a-E18N 0

E18a-,F19N

S23a-G24N

4.6 P22a-S23N / siOa-

E25 N’ V2’N

F19a-S20N’

8.6 8.4

w2 (p.p.m.)

(0 I

6.8 0.4

W2(pp.mi

(bl

8.0

Figure 7. Portion of 500 MHz NOESY spectra of’ Pc5 (a) in 90% ‘H,O/lO% ‘H,O, pH4.7. 278K, (b) in 50(& d3-TFE/‘H,O, pH 5.0, 288K. The mixing time r was 200ms. The NH-NH and NHCH regions are shown and the sequential connectivities are indicated.

and daN(i,i + 1) NOE connectivities are unchanged obtained for the myohemerythrin peptides (accom- on addition of TFE; the &(i,i+ 1) NOES remain panying paper) indicate that TFE does not indiscri- very weak compared, for example, t’o those minately induce helix in any peptide sequence: an observed for the myohemerythrin peptides in water intrinsic propensity for helix formation appears to

solution (accompanying paper) or in TFE (Dyson et be a prerequisite for induction and stabilization of al., 198%). These results together with those helical conformations by TFE.

Table 3 Resonance assignments for PCS in 50% d3-TFE/H,O, 2888, pH 5.0

Amino acid residue

NH 2 (‘“H C8H CYH (“H Other

SIX17 4.20 Glu18 8.99 4.37 Phel9 8.20 470 Ser2O 7.93 4-52 VA21 804 4.41 I’m22 4.49 SW23 S.25 4.4x

Gly24 %40 404 Glu25 &19 442 Lys26 8ao 4.29

4.05

2.00 2.35 323.3m 7.29 736,7.29 (C’H,#H) 3% 2.15 1.01, 1.01 *33,2.03 2.12 392.3.72 402,3,90

2.lti,2.01 2.47 1.91,1.75 1.46 1.74 3.05 (C’H,)

Folding of Plastocyanin Fragments 831

daN (/;iCI f

dNN (i,i+l)

d@ (i,i+l I

daN (i,i+i)

dNN (ii-l-I)

dpN (i,i+l)

20 25

SEFSVPSGFK

*W

m *m

Wd -

(0)

20 25 SEFSVPSGEK

Figure 8. Schematic diagram showing the intensity of NOE connectivities observed for Pc5 in (a) 90% ‘H,O/lO% ‘H,O. pH4.7 and (b) 50% d3-TFE/‘H,O, pH 5.0.

The chemical shifts of the amide protons of Pc5 differ significantly in water and 50% TFE (Fig. 7), to a greater extent than would be expected for the slight’ difference in the temperature and pH between the two spectra. With the exception of Glu18, the amide proton resonances shift upfield by 014 to 0.41 p.p.m., yet t,here is no indication from either c.d. or NOESP spectra that helical structures have been induced. The C”H chemical shifts, which are rather sensitive to helix formation in peptides, are relatively unaffected by addition of TFE. Thus, upfield shifts of amide proton resonances upon addi- tion of helix-stabilizing organic solvents such as TFE are not. useful as diagnostics of helix formation.

(f, IsomerGm oj’ proline residues

French bean plastocyanin contains two cis- proline residues, at positions 16 (involved in a type VI turn) and 36 (loop region) (Chazin & Wright, 1988; Moore et al., 1991). The same cis peptide bonds are found in the poplar (PopJus nigra) pro- tein (Guss & Freeman, 1983). The peptide sequences in the vicinity of the two cis-proline residues are quite different: for Prol6, FVPS, and for Pro36, GFPH. The observed behavior of these sequences in short peptides is also different.

Samples of peptides Pc7 and Pc8 (containing the GFPH sequence) purified by h.p.1.c. show evidence of cis-trans isomerism: two distinct sets of ‘H reson- ances are observed, with the chemical shifts of residues close to the proline residue in the peptide

sequence showing the most difference between the two species. The major species ( -800,b) contains proline in the tram configuration, as indicated by NOES between the C”H of Phe35 and the C’H of Pro36 in both peptides. The minor species ( u 20%) contains a cis peptide bond at the proline residue. indicated by an NOE cross-peak (Fig. 9) between the C”H resonances of the Phe and Pro of the minor species (Wiithrich et al., 1984). An aromatic residue preceding proline predisposes it to the formation of a proportion of cis isomers in solution (Grathwohl &r Wiithrich, 1976; Dyson et aE., 1988a). There is no evidence of a cis-proline-containing species in the spectra of peptide Pc3, which contains FVPS, consistent with its Val-Pro sequence.

The two eis-proline residues in native plasto- cyanin thus appear to arise differently: Pro36, preceded by an aromatic residue. has a predisposi- tion in the unfolded state towards the cis configura- tion; this conformation is stabilized in the folded protein. Pro16 has no sequence-derived predisposi- tion towards the cis conformation. In this case the cis peptide bond is dictated by the tertiary structure of the protein. Proline cis-trans isomerism has been postulated as the slow step in the folding reactions of some proteins (Brandts et aZ.: 1975). For plasto- cyanin, this may well also be the case; no experi- mental folding dat)a on plastocyanin are available at this time.

The difference in the surrounding structure in the intact plastocyanin molecule at the two positions is also worthy of note: a type VI turn for Prol6, and a loop structure involved with copper ligation for Pro36, arguing further for a difference in the provc- nance of the cis configuration: the &-Pro36 con- figuration may well be necessary to preserve the geometry of t,he copper site and the configuration of the His37 ligand. Both of these proline residues are conserved in the sequences of higher plant, and algal plastocyanins (Sykes, 1985). Phe35 is also conserved except for Anabaena variabilis plastocyanin, where it is substituted with a Pro, another residue that can give rise to significant populations of &s-proline in peptides (Cung et al., 1985; St,ewart, et al., 1990). The conservation of Va115 is less strict: this position cont’ains Leu, Ile and Glu, consistent once again with the idea that the cis peptide bond at this position is dictated by the requirements of the folded structure for a Type VI turn, rat,her than by the local sequence.

(g) Folding initiation structures in solution

In the preceding discussion, we have focused on three regions of plastocyanin which, in the form of short peptide fragments, show some indication of slight conformational preferences for folded con- formations. These regions are: the hydrophobic cluster involving residues 27 to 29 (Pc6 and Pc7), the region of high concentration of acidic groups (residues 42 to 45, Pc9) and the reverse turn between residues 65 to 68 (Pc13 and its analogs). In general. however, the peptides derived from the

832 II. J. Dyson et al.

3.6

4.2

4.2 4-o 3-8

w2tp.p.m.)

3-6 3-4

Figure 9. Portion of a 3OOMHz phase-sensitive ROESY spectrum of Pc7 in 90% ‘H,O/lO% ‘H,O. pH 4.1, mixing time 200 ms, showing the NOE connectivities between the C”H of Phe35 and Pro36 in the minor (c&s) form of the peptide.

French bean plastocyanin sequence are relatively devoid of folded backbone conformations and adopt random dihedral angles, predominantly in the /I region of (r$,I(/) space. Given that plastocyanin is a p-sandwich protein, with only one small stretch of helix, it is perhaps not surprising that the majority of the molecule, even in the form of small fragments, prefers /?-backbone conformations. However, the intact protein does contain several well-defined p-turns in solution, as well as loops terminating the p-strands, and a helix between residues .52 to 56. The present study shows that the preferences of these interstrand peptides for backbone conforma- tions in other than the Bregion of (4,$) space are generally small. Nevertheless, the weak d,,(i,i f 1) NOE connectivities that are observed, as well as the lowest temperature coefficients, do occur generally in the turn, helix and loop regions, and are absent’ from the peptides corresponding to the P-strands, with the exception of the short strand encompassing residues 17 to 21. The regions of the peptides that show a detectable population of backbone dihedral angles in t,he a region of (4,$) space, as evidenced by the observation of sequential d,,(i,i+ 1) NOE

connectivities, are mapped onto the plastocyanin structure in Figure 10.

A similar study of peptide fragments of a four- helix bundle protein (accompanying paper) showed pronounced conformational preferences for helix and nascent’ helix in peptides that correspond to helices in the folded protein. These peptides and those representing the inter- and extra-helix loop regions populate, to a significant extent, dihedral angles in the a region of (4,II/) space. Thus, it appears that there is a measurable prepartitioning of the conformational space populated by the poly- peptide backbone in the unfolded state that is related to the secondary structure present in the folded protein.

Why should the peptide sequences derived from the helical protein myohemerythrin exhibit rela- tively strong propensities for pre-formed secondary structure whereas the peptide sequences derived from the b-sandwich protein plastocyanin show rather little tendency to adopt secondary struc- tures, even in the turn regions between the p-strands? A key to this paradox may lie in theo- retical simulations of protein folding using on-

Folding qf Plastocyanin Fragments 833

Figure 10. Regions of the plastocyanin molecule for which the peptide fragments show significant populations of backbone conformations in the LX region of (+,tj) space, inferred from the relative sizes of the d,,(i,i+ 1) and &(i.i+ 1) NOE connectivities, mapped onto a represen- tation of the structure of French bean plastocyanifi (Moore et al.. 1991). Red represents the regions for which the d,,(i,i+l) to d,,(i,i+l) ratio exceeds -@5 (i.e. the d,,(i,i+ 1) NOE is relatively strong); yellow represents the regions for which the d,,(i,i + 1) NOE is present, but the d,,(i,i+ 1) to d,,(i,i+ 1) ratio is less than 0.5. White regions indicate part,s of the sequence where no d,,(i,i + I ) NOES are observed in the peptide fragments.

lattice Monte Carlo calculations (for a review, see Skolnick & Kolinski, 1989). A unique and topologi- tally correct conformation for a protein can be folded repeatedly by these methods, by specifying only in the most general terms the hydrophobic and hydrophilic character of the amino acid side-chains and, most importantly, a slight conformational preference for turns at one or two specific sites in the chain. Specification of these small turn prefer- ences is essential for reproducible formation of the unique and correctly folded structure. The folding topology of plastocyanin has been correctly repro- duced by these methods, starting from unfolded states (Skolnick & Kolinski, 1990). In general terms. Monte Carlo simulations suggest that the turn propensity required to fold correctly fhe P-barrel topology (0.30/,,) is considerably less than that required for the reproducible folding of a four helix bundle (5 to 8%) (Sikorski & Skolnick, 1990: Skolnick & Kolinski, 1991), in exact parallel to the

conformational preferences observed by n.m.r. for the peptides derived from plastocyanin and myo- hemerythrin. The persuasive correlation between experiment and theory in this case leads us to suggest that protein folding is initiated in slightly different ways for these two simple protein structure motifs. For the helical protein, there is apparently a significant requirement for pre-formed secondary structure, turns and the beginnings of helix, for correct folding. The apparent lack of a requirement for strong turn propensities for correct folding of plastocyanin implies that turn formation in this P-sandwich protein, while not totally irrelevant for initiation of folding, is to a considerable extent driven by the folding process and that the turns are stabilized by the formation of the final folded structure.

We thank Drs John Tainer and Elizabeth Getzoff for help with the original peptide design work, Drs Mark Rance, *John Osterhout and Jeff Skolnick for helpful discussions, Dr R. Arlinghaus, Johnson & Johnson Biotechnology Center, for synthesizing the original set of peptides for structural and immunological character- ization, Linda Tennant, Vicki Feher and Steve Schiebel for help with peptide purification and Garry Gippert and Michael Pique for help with computer graphics. This work was supported by grant GM38794 from the National Institutes of Health.

References

Billeter. M., Braun, W. & Wiithrich. K. (1982). Sequential resonance assignments in protein ‘H nuclear magnet’ic resonance spectra. (‘omput’ation of sterically allowed proton-proton distances and stat)istical analysis of proton-prot,on distances in single crystal protein conformations. .I. Mol. Biol. 155, 321-346.

Bothner-By, A. A., Stephens, R. I,.. Lee. J. M.. Warren, (1. D. & Jeanloz, R. W. (1984). Structure determination of a tetrasaccharide: t,ransient, nuclear Overhauser effects in the rotating frame. J. Ampr. (Them. 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. Hiochrmistry, 14, 49534963.

Braun, W., Wider. G., Lee, K. H. & Wiithrich, K. (1983). Conformation of glucagon in a lipid-water interphase by ‘H nuclear magnetic resonance. .I. Mol. Hiol. 169, 921-948.

Bundi, A., Andreatta, R. H., & Wiithrich, K. (1978). Characterisation of a local structure in the synthetic parathroid hormone fragment 1-34 by ‘H nuclear- magnetic-resonance techniques. Eur. .I. Riochem. 91, 201-208.

Bycroft, M., Matouschek, A., Kellis, J. T., Serrano, L. & Fersht, A. R. (1990). Detection and characterizat,ion 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-1 envelope glycoprotein ~~120. Biochemistry, 30, 9187-9194.

834 II. J. Dyson et al.

Chazin, W. J. & Wright, P. E. (1988). Complete assignment of the ‘H nuclear magnetic resonance spectrum of French bean plastocyanin. Sequential resonance assignments, secondary structure and global fold. J. Mol. Biol. 202, 623-636.

Chou, P. Y. & Fasman, G. D. (1978). Prediction of the secondary structure of proteins from their amino acid sequence. Adva,n. Enzymol. 47, 45-147.

Collyer, C. A., Guss. J. M., Sugimura, Y., Yoshizaki, F. & Freeman, H. C. (1990). Crystal structure of plastocyanin from a green alga, Enteromorpha prolifera. J. Mol. Biol. 211, 617-632.

Cung, M. T., Vitoux, B., Marraud, M., Giessner-Prettre & Aubry, A. (1985). 2D NMR study of Pro-Pro sequences. A proposed NMR correlation concerning the Y angle. In Peptides Structure and Function. Proceedings of the 9th American Peptide Symposium (Deber, C. M., Hruby, V. J. & Kopple, K. D.. eds). pp. 125-128, Pierce Chemical Co: Rockford, IL.

Dyson, H. J. & Wright, P. E. (1991). Defining solution conformations of small linear peptides. Annu. Rev. Biophys. Biophys. Chem. 20, 519538.

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-II reverse turn. ivature (London), 318, 480483.

Dyson, H. J., Rance, M., Houghten. R. A., Lerner, R. A. & Wright, P. E. (1988a). Folding of immunogenic peptide fragments of proteins in water solution I. Sequence requirements for the formation of a reverse turn. J. Mol. Biol. 201, 161-200.

Dyson, H. ,J., Rance, M.; Houghten, R. A.; Wright, P. E. & Lerner, R. A. (19886). Folding of immunogenic peptide fragments of proteins in water solution. TT. The nascent helix. J. Mol. Biol. 201, 201-218.

Dyson, H. J., Lerner. R. A. & Wright. P. E. (1988c). The physical basis for induction of protein-reactive antipeptide antibodies. An,nu. Rev. Biophys. Biophys. Chem. 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. 78287837.

Dyson, H. J., Norrby, E.. Hoey, K., Parks, D. E.. Lerner. R. A. & Wright, P. E. (1992a). Immunogenic peptides corresponding to the dominant antigenic region Alas97 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., Merutka, G., Waltho, *J. P., Lerner, R. A. & Wright, P. E. (19925). Folding of peptide fragments comprising the complete sequence of proteins: models for initiation of protein folding. I. Myohemerythrin. .J. Nol. Biol. 226. 795-817.

Evans, P. A., Topping, K. D., Woolfson, D. N. & Dobson. C. M. (1991). Hydrophobic clustering in nonnative states of a protein: interpretation of chemical shifts in NMR spectra of denatured states of lysozyme. Proteins, 9, 248-266.

Goodman. E. M. & Kim, P. S. (1990). Tests of isolated P-sheet formation in a cyclic peptide from BPTI. In Current Research in Protein Chemistry (Villafranca. ,J. J., ed.), pp. 301-308, Academic Press, San Diego.

Gooley, P. R. & McKenzie, N. E. (1988). Location of an a-helix in fragment 96-133 from bovine somatotropin

by ‘H NMR spectroscopy. Biochemistry, 27. 40324040.

Grathwohl, C. 6 Wiithrich, K. (1976). The X-Pro peptide bond as an NMR probe for conformational studies of linear peptides. Biopolymers, 15, 2025-2041.

Guss, tJ. M. & Freeman, H. C. (19831. Structure of oxidized poplar plastocyanin at 1.6 A resolution. .I. Mol. Biol. 169, 521-563.

Hartman, R., Schwaner, R. C. & Hermans. ,J. (1974). Beta poly(q-lysine): a model system for biological self-assemblv. J. Mol. Biol. 90, 415--d9Q

Jeener, J., Meier, B. H., Bachmann. P. & Ernst. R. R. (1979). Investigation of exchange processes by two- dimensional NMR spectroscopy. J. Chem. Phys. 71, 454-553.

Lark, I,. R., Berzofsky, J. A. & Gierasch, L. M. (1989). T-cell antigenic paptides from sperm whale myoglobin fold as amphipathic helices: a possible determinant for immunodominance? Peptide Res. 2. 314-321.

Leist, T. 12: Thomas, R. M. (1984). Synthesis and physicochemical characterization of major fragments of human leucocyte interferon ~1,. Biochemistry, 23, 2541-2547.

Lim, V. I. (1974). Structural principles of the globular organization of protein chains. A stereochemical theory of globular protein secondary structure. J. Mol. Biol. 88, 857-872.

Lu, Z.-X., Fok. K.-F., Erickson, B. W. & Hugli, T. E. (1984). Conformational analysis of COOH-terminal segments of human C’Ra. .I. Hiol. (‘hpm. 259. 7367-7370.

Milne. P. K., Wells, tJ. R. E. & Ambler. K. 1’. (1974). The amino acid sequence of plastocyanin from French bean (Phaseolus vulgaris). Biochem. .J. 143. 691-701.

Miranker. A.. Radford, S. E., Karplus, MM. 8r Dobson, C. M. (1991). Demonstration by NMR of folding domains in lysozyme. Nature (London). 349. 633-636.

Moore, ,J. M., Case, D. A., Chazin, W. J., Gippert. G. P.. Have]. T. F., Powls, R. &. Wright. P. E. (1988). Three-dimensional solution structure of plastocyanin from the green alga Scenedesmus or’,liqwti.s. Science. 240, 314-317.

Moore, .J. 11.. LePre, C. A., (iippert, G. l’., (Ihazin. W’. *J.. Case. D. A. 8r Wright. I’. E. (1991). High resolution st,ructure of French bean plastocyanin and comparison wit,h the crystal structure of poplar plastocyanin. .I. Mol. Riol. 221. 5333555.

Muga, A., Surewicz, W. K., Wong, P. T. T., Mantsch. H. H.. Singh, V. K. & Shinohara. T. (1990). Structural studies with the uveopathogenic peptide M derived from retinal S-antigen. Biochemistry, 29.

2925-2930. Narita, M., Doi. M., Nakai, T. & Takegahara, H. (1988).

Synt,hesis and solubility properties of peptide fragments of human hemoglobin a-chain (123 136). lnt. ,I. Pppt. Prot. Res. 32. 200-207.

Nelson, ,I. W. bi Kallenbach. N. R’. (1989). Persistence of the a-helix stop signal in the S-peptidr in trifluoroethanol solutions. Bioch,em~istry 28. 5256-5261.

Nozaki. 1’. & Tanford, (‘. (1971). The solubility of amino acids and t,wo glycine peptides in aqueous ethanol and dioxane solutions. ,I. Bid. (‘hwn. 244. 2211-2'17.

Osterman, I). (:. & Kaiser. E:. T. (1985). Design and characterization of peptides wit,h amphiphilic /&strand structures. .I Cull. Biochem. 29, 57-72.

Otting. (:. \Vidmvr. H.. \Vagner. G:. B Wiithrich. K. (19%). Origin oft, and t2 ridges in 2D N!NR spectra and pt~w~~~*rtv for suppression. ./. Ncryrc Krson. 66. IX7 l!G.

Ranw. 11.. Swrnsrr~ 0. W. Kodenhausen. (i.. \Vagner. (i.. Ernst, R. It. K: \Viithrich. K. (1983). Improved spt~(~tr;tl wsolution in (‘OSY ‘H R’YlR spectra of protrins c+tr douhlr quantum filtering. Hiochrnt. Hioph~p. Krs. ~‘07//7,111% 117. 479sG3.i.

liodt~r. H.. Elii\r. (:. A. & lC:nglander. S. LV. (1988). Struc%ural c,h;rlac,terization of folding intermediatrs in cytjocahrorrrc, I’ by H-ruvhange labelling and proton SYI R. Snlurr, (Lorrdo~~). 335, 700-704.

Sikoraki. A. & Skolnick. ,J. (1990). Dynamic Monte Carlo simulations of globular protein folding/unfolding pat,hu-ayn. JI. r-Hrlica! motifs. .J. Mol. Hid. 215. I x:3-- 1 !M

Skolnic*k. .I. &, Kolinski. A. (1989). (‘omputer simulations (II’ globular l)rotein folding and trrtiar?, structurr. .-lnrr~r. Ifrr. I’h!ls. (‘hum 40. 207-235.

Sko1nic.k. .I. & Kolinski. A. (1990). Simulations of thv folding of a globular prot,rin. SciPncr. 250. I lPl-11%.

Sko1nic.k. ,I. & Kolinski. A. (1991). Dynamic Monte C’arlo simulations of’ a IWW lattirr model of globular Jjrotein folcling. strut+ urc and tlynanlics. .J. .Wol. Rio/. 221. 4!)!I--.x1 I

Struart. I). E.. Sarkar. A & \Vamplrr. .J. E. (1990). Oc~c~urrt~nct~ and role of cix peptidr bonds in protein ntr’uc+urtx .I .Ilo/. Hid. 214. ZX-P60.

Sykw. .\. (:. (l!M). Strrlcttlrr and rlrc+ron-transfer

wactivity of the blue copper prot,rin pIast,ocyanin.

(‘hrnr. Ah. Krr. 14. d83-315. Timashrff, S. Xv. (1970). Protein-solvent intrracstions and

protein c.onformation. Arc. C’hcnr. Krs. 3. t&CiH. I:dyaonkar. ,I. H. B Baldwin. R. L. (1988). SMR evidence

for an early framework intrrmediate on the folding pat,hway of ribonurlease A. S~t/rrr f/,o~~on). 335. W4-fiY!)

I:dgaonkar. ,I. K. k Hald\vin. R. I,. (19X8). Early folding intrrmrdiat,e of ribonuclease X. I’m Sfrf. .-Iwd. A?;.. I..S..-l. 87. xl!)7-8ml.

\‘anGert. A. 1,. (1968). Calibration of the methanol and gly~*ol nuc&ar magnetic resonance thermometers wit,h a static, thermistor probr. .-lntr/. C’hPm. 40. .)zyy -5->yc). IL I

\Valtho. .I. I’.. Frhtbr. Y. A.. Lrrnrr. R. A. & \Vright. I’. E. (19X9). (‘onformation of a T cell stimulating peptidr itr aqueous solution. FE&\’ Lrttrrs. 250. 1C)O-404.

Williamson. M. I’.. Hall. M. .J. & Handa. K. K. (19%) ‘H SMR assignment and swondary structuw of a Ilrrj/Ps .xin/j//rs virus glycoprotrin I)-1 antigetlic domain. /C//r. .I. Wiochrm. 158. 3”7-.Xti.

Wright. I’. E.. I)yson. H. J. bt Lrrnrr. 12. .\. (198X). (‘onformation of peptide fragments of protrins in ;lc~“twls solution: implications for initiation of i)r.otcin folding, J~ioch~rnistry, 27. 7167 7 I iT,.

\I’iithricah. K.. t<illrt,er. 11. Hi Hraun. 1V. (1984). I’olypvptide src.ondarg structurr drtrrnlination bj nuvlrar magiirtic resonance observation of short l’roton-J)rotoi, distances. .I. M21ol. Iliol. 180. 7 I ;,-7N.