Helical Conformations of Hexapeptides Containing N ...

Helical Conformations of Hexapeptides Containing N-Terminus DiprolineSegments

Kantharaju,1 Srinivasarao Raghothama,2 Subrayashastry Aravinda,3 Narayanaswamy Shamala,3

Padmanabhan Balaram1

1Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India

2NMR Research Centre, Indian Institute of Science, Bangalore 560012, India

3Department of Physics, Indian Institute of Science, Bangalore 560012, India

Received 24 September 2009; revised 28 November 2009; accepted 23 December 2009

Published online 27 January 2010 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.21395

This article was originally published online as an accepted

preprint. The ‘‘Published Online’’ date corresponds to the

preprint version. You can request a copy of the preprint by

emailing the Biopolymers editorial office at biopolymers@wiley.

com

INTRODUCTION

The conformational properties of proline residues in

peptides and proteins have been the subject of intense

study over the past four decades.1–6 The covalent

constraints imposed by pyrrolidine ring formation

limits backbone torsional freedom to Ramachandran

Helical Conformations of Hexapeptides Containing N-Terminus DiprolineSegments

ABSTRACT:

The role of N-terminus diproline segments in facilitating

helical folding in short peptides has been investigated in a

set of model hexapeptides of the type Piv-Xxx-Yyy-Aib-

Leu-Aib-Phe-OMe (Piv, pivaloyl). Nine sequences have

been investigated with the following N-terminus

dipeptide segments: DPro-Ala (4) and Pro-CPro (5, C,

pseudoproline), Ala-Ala (6), Ala-Pro (7), Pro-Ala (8),

Aib-Ala (9), Ala-Aib (10). The analog sequences Piv-

Pro-Pro-Ala-Leu-Aib-Phe-OMe (2) and Piv-Pro-Pro-

Ala-Aib-Ala-Aib-OMe (3) have also been studied. Solid

state conformations have been determined by X-ray

crystallography for peptides 4, 6, and 8 and compared

with the previously determined crystal structure of

peptide 1 (Boc-Pro-Pro-Aib-Leu-Aib-Val-OMe); (Rai et

al., JACS 2006, 128, 7916–7928). Peptides 1 and 6 adopt

almost identical helical conformations with unfolding of

the helix at the N-terminus Pro (1) residue. Peptide 4

reveals the anticipated DPro-Ala type II’ b-turn, followed

by a stretch of 310-helix. Peptide 8 adopts a folded

conformation stabilized by four successive 4?1

intramolecular hydrogen bonds. Ala (2) adopts an aL

conformation, resulting in a type II b-turn conformation

followed by a stretch of 310-helix. Conformational

properties in solution were probed using solvent

perturbation of NH chemical shifts which permit

delineation of hydrogen bonded NH groups and nuclear

Overhauser effects (NOEs) between backbone protons,

which are diagnostic of local residue conformations. The

results suggest that, continuous helical conformations are

indeed significantly populated for peptides 2 and 3.

Comparison of the results for peptides 1 and 2, suggest

that there is a significant influence of the residue that

follows diproline segments in influencing backbone

folding. # 2010 Wiley Periodicals, Inc. Biopolymers (Pept

Sci) 94: 360–370, 2010.

Keywords: proline peptides; diproline segments; crystal

structures; peptide helices; peptide conformations; NMR

of peptides

Correspondence to: Prof. N. Shamala, Department of Physics, Indian Institute of

Science, Bangalore 560 012, India; e-mail: [email protected] or Prof. P.

Balaram, Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560

012, India; e-mail: [email protected]

Contract grant sponsors: Council of Scientific and Industrial Research, India;

Department of Biotechnology, India

VVC 2010 Wiley Periodicals, Inc.

360 PeptideScience Volume 94 / Number 3

/ values of �6086 208 in L-Pro and +608 6 208 in D-Pro.7–9

Alkylation at the amino group also reduces the free energy dif-

ference between the cis and trans conformations about the am-

ide bond in proline peptides, with the Xxx-Pro bond having a

significantly greater propensity to adopt cis conformations (x¼ 08), as compared to peptide bonds between nonproline resi-

dues.10–14 The conformations of peptides containing Pro resi-

dues are specified by the value of x (1808 trans, 08 cis) and the

Ramachandran angles / and w. A vast body of crystal struc-

tures of proteins and peptides establishes three conformational

clusters for Pro residues corresponding to w & �308 (helical,

aR), +708 (C7, c-turn), and & +1208 (polyproline, PII) confor-

mations.7–9,15–17 Several analysis of the database of protein

crystal structures have led to the conclusion that proline resi-

dues facilitate b-turn conformations, preferentially occupying

the i+1 position.18–20 Proline residues are incompatible with

b-sheet conformations, interrupting cross-strand hydrogen

bonding patterns, although Pro residues are uncommon in b-

bulges.21,22 The amino acid Pro is sometimes described as a

‘‘helix breaker,’’ a characterization that is strictly inappropriate.

Indeed, Pro residues can be accommodated at internal posi-

tions in helices, with a resultant bend in the helix backbone.23–

29 In helices, hydrogen bond interruption appears to be of lim-

ited consequence. Interestingly, Pro residues have a high pro-

pensity to occur at the N-terminus end of helices.30–34 This is

a consequence of the fact that the dihedral angle / is con-

strained to the right value for a 310/a -helix and the amino ter-

minus residues (two residues for a 310-helix and three residues

for an a-helix) are not involved in hydrogen bonding. Dipro-

line (Pro-Pro) segments have therefore been suggested to be

potential nuclei for initiating helical folding in peptides.35

Covalently constrained diproline surrogates have been shown

by Kemp et al. to be effective templates for inducing helical

conformations in short acyclic sequences.36–40 In an earlier

study from this laboratory, we examined the conformational

preferences of an amino terminal diproline segment in

designed synthetic hexapeptides.41 The model peptide Piv-

Pro-Pro-Aib-Leu-Aib-Phe-OMe (1) was shown to adopt, in

crystals, a helical conformation over the segment residues 2–5,

with the N-terminal Pro(1) residue adopting a PII conforma-

tion. Solution NMR studies in a poorly interacting solvent like

CDCl3 suggested this partially unravelled helical conformation

was maintained in solution, although NOE evidence also sug-

gested an appreciable population of continuous helical struc-

tures. The impetus for the design of peptide 1 was the observa-

tion that the model sequence Piv-Pro-Pro-Ala-NHMe favored

an incipient 310-helical conformation in CDCl3, stabilized by

two successive intramolecular 4?1 hydrogen bonds.35,42 Our

experimental studies however suggest that in the longer pep-

tide sequence, the Pro(1) residue favored a semi-extended

(PII) conformation. To further probe the role of sequence

effects, we have systematically investigated nine related peptide

sequences in which the residues have been varied at positions

1, 2, and 3. The sequences reported are:

Piv-Pro-Pro-Aib-Leu-Aib-Phe-OMe (1)

Piv-Pro-Pro-Ala-Leu-Aib-Phe-OMe (2)

Piv-Pro-Pro-Ala-Aib-Ala-Aib-OMe (3)

Piv-DPro-Ala-Aib-Leu-Aib-Phe-OMe (4)

Piv-Pro-CPro-Aib-Leu-Aib-Phe-OMe (5) (CPro ¼pseudoproline)

Piv-Ala-Ala-Aib-Leu-Aib-Phe-OMe (6)

Piv-Ala-Pro-Aib-Leu-Aib-Phe-OMe (7)

Piv-Pro-Ala-Aib-Leu-Aib-Phe-OMe (8)

Piv-Aib-Ala-Aib-Leu-Aib-Phe-OMe (9)

Piv-Ala-Aib-Aib-Leu-Aib-Phe-OMe (10)

In all cases the segment corresponding to residues 3–5 has

a strong tendency to adopt helical conformations, because of

the presence of at least one or two helix promoting Aib resi-

dues.7,8 Peptide 4 provides an opportunity to establish the role

of the configuration of the N-terminal proline residue, while

peptide 5 was designed to restrict the peptide bond between

residues 1 and 2 to the cis conformation.43–48 The present

investigation, allows us to analyze the interplay between the

conformation directing influences of the N-terminal segment

and the C-terminal segment in a short peptide.

MATERIALS AND METHODS

Peptide SynthesisPeptides were synthesized by classical solution phase chemistry using a

racemization free fragment condensation strategy using the tert-butylox-

ycarbonyl (Boc) or pivaloyl (Piv) group and the methyl ester for

blocking, N- and C-termini, respectively. Peptide 5 Piv-Pro-CPro-Aib-

Leu-Aib-Phe-OMe was prepared by the fragment condensation of

Piv-Pro-CPro-OH and H2N-Aib-Leu-Aib-Phe-OMe. The dipeptide

Piv-Pro-CPro-OH was synthesized by the coupling of Piv-Pro-OH and

the L-2,2-dimethyl-1,3-thiazolidine-4-carboxylic acid (readily prepared

by refluxing the mixture of L-cysteine hydrochloride monohydrate (7 g,

0.04 mol), 100 mL of 2,2-dimethoxypropane and 500 mL of dry ace-

tone for about 2 h. after cooling to room temperature, the resulting

white plates were collected by simple filtration),49 by using a O-benzo-

triazole-N,N,N0,N0-tetramethyluronium-hexafluorophosphate (HBTU)/

HOBT and diisopropylethylamine (DIPEA) as coupling agents were

used. All the intermediates were characterized by ESI MS on Esquire

3000 Bruker Daltonics mass spectrometer. The target peptides were

purified using reverse-phase medium pressure liquid chromatography

(C18, 40–60 l) and high performance liquid chromatography (HPLC)

on a reversed phase C18 column (5–10 l, 7.8–250 mm) using metha-

nol/water gradients. The target peptides were characterized by mass

spectrometry and by complete assignment of 500 MHz 1H NMR

Helical Conformations of Hexapeptides 361

Biopolymers (Peptide Science)

spectra. ESI MS/(m/z): 2, 727.4 [M+H]+ (calcd: 726.4 Da); 749.3

[M+Na]+; 3, 623.5 [M+H]+ (calcd: 622.4 Da); 645.5 [M+Na]+; 4, 7,

and 8, 715.4 [M+H]+ (calcd: 714.2 Da); 737.4 [M+Na]+; 753.3

[M+K]+; 5, 787.4 [M+H]+ (calcd: 786.2 Da); 809.4 [M+Na]+; 825.3

[M+K]+; 6, 689.4 [M+H]+ (calcd: 688.3 Da); 711.4 [M+Na]+; 727.6

[M+K]+; 9 and 10, 703.5 [M+H]+ (calcd: 702.2 Da); 725.7 [M+Na]+;

741.3 [M+K]+.

NMR Spectroscopy. NMR spectra were recorded on Bruker

AV700 MHz and DRX500 spectrometers. The 1D and 2D spectra

were recorded at a peptide concentration of *5 mM in CDCl3 at

300 K. Delineation of exposed NH groups was achieved by titrating

CDCl3 solutions with low concentrations of DMSO-d6.

Residue-specific assignments were obtained from TOCSY experi-

ments, while ROESY spectra permitted sequence-specific assign-

ments. All 2D experiments were recorded in phase-sensitive mode

using the TPPI (time proportional phase incrementation) method.

A dataset of 1024 3 450 was used for acquiring the data. The same

dataset was zero-filled to yield a data matrix of size 2048 3 1024

before Fourier transformation. A spectral width of 6000 and 8700

Hz was used in both dimensions at 500 and 700 MHz, respectively.

Mixing times of 100 and 200 ms were used for TOCSY and ROESY,

respectively. Shifted square sine bell windows were used while proc-

essing. All processing was done using BRUKER TOPSPIN software.

X-Ray Diffraction. Crystals of peptides 4, 6, and 8 were grown

by slow evaporation of methanol/water. The X-ray diffraction data

were collected on a Bruker AXS SMART APEX CCD diffractometer

using MoKa radiation. The crystal structures were solved by direct

methods using SHELXS-97.50 The structures were refined isotropi-

cally followed by full matrix anisotropic least-squares refinement

using SHELXL-97.51 The solvent molecules in peptide 6 were

located from a difference Fourier map. All the hydrogen atoms were

fixed geometrically in idealized positions and allowed to ride with

the C or N atom to which each was bonded, in the final cycle of

Table I Crystal and Diffraction Parameters of Peptides 4, 6, and 8 (Piv-Xxx-Yyy-Aib-Leu-Aib-Phe-OMe)

Peptide 4 Peptide 6 Peptide 8

Xxx ¼ DPro; Yyy ¼ Ala Xxx ¼ Yyy ¼ Ala Xxx ¼ Pro; Yyy ¼ Ala

Empirical formula C37 H58 N6 O8 C35 H56 N6 O8 n 3H2O C37 H58 N6 O8

Crystal habit Clear, transparent Clear, transparent Clear, transparent

Crystal size (mm) 0.33 3 0.16 3 0.11 0.51 3 0.24 3 0.11 0.56 3 0.11 3 0.10

Crystallizing solvent Methanol/water Methanol/water Methanol/water

Space group P21 P1 P212121

Cell parameters

a (A) 9.243(3) 9.613(1) 11.078(1)

b (A) 20.320(6) 10.916(1) 11.406(1)

c (A) 10.839(3) 11.125(1) 32.43(4)

a (deg) — 78.5(2) —

b (deg) 90 76.1(2) —

c (deg) — 74.6(2) —

Volume (A3) 2035.7(10) 1080.7(2) 4291.0(6)

Z 2 1 4

Molecules/asym.unit 1 1 1

Cocrystallized solvent None three water molecules None

Molecular weight 714.89 742.91 714.89

Density (g cm�3)(cal) 1.166 1.141 1.159

F (000) 772 402 1544

Radiation MoKa; (k ¼ 0.71073 A) MoKa; (k ¼ 0.71073 A) MoKa; (k ¼ 0.71073 A)

Temperature (8C) 21 21 21

h range (8) 1.88–26.52 1.91–26.86 1.26–26.84

Scan type x – xMeasured reflections 19,785 10,950 30,926

Unique reflections 4042 4182 4663

Observed reflections [|F|>4r(F)] 3520 4046 4053

Final R (%) 4.85 3.88 5.34

Final wR2 (%) 13.19 11.05 13.02

Goodness-of-fit (S) 1.036 0.989 1.218

Dqmax (e A�3

) 0.52 0.29 0.37

Dqmin (e A�3

) �0.22 �0.18 �0.15

No. of restraints/parameters 1/460 3/493 0/460

Data-to-parameter ratio 7.6 : 1 8.2 : 1 8.8 : 1

362 Kantharaju et al.


refinement. The water hydrogen atoms in 6 were located from a dif-

ference Fourier map. The final R factors were 4.85, 3.88, and 5.34%

for peptides 4, 6, and 8, respectively. The crystal and diffraction

parameters for peptides 4, 6, and 8 are summarized in Table I. The

crystallographic coordinates for the structures are deposited at the

Cambridge Crystallographic Data Centre with deposition numbers

CCDC 748694 (4), CCDC 748693 (6), and CCDC 748695 (8). These

data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/

retrieving.html (or from the Cambridge Crystallographic Data

Centre, 12 Union Road, Cambridge CB21EZ, UK; fax: (+44) 1223-

336-033; or e-mail: [email protected]).

RESULTS AND DISCUSSIONThe purpose of the present study was to establish the molec-

ular conformations of peptides 2–10 in solution and in the

solid state. Specifically, our intention was to establish the

number of intramolecular hydrogen bonds formed in poorly

solvating media like CDCl3, with the hope of establishing the

presence of continuous helical conformations incorporating

the N-terminal segment in a helix promoting turn. In spite

of considerable effort diffraction quality crystals were

obtained only for peptides 4, 6, and 8, while structure 1 has

been previously reported.41

Solid State Conformations

Figure 1 compares the molecular conformation determined

for peptides 1, 4, 6, and 8. The backbone torsion angles for

peptides 4, 6, and 8 are summarized in Table II. For compari-

son the torsion angle of peptide 1 is also given in the Table

II. The superposition of the structures of peptides 1 (Pro-

Pro) and 6 (Ala-Ala) reveals an almost identical backbone

conformation, with an RMSD value of 0.16 A. The confor-

mation of peptide 6 reveals a PII conformation for the Ala(1)

residue, followed by a 310/a-helix formed by the segment,

residues 2–5. Three intramolecular hydrogen bonds are pres-

ent. The Ala(1)CO:::HNLeu(4) interaction is of the 4?1

type (310-helix), while the Ala(1)CO:::HNAib(5) and Ala(2)-

CO:::HNPhe(6) interactions are of the 5?1 type (a-helix).

Interestingly, in both peptides 1 and 6 formation of a contin-

uous helix is not observed, with partial unraveling due to the

PII conformation of the N-terminus residue. The possibility

that intermolecular interactions in the solid state may con-

tribute to this feature merits consideration.

Peptide 4 (DPro-Ala) adopts a solid state conformation in

which four successive 4?1 intramolecular hydrogen bonds

are formed. The DPro(1)-Ala(2) segments form the antici-

pated type II’ b-turn structure placing an LAla(2) residue in

the aR region of /, w space. The segment residue 2–5 adopts

a right handed 310-helical conformation.

Peptide 8 (Pro-Ala) forms a folded conformation stabi-

lized by a four successive 4?1 intramolecular hydrogen

bonds. The Pro(1)-Ala(2) segment adopts a type II b-turn

conformation, with the LAla residue exhibiting an unusual aL

conformation (positive values of the Ramachandran angles

/, w). The 4?1 hydrogen bond with Piv(0)CO:::HNAib(3)

FIGURE 1 Molecular conformation in crystals of peptides 1, 4, 6, and 8. All the hydrogen bonds

are shown as dotted lines.



is characterized by a relatively long N:::O distance of 3.35 A.

The segment residues 2–5 forms a left handed 310-helix, with

both LAla(2) and L Leu(4) lying in the aL region of the Rama-

chandran map. For L-residues an energy difference of *1

kcal mol�1 has been estimated between the aR and aL confor-

mations, with the left handed structure being energetically

less stable.52,53 While L-residues occur relatively infrequently

in the aL region in the crystal structures of peptides and pro-

teins,54 there are examples of aL conformations previously

reported in peptide helices.55–57 Nevertheless, the observed

conformation of peptide 8 was surprising, since all four chi-

ral residues in the hexapeptide have the L-configuration.

The crystal structures of peptides 1 (Pro-Pro), 6 (Ala-

Ala), and 8 (Pro-Ala) do not reveal type I/III, 310-helical

turns for the amino terminus dipeptide segments. This is

indeed a conformation (/ ¼ �60.08, w ¼ �30.08), which

may have been populated in peptides 6 and 8 and was antici-

pated in peptide 1. Somewhat surprisingly, peptide 8 adopted

an opposite screw sense for the helical segment spanning res-

idues 2–5. In the case of short peptides, which can populate

several energetically accessible conformations, crystal struc-

tures often provide a definitive characterization of one of the

multiple conformational states. Exceptions, of course, are

cases where polymorphs are formed trapping molecules in

distinct structures. To further probe conformational propen-

sities for peptides 2–10, we turn to 1H NMR studies in

CDCl3 solutions.

Conformations in Solution

The choice of a noninteracting solvent like CDCl3 was

intended to avoid solvent competition for hydrogen bonding

sites, thereby promoting formation of folded conformations.

Resonance assignments were readily achieved by using a

combination of TOCSY and ROESY experiments. Figure 2

schematically summarizes the observed dispersion of NH

Table II Torsion Angles (Deg) in Piv-Xxx-Yyy-Aib-Leu-Aib-Phe-OMe

Residues Peptide 1 Peptide 4 Peptide 6 Peptide 8

Xxx (1) Xxx ¼ Pro Xxx ¼ DPro Xxx ¼ Ala Xxx ¼ Pro

/ �62.4 (�63.9) 56.6 �56.8 �63.4

w 160.2 (160.7) �136.6 149.7 135.2

x �177.7 (�178.3) �171.1 175.8 172.8

Yyy (2) Yyy ¼ Pro Yyy ¼ Ala Yyy ¼ Ala Yyy ¼ Ala

/ �50.7 (�53.3) �64.4 �54.3 59.9

w �42.1 (�42.4) �24.6 �39.6 16.6

x �178.2 (�178.5) �178.0 �177.3 �169.8

Aib (3)

/ �55.8 (�54.8) �57.6 �57.1 50.1

w �31.8 (�38.1) �27.1 �30.3 31.4

x 179.2 (�177.3) �176.3 177.1 �178.9

Leu (4)

/ �94.6 (�86.6) �65.8 �87.2 51.4

w �41.3 (�42.9) �13.8 �51.8 29.4

x �178.5 (�179.5) 172.8 �172.5 �178.9

v1 �64.5 (�76.8) �60.8 �71.7 �58.8

v2 169.6, �68.6 (172.7, �69.3) �61.4, 176.3 �75.7, 161.5 �49.4, �175.8

Aib (5)

/ �58.6 (�60.3) �58.0 �61.4 55.4

w �42.8 (�42.1) �27.4 �38.4 34.9

x �175.2 (�171.7) �178.1 �173.4 170.4

Phe (6)

/ �106.7 (�114.2) �98.1 �109.4 �53.3

w �5.7 (31.0) �74.8 �38.9 138.5

x 178.6 (�176.1) �175.5 �177.0 169.8

v1 �56.4 (�62.3) �79.5 �70.6 170.0

v2 �66.2, 109.8 (�62.3, 117.5) 16.3, �161.9 �91.8, 86.9 69.2, �109.6

The hydrogen bond parameters in 4: N(3):::O(0) ¼ 3.212A; ffN-H:::O ¼ 142.58; N(4):::O(1) ¼ 3.217A; ffN-H:::O ¼ 162.18; N(5):::O(2) ¼ 3.105A; ffN-

H:::O ¼ 165.48; N(6):::O(3) ¼ 3.097A; ffN-H:::O ¼ 164.38; in 6: N(4):::O(1) ¼ 2.960A; ffN-H:::O ¼ 140.48; N(5):::O(1) ¼ 3.014A; ffN-H:::O ¼ 174.08;N(6):::O(2) ¼ 2.886A; ffN-H:::O ¼ 160.88; and in 8: N(3):::O(0) ¼ 3.346A; ffN-H:::O ¼ 152.88; N(4):::O(1) ¼ 2.886A; ffN-H:::O ¼ 165.28; N(5):::O(2) ¼2.941A; ffN-H:::O ¼ 161.38; N(6):::O(3) ¼ 2.986A; ffN-H:::O ¼ 163.28.



resonances in peptides 1–10. The involvement of an NH

group in intramolecular hydrogen bonds was probed by

monitoring changes in chemical shifts of NH resonances

upon addition of (CD3)2SO, to solutions of peptides in

CDCl3. Ideally, in a solvent titration experiment, solvent

exposed NH groups show pronounced downfield shifts with

increasing DMSO-d6 concentration, while intramolecularly

hydrogen bonded NH groups are shielded from the solvent

medium and therefore exhibit limited solvent dependence of

chemical shifts. Figure 3 presents solvent titration curves

obtained for peptides 2–10. The degree of solvent sensitivity

of NH chemical shifts is compared for the entire peptide se-

ries in Figure 4. The parameter Dd represents the difference

in chemical shifts between solutions in CDCl3 and solvent

mixtures containing 20–30% (v/v) DMSO-d6. Inspection of

Figure 4 reveals that solvent exposed NH groups have down-

field shifts of the order of 1–2 ppm, while shielded NH

groups exhibit Dd values <0.4 ppm. Intermediate values

between 0.4 and 0.8 ppm are observed for a few NH groups.

Figure 5 provides a correlation of the observed NH chemical

shifts and Dd values for residue 3 (Aib/Ala) in peptides 1–10.

If residues 1 and 2 adopt conformations compatible with a

4?1 hydrogen-bonded turn, residue 3 NH would be

expected to be involved in an intramolecular interaction. Fig-

ure 5 clearly reveals two outliers, peptides 1 (Pro-Pro) and 7

(Ala-Pro). In both these cases, residue 3 NH is undoubtedly

solvent exposed, precluding formation of a turn at the N-ter-

minus. This observation is consistent with the observed solid

state conformation of peptide 1. In contrast, peptide 6 (Ala-

Ala) is characterized by a very low Dd value for Aib(3) NH

suggesting that a significant population of a continuous heli-

cal conformations are indeed present in solution. The NMR

data points to the possibility that in the short sequence mul-

tiple conformations may be present in solution, with the

crystal structure providing a view of only one of the energeti-

cally accessible conformations. Comparison of the distribu-

tion of Dd values in Figure 4 provides an initial estimate of

the number and identity of the solvent-shielded (intramolec-

ularly hydrogen bonded) NH groups in peptides 1–10. We

first consider the data for peptides 4 and 5, which are not

expected to form continuous right handed 310/a-helical

structures. In peptide 4 the presence of the DPro residue at

position 1 locks the N-terminus segment DPro-Ala, into a

type II’ conformation, facilitating right handed helix forma-

tion for the segments 2–5. This is a consequence of the

requirement for the i+2 residue [Ala(2)] in a type II’ b-turn

to adopt an aR conformation. Indeed, the observed Dd values

strongly support of the presence of four intramolecular

hydrogen bonds, with Aib(3), Leu(4), Aib(5), and Phe(6)

showing extremely low Dd values. This, four hydrogen

bonded, structure is an agreement with the results from X-

ray diffraction, which reveals a type II’ b-turn followed by a

right handed 310-helical structure (see Figure 1). Peptide 5

contains the pseudoproline (L-2,2-dimethyl-1,3-thiazolidine-

4-carboxylic acid) residue at position 2. The presence of a

gem-dimethyl group at the Cd atom of the thiazolidine ring

makes the Pro-CPro cis imide configuration energetically

favorable relative to the trans form.45 The cis geometry of the

Pro-CPro peptide bond is conclusively established by the ob-

servation of the strong NOE (daa) between Pro(1) CaH and

CPro(2) CaH protons (see Figure 6). The extremely low Ddvalue obtained for Aib(3) NH is consistent with the forma-

tion of a 4?1 hydrogen bond type VI b-turn20 with Pro(1)

[/ ¼ �60.08, w ¼ 120.08] and CPro(2) [/ ¼ �90.08, w ¼0.08] at the i+1 and i+2 positions, respectively (see Figure 6).

FIGURE 2 Schematic representation of NH chemical shifts of

peptides 1–10.



FIGURE 3 Plot of NH chemical shifts (ppm) in peptides 2–10 as a function of DMSO-d6 con-

centration in CDCl3 solution.

FIGURE 4 Distribution of Dd (ppm) resonances in peptides 1–

10. Structure and hydrogen bonds are shown for crystallographically

characterized examples.

FIGURE 5 Plot of Dd (ppm) vs. NH chemical shifts (ppm) for

residue 3 (Aib/Ala) in peptides 1–10.



The relatively high Dd value observed for Aib(5) NH is

clearly indicative of pronounced solvent exposure, indicative

of disruption of the helix over residues 2–5.

The data in Figure 4 reveals that, peptide 3 (Piv-Pro-Pro-

Ala-Aib-Ala-Aib-OMe) appears to be clear case where the NH

groups of residues 3–6 are solvent-shielded, supporting con-

tinuous helical conformations over the entire length of the

peptide. It is important to note that the closely related peptide

2 also exhibits relatively low Dd values for these four NH

resonances. Peptide 9 (Piv-Aib-Ala-Aib-Leu-Aib-Phe-OMe)

was designed as a ‘‘control’’ sequence, lacking the N-terminus

Pro residues. As anticipated, NH resonances of residues 3–6 in

peptide 9 exhibit very low Dd values, consistent with continu-

ous 310-helical conformations over the entire length, residues

1–6. Switching the two residues at the N-terminus in 9 yields

peptide 10, which has an appreciably larger Dd value for

Aib(3) NH, suggestive of partial unfolding at the N-terminus.

It is noteworthy that peptide 7 (Ala-Pro) yields the largest Ddvalue for Aib(3) NH, suggesting an almost complete absence

of contributions from helical turns at the N-terminus.

The present study was initiated on the premise that the N-

terminus diproline segment may facilitate helical folding in

short peptides. This expectation was based on the two hydro-

gen bonded, incipient 310-helical conformation proposed for

the model peptide Piv-Pro-Pro-Ala-NHMe in solution.35

The results presented above, on hexapeptides containing a

Pro-Pro segment at the N-terminus, suggest that the nature

of the residue following the diproline segment may have a

significant effect on conformational preferences. The

observed differences, between peptide 1 on the one hand and

peptides 2 and 3 on the other, suggest that the Ala residue at

position 3 appears superior to an Aib residue in promoting

310-helical folding at the N-terminus. A similar effect was

noted in the model tripeptides Piv-Pro-Pro-Xxx-NHMe

where the peptide Xxx ¼ LAla showed a significantly greater

tendency to favor incipient 310-helical conformations.42 This

was unanticipated since Aib residues have been well estab-

lished as strong promoters of helical conformations, with a

distinct propensity to favor 310-helical structures.58,59 This

interpretation is based on the differences in the degree of

FIGURE 6 Partial 500 MHz ROESY spectra of peptide 5 in CDCl3, showing the CaH$CaH,

CaH$NH and NH$NH Regions.



solvent exposure of the residue 3 NH in peptide 1 as com-

pared to peptides 2 and 3 (Figures 4 and 5). To further exam-

ine conformational choices in peptides 1–10, we turn to a

comparison of nuclear Overhauser effects (NOEs) between

peptide backbone protons.

For amino acid residues adopting conformations in the

helical region of /, w space, the distances between the NH

protons of the preceding and succeeding peptide units

(dNNi,i+1) are short (2.8 A)60 resulting in the observation of

relatively intense NOEs. The observation of successive

dNNi,i+1 NOEs along the polypeptide sequence, is diagnostic

of the formation of a helical segment, especially when the

presence of solvent-shielded NH groups is established.

Figure 7 compares the NOEs between backbone protons

(CaH, NH) observed in peptide 2 (Piv-Pro-Pro-Ala-Leu-Aib-

Phe-OMe), peptide 3 (Piv-Pro-Pro-Ala-Aib-Ala-Aib-OMe),

and peptide 4 (Piv-DPro-Ala-Aib-Leu-Aib-Phe-OMe). In pep-

tide 2, which contains the N-terminus diproline segment, suc-

cessive dNNi,i+1 NOEs are observed over the segment residues

3–6. In addition, the CaH$NH NOEs, daN (1/3, 2/5, and 3/5)

are also diagnostic of a significant population of a folded heli-

cal conformations over the entire length of the peptide.

Peptide 3 which differs from 2 only in the nature of the

C-terminus residue 6 (Aib instead of Phe) provides further

evidence for a continuous helix in short peptide containing

N-terminus diproline segments. Successive dNNi,i+1 and

daNi,i+2 and daNi,i+3 are observed, strongly supporting a sig-

nificant population of folded helical structures. These NOE

results are completely consistent with the earlier observations

derived from Figures 4 and 5 that, the NH group of residues

3–6 are strongly solvent-shielded, indicating their involve-

ment in intramolecular hydrogen bonds. Peptide 4 differs

from peptide 8 at the N-terminus Pro residue (DPro in 4 and

Pro in 8). The partial ROESY spectrum of peptide 4 reveals

NOEs of daNi,i+1, dNNi,i+1 and long range NOEs daN (1/3, 2/4,

and 4/6) (see Figure 7). Further, the solvent titration shows

that the residue Ala(2) is solvent-exposed and residues 3–6

are strongly solvent-shielded (see Figure 3), supporting a

folded helical conformation. The NOEs obtained between

backbone protons for peptides 1, 6, 7, and 8 are presented in

Figure 8 in a manner that permits comparison of the relative

intensities of the daNi,i+2 and daNi,i+3 NOEs, which are diag-

nostic of helical conformations. Inspection of Figure 8 shows

the complete absence of NOEs between Ala(1) CaH and

Aib(3) NH or Leu(4) NH resonances in peptide 7(Piv-Ala-

Pro-Aib-Leu-Aib-Phe-OMe). Indeed, peptide 7 shows the

largest Dd value for Aib(3) NH, which has been earlier inter-

preted to indicate largely unfolded conformers at the N-ter-

minus. An interesting feature of the spectrum of peptide 8 is

the relatively broad resonances observed for Ala(2) CaH and

NH protons and the extremely weak intraresidue (daNi,i)

NOEs between these protons. This feature is suggestive of a

conformational exchange process involving residue 2. Inter-

estingly, the crystal structure of peptide 8 (see Figure 1),

reveals an unanticipated left handed helical (aL) conforma-

tion at the Ala(2) residue. It is indeed, conceivable that

exchange processes that interconvert multiple conforma-

tional states lead to observed line broadening.

FIGURE 7 Partial 500 MHz ROESY spectra of peptides 2 (right), 3 (middle), and 4 (left), show-

ing the CaH$NH and NH$NH regions.



CONCLUSIONSThe results presented above suggest, the short peptide-con-

taining N-terminus diproline segment can indeed adopt con-

tinuous helical conformations with the nature of residue 3

having a significant influence on the helical populations. The

Ala residue appears to strongly support helical folding in N-

terminus Pro-Pro-Xxx sequences. The observation that Aib at

position 3 appears to be less effective than Ala in stabilizing

helical conformations at the diproline segment was unantici-

pated. The origin of this effect is not readily apparent. The

crystal structure observation of a left handed helix promoted

by an N-terminus type II turn in peptide Pro-Ala(4) was also

unanticipated. The results of the present study emphasize the

role of certain sequence effects in determining the conforma-

tional property of an N-terminus diproline segment. The role

of specific peptide segments in the nucleation and stabilization

of folded structures merits further consideration.

S. Aravinda thanks the Council of Scientific and Industrial Research

for a Research Associateship.

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