Chain Length Effects on Helix-Hairpin Distribution in Short Peptides withAib-DAla and Aib-Aib Segments

Appavu Rajagopal,1 Subrayashastry Aravinda,2 Srinivasarao Raghothama,3

Narayanaswamy Shamala,2 Padmanabhan Balaram1

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

2Department of Physics, Indian Institute of Science, Bangalore 560012, India

3NMR Research Centre, Indian Institute of Science, Bangalore 560012, India

Received 23 November 2010; revised 14 February 2011; accepted 18 February 2011

Published online 7 March 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.21613

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

Hydrogen bonds between backbone donor (NH) and

acceptor (CO) groups are important stabilizing

interactions in peptide and protein structures.1,2

The b-turn, in which 10 atoms form a hydrogen

bonded ring, is the one of the best characterized

ABSTRACT:

The Aib-DAla dipeptide segment has a tendency to form

both type-I0/III0 and type-I/III b-turns. The occurrence of

prime turns facilitates the formation of b-hairpin

conformations, while type-I/III turns can nucleate helix

formation. The octapeptide Boc-Leu-Phe-Val-Aib-DAla-

Leu-Phe-Val-OMe (1) has been previously shown to form

a b-hairpin in the crystalline state and in solution. The

effects of sequence truncation have been examined using

the model peptides Boc-Phe-Val-Aib-Xxx-Leu-Phe-

NHMe (2, 6), Boc-Val-Aib-Xxx-Leu-NHMe (3, 7), and

Boc-Aib-Xxx-NHMe (4, 8), where Xxx ¼ DAla, Aib. For

peptides with central Aib-Aib segments, Boc-Phe-Val-

Aib-Aib-Leu-Phe-NHMe (6), Boc-Val-Aib-Aib-Leu-

NHMe (7), and Boc-Aib-Aib-NHMe (8) helical

conformations have been established by NMR studies in

both hydrogen bonding (CD3OH) and non-hydrogen

bonding (CDCl3) solvents. In contrast, the corresponding

hexapeptide Boc-Phe-Val-Aib-DAla-Leu-Phe-Val-NHMe

(2) favors helical conformations in CDCl3 and b-hairpin

conformations in CD3OH. The b-turn conformations

(type-I0/III) stabilized by intramolecular 4?1 hydrogen

bonds are observed for the peptide Boc-Aib-DAla-NHMe

(4) and Boc-Aib-Aib-NHMe (8) in crystals. The

tetrapeptide Boc-Val-Aib-Aib-Leu-NHMe (7) adopts an

incipient 310-helical conformation stabilized by three

4?1 hydrogen bonds. The peptide Boc-Val-Aib-DAla-

Leu-NHMe (3) adopts a novel a-turn conformation,

stabilized by three intramolecular hydrogen bonds (two

4?1 and one 5?1). The Aib-DAla segment adopts a

type-I0 b-turn conformation. The observation of an NOE

between Val (1) NH$HNCH3 (5) in CD3OH suggests,

that the solid state conformation is maintained in

methanol solutions. # 2011 Wiley Periodicals, Inc.

Biopolymers (Pept Sci) 96: 744-756, 2011.

Keywords: type-I0-turn; helix-b-hairpin transitions;peptide conformation; hydrogen bonding; nuclearOverhauser effects; a-turns

Chain Length Effects on Helix-Hairpin Distribution in Short Peptideswith Aib-DAla and Aib-Aib Segments

Correspondence to: Narayanaswamy Shamala; e-mail: shamala@physics.iisc.ernet.in

(and) Padmanabhan Balaram; e-mail: pb@mbu.iisc.ernet.in

Contract grant sponsor: Department of Biotechnology, India, in the area of

Molecular Diversity and Design

VVC 2011 Wiley Periodicals, Inc.

744 PeptideScience Volume 96 / Number 6

elements in polypeptide structures.3–6 This conformational

feature is determined by the backbone torsion angles (/, w)of two residues (i and i + 1) in peptides.7,8 The type I/III b-turn, which is formed when both residues i and i + 1 lie in

the right handed helical (aR) region of Ramachandran space,

is the conformational element which, when repeated, leads to

the formation of a 310-helical segment in a polypeptide

chain. For example, the repetitive type-III consecutive b-turnstructure corresponds to a single turn of a 310-helix.

9–11 The

type-I0/III0 (prime) turn is formed when residues i/i + 1

adopt left handed helical (aL) conformations in which both

/ and w have positive values. This prime turn nucleates b-hairpin formation when placed centrally in a polypeptide

segment consisting of L-Residues.12,13 Conformationally con-

strained residues have been developed as a strategy to con-

struct obligatory turn segments of both type-I/III and type-

I0/III0 categories. In particular, Aib-LXxx segments may be

used to preferentially populate local type-I/III conforma-

tions, while Aib-DXxx segments show a preference for type-I0/III0 b-turn structures.14 The formation of right handed 310/a-helices in peptides containing Aib-LXxx sequence has been

widely demonstrated.15–20 The b-hairpin structure of an oc-

tapeptide containing a central Aib-DAla type-I0 b-turn has

been characterized in crystals by X-ray diffraction.21 More

recently, the obligatory type-I0 b-turn template, Aib-DPro has

permitted crystallographic characterization of the b-hairpinconformation in the octapeptide, Boc-Leu-Val-Val-Aib-DPro-

Leu-Val-Val-OMe.22 The achiral dipeptide segment Aib-Gly

has also been used to stabilize b-hairpin folding, with several

model sequences investigated by isotope-edited IR spectros-

copy.23,24 An earlier study from this laboratory addressed the

conformation directing effect of the Xxx residue in the octa-

peptide Boc-Leu-Phe-Val-Aib-Xxx-Leu-Phe-Val-OMe. For

Xxx ¼ DAla, the b-hairpin conformation appears strongly

favored in hydrogen bonding solvents like CD3OH and

DMSO-d6, whereas in poorly interacting solvents like CDCl3,

NMR evidence favors a mixture of a-helical and b-hairpinconformations. For Xxx ¼ Aib, helical conformations are

exclusively observed in CDCl3, while in CD3OH and DMSO-

d6 the NMR data supports a mixed population of a-helicesand b-hairpins.25 Evidence for similar conformational equili-

bria was also obtained for the octapeptide Boc-Leu-Val-Val-

Aib-Gly-Leu-Val-Val-OMe26 (see Table I). For an eight resi-

due protected peptide, formation of a 310-helix results in six

intramolecular (C10) hydrogen bonds, while an a-helix yieldsfive intramolecular (C13) hydrogen bonds. b-Hairpin results

in four cross-strand hydrogen bonds. Thus, in solvents like

CDCl3 which do not compete for backbone hydrogen bond-

ing sites, helix formation is likely to be favored, while b-hair-pins may be promoted in solvents like CD3OH, which can

Table I Designed Peptide Sequences

No. Peptide Sequence Observed Conformations References

1 Boc-Leu-Phe-Val-Aib-DAla-Leu-Phe-Val-OMe (1) NMR: Helix in CDCl3; b-hairpin in CD3OH and DMSO-d6 25

X-ray: b-hairpin, Type I0 b-turn 21

2 Boc-Phe-Val-Aib-DAla-Leu-Phe-NHMe (2) NMR: Helix in CDCl3; Helix + b-hairpin in CD3OH Present study

3 Boc-Val-Aib-DAla-Leu-NHMe (3) NMR: Helix in CDCl3; b-hairpin in CD3OH Present study

X-ray: a-turn4 Boc-Aib-DAla-NHMe (4) X-ray: Type-I0 b-turn Present study

5 Boc-Leu-Phe-Val-Aib-Aib-Leu-Phe-Val-OMe (5) NMR: Helix in CDCl3; Helix + b-hairpin in

CD3OH and DMSO-d6

25

6 Boc-Phe-Val-Aib-Aib-Leu-Phe-NHMe (6) NMR: Helix in CDCl3 and CD3OH Present study

7 Boc-Val-Aib-Aib-Leu-NHMe (7) NMR: Helix in CDCl3 and CD3OH Present study

X-ray: 310-helix

8 Boc-Aib-Aib-NHMe (8) X-ray: Type-III b-turn Present study

9 Boc-Leu-Val-Val-Aib-Gly-Leu-Val-Val-OMe (9) NMR: Helix in CDCl3 and CD3CN; b-hairpin in

CD3OH and DMSO-d6

26

10 Boc-Leu-Val-Val-DPro-LPro-Leu-Val-Val-OMe (10) NMR: b-hairpin, Type-II0 b-turn 25

11 Boc-Leu-Phe-Val-DPro-LPro-Leu-Phe-Val-OMe (11) NMR: b-hairpin, Type-II0 b-turn 25

X-ray: b-hairpin, Type-II0 b-turn12 Boc-Leu-Val-Val-DPro-Aib-Leu-Val-Val-OMe (12) X-ray: b-hairpin, Type-II0 b-turn 25

13 Boc-Leu-Val-Val-Aib-DAla-Leu-Val-Val-OMe (13) NMR: Helix in CDCl3; b-hairpin in CD3OH 25

14 Boc-Leu-Val-Val-Aib-DVal-Leu-Val-Val-OMe (14) NMR: b-hairpin in CDCl3 and CD3OH 25

15 Boc-Leu-Phe-Val-Aib-Gly-Leu-Phe-Val-OMe (15) NMR: Helix in CDCl3; b-hairpin in CD3OH 25

16 Boc-Leu-Val-Val-Aib-DPro-Leu-Val-Val-OMe (16) NMR: b-hairpin in CDCl3, CD3OH and DMSO-d6 25

X-ray: b-hairpin, Type-I’ b-turn 22

17 Boc-Leu-Phe-Val-Aib-Ala-Leu-Phe-Val-OMe (17) NMR: Helix in CDCl3 and DMSO-d6 25

Chain Length Effects on Helix-Hairpin Distribution 745

Biopolymers (Peptide Science)

provide interacting partners for exposed donor and acceptor

groups that project outwards, in extended strands (see Figure

1). In the case of shorter four residue peptide sequences, both

incipient 310-helical and b-hairpin conformations may be

present. Such sequences may be of relevance in studies of con-

formational dynamics of peptide by NMR methods.27 To

explore the effect of peptide chain length on conformational

properties, determined by central segments which can

undergo type-III to I0 transitions, we have carried out studies

on truncated peptides of the parent sequences Boc-Leu-Phe-

Val-Aib-Xxx-Leu-Phe-Val-OMe, Xxx ¼ DAla (1) and Aib.(5)

The choice of the parent peptide with the aromatic Phe resi-

dues at positions 2 and 7 was made in order to address the

possibility of weak aromatic interactions contributing to hair-

pin stability.28 We describe in this report, conformational

studies in solution for the truncated peptide sequences, Boc-

Phe-Val-Aib-DAla-Leu-Phe-NHMe (2), Boc-Val-Aib-DAla-

Leu-NHMe (3), Boc-Phe-Val-Aib-Aib-Leu-Phe-NHMe (6),

and Boc-Val-Aib-Aib-Leu-NHMe (7). In addition, we report

the crystal structure of turn segments, Boc-Aib-DAla-NHMe

(4), Boc-Aib-Aib-NHMe (8) and the truncated tetrapeptide

models Boc-Val-Aib-DAla-Leu-NHMe (3) and Boc-Val-Aib-

Aib-Leu-NHMe (7).

MATERIALS AND METHODS

Peptide SynthesisPeptides 1 and 5 were synthesized as previously reported.22 Peptides

2, 3, 4, 6, 7, and 8 were prepared by standard solution phase meth-

ods. The t-butyloxycarbonyl (Boc) and methyl ester were used for

N- and C-terminal protection, respectively. Peptide bond formation

was achieved by using N,N0-dicyclohexylcarbodiimide (DCC) and

1-hydroxybenzotriazole (HOBt). The conversion of C-terminal

methyl esters to N-methyl amides was carried out by saturating pep-

tide ester solutions in dry tetrahydrofuran (THF) with methylamine

gas. Racemization was minimized by condensing Boc protected

amino acids to the growing C-terminus. The crude peptide products

obtained after standard work-up procedures were purified by silica

gel (230–400 mesh) chromatography. The hexapeptides 2 and 6

were purified by medium pressure liquid chromatography (reverse

phase, C18, 40–60 l), using methanol/water mixtures for elution.

The peptides 2 and 6 were further purified by reverse phase HPLC

(C18, 10 l, 10 mm–250 mm), using methanol–water gradients. All

the peptides were characterized by electrospray ionization mass

spectrometry (ESI-MS) on a Bruker Daltonics Esquire-3000 instru-

ment and by complete assignment of the 500 MHz 1H NMR spectra

(Bruker AV500). Mass spectral data (m/z): Peptide 2, 794.1 [M +

H]+ (Mcal ¼ 793 Da); 816.1 [M + Na]+; 832.0 [M + K]+; 3, 500.2

[M + H]+ (Mcal ¼ 499 Da); 522.2 [M + Na]+; 4, 288.2 [M + H]+

(Mcal ¼ 287 Da); 310.2 [M + Na]+; 6, 808.1 [M + H]+ (Mcal ¼ 807

Da); 830.1 [M + Na]+; 846.1 [M + K]+; 7, 514.3 [M + H]+ (Mcal ¼513 Da); 536.2 [M + Na]+; 8, 302.2 [M + H]+ (Mcal ¼ 301 Da);

324.2 [M + Na]+.

NMR SpectroscopyExperiments were carried out on Bruker AV700 and AV500 spec-

trometers. All spectra were recorded at a peptide concentration of

*5 mM in CDCl3 and CD3OH at 300 K. Delineation of exposed

NH groups was achieved by titrating CDCl3 solutions with low con-

centrations of DMSO-d6. TOCSY and ROESY experiments were

recorded in phase sensitive mode using the TPPI (time proportional

phase incrementation) method. A data set of 1024 3 450 was used

for acquiring the data. The same data set 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 processing, using BRUKER TOPSPIN

software.

X-Ray DiffractionCrystals of peptides 3, 4, 7, and 8 were grown by slow evaporation

from methanol/water mixtures. X-ray diffraction data were collected

on a Bruker AXS KAPPA APEX II CCD diffractometer using MoKa

radiation. The crystal structures were solved by direct methods

using SHELXS-97.29 The structures were refined isotropically fol-

lowed by full matrix anisotropic least-squares refinement using

SHELXL-97.29 The solvent molecules in peptides 3, 4, and 8 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

refinement. The water hydrogen atoms in 8 were located from a dif-

ference Fourier map. The final R factors were 6.73%, 8.58% 3.53%,

and 4.12% for peptides 3, 4, 7, and 8, respectively. The crystal and

diffraction parameters for peptides 3, 4, 7, and 8 are summarized in

Table II. The crystallographic coordinates for the structures are de-

posited at the Cambridge Crystallographic Data Centre with deposi-

tion numbers CCDC 801464 (3) 801463, (4) 801466 (7), and

801465 (8). These data can be obtained free of charge via

www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge

FIGURE 1 Schematic representation of the solvent dependent

conformational equilibrium between an a-helix (left) and a

b-hairpin (right).

746 Rajagopal et al.

Biopolymers (Peptide Science)

Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ,

UK; fax: (+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk).

RESULTS

NMR Studies in Solution

The nature of the peptide conformations in solution was

probed using backbone nuclear Overhauser effects (NOEs)

to establish local residue conformations and by solvent de-

pendence of amide NH chemical shifts, for differentiating

between internally hydrogen bonded and solvent exposed

NH groups. NMR data were collected in two different solvent

systems CDCl3 and CD3OH. In the case of the aprotic solvent

CDCl3, conformations which maximize intramolecular

hydrogen bonding were expected to be favored, while

CD3OH is anticipated to promote conformers which have a

greater number of solvent exposed backbone NH groups.

The NMR parameters obtained for the truncated tetra

and hexapeptides 2, 3, 6, and 7 are compared with those of

the parent octapeptide Boc-Leu-Phe-Val-Aib-DAla-Leu-Phe-

Val-OMe (1) and Boc-Leu-Phe-Val-Aib-Aib-Leu-Phe-Val-

OMe (5). The central Aib-DAla segments is anticipated to

favor a type-I0 b-turn conformation, while the Aib-Aib seg-

ment has an equal propensity to adopt type-I/III or type-I0/III0 b-turn conformations. This study is designed to probe

contextual effects on conformational choice at the central

segments. While a central type-III b-turn may serve to nucle-

ate and stabilize a-helical conformations in short peptides,

the type-I0 b-turn is expected to promote b-hairpin confor-

mations. The chemical shift values for backbone NH and

CaH protons in CDCl3 and CD3OH are summarized in Table

III for peptides 2, 3, 6, and 7. Corresponding values for the

parent peptides Boc-Leu-Phe-Val-Aib-DAla-Leu-Phe-Val-

OMe (1) and Boc-Leu-Phe-Val-Aib-Aib-Leu-Phe-Val-OMe

(5) are also listed for comparison.

Figures 2 and 3 provide a comparison of key backbone

NOEs obtained for the hexapeptides 2 and 6, in CD3OH sol-

utions. In b-hairpin structures cross-strand (non-sequential)

NOEs may be anticipated, while a-helices are characterized

by a succession of sequential NiH$Ni+1H NOEs. Figure 2a

reveals that in the hexapeptide Boc-Phe-Val-Aib-DAla-Leu-

Phe-NHMe (2) a relatively intense NOE is observed between

Val (2) NH and Leu (5) NH. Furthermore, an appreciable

Table II Crystal and Diffraction Parameters of Peptides 3, 4, 7, and 8

Peptide 3 Peptide 4 Peptide 7 Peptide 8

Empirical formula C24H45N5O6 � H2O C13H25N3O4 � 0.33H2O C25H47N5O6 C14H27N3O4 � H2O

Crystal habit Clear Clear Clear Clear

Crystal size (mm) 0.313 0.253 0.13 0.263 0.1 3 0.04 0.66 3 0.34 3 0.14 0.6 3 0.25 3 0.04

Crystallizing solvent Methanol/water Methanol/water Methanol/water Methanol/water

Space group C2 P212121 P21 P21Cell parameters

a (A) 17.674(1) 10.526(2) 10.244(8) 8.653(1)

b (A) 11.508(8) 21.781(4) 11.164(8) 12.517(2)

c (A) 16.693(1) 22.489(4) 13.954(1) 8.861(1)

a, b, c (deg) 90, 108.1(1), 90 90, 90, 90 90, 104.3(1), 90 90, 94.6(2), 90

Volume (A3) 3228.2(4) 5155.8(2) 1546.4(2) 956.7(2)

Z/Z’ 4/1 12/3 2/1 2/1

Cocrystallized solvent 1 water 1 water None 1 water

Molecular weight 515.65 292.69 513.6 319.4

Density (g/cm3)(cal) 1.06 1.131 1.103 1.109

F (000) 1120 1904 560 348

Radiation MoKa MoKa MoKa MoKa

h range (o) 2.14 to 25.0 2.04 to 27.1 2.1 to 26.8 2.82 to 26.2

Scan type x x x xMeasured reflections 15504 20866 11697 6690

Unique reflections 2995 6264 3216 1857

Observed reflections [|F|>4r(F)] 2672 1900 2988 1688

Final R/wR2 (%) 6.73/18.1 8.58/14.6 3.53/9.93 4.12/10.8

Goodness of fit (S) 1.11 0.93 1.07 0.968

Dqmax/Dqmin (e/A3) 0.69/�0.17 0.22/�0.27 0.12/�0.13 0.17/�0.14

No. of restraints/parameters 1/325 0/550 1/325 1/267

Data to parameter ratio 8.2:1 3.5:1 9.2:1 6.3:1

Chain Length Effects on Helix-Hairpin Distribution 747

Biopolymers (Peptide Science)

TableIII

NMRParam

etersforPeptides

NH

CaH

Dd,ppm

b,c

(dd/dT,ppm/k)

NH

CaH

Dd,ppm

d

(dd/dT,ppm/k)

NH

CaH

Dd,ppm

d

(dd/dT,ppm/k)

d(ppm)aCDCl 3(C

D3OH)

d(ppm)aCDCl 3(C

D3OH)

d(ppm)CDCl 3(C

D3OH)

Peptide1

Peptide2

Peptide3e

Leu(1)

4.85

3.90

1.21

(6.24)

(4.10)

(�5.57)

Phe(2)

6.70

4.49

0.48

Phe(1)

5.94

4.32

0.58

(8.16)

(5.44)

(�7.79)

(6.75)

(4.64)

(�7.80)

Val(3)

7.22

3.90

0.11

Val(2)

7.20

3.85

1.37

Val(1)

5.19

3.54

0.23

(8.99)

(4.15)

(�7.87)

(8.51)

(4.08)

(�12.8)

(6.80)

(3.76)

(�8.67)

Aib(4)

7.29

—0.61

Aib(3)

7.68

—0.58

Aib(2)

6.36

—1.92

(8.79)

(�)

(�9.13)

(8.54)

(�)

(�9.41)

(�)

(�)

(�9.38)

DAla(5)

7.42

3.98

0.15

DAla(4)

7.57

3.99

0.34

DAla(3)

7.14

4.32

0.41

(7.92)

(4.27)

(�9.54)

(7.75)

(4.18)

(�7.63)

(7.80)

(4.26)

(�4.11)

Leu(6)

7.50

4.28

0.19

Leu(5)

7.76

4.10

0.37

Leu(4)

7.19

4.42

0.41

(8.21)

(4.77)

(�3.89)

(8.12)

(4.46)

(�5.07)

(7.91)

(4.38)

(�2.76)

Phe(7)

7.50

4.85

0.31

Phe(6)

7.56

4.64

0.12

NH(5)

6.90

—0.52

(8.74)

(4.46)

(�10.5)

(8.35)

(4.27)

(�12.5)

(7.92)

(�)

(�5.50)

Val(8)

7.19

4.45

0.01

NH(7)

7.19

—0.28

(8.14)

(4.12)

(�5.84)

(7.54)

(�)

(�7.66)

Peptide5

Peptide6

Peptide7

Leu(1)

5.08

3.86

1.19

(6.92)

(3.97)

(�3.77)

Phe(2)

6.70

4.41

0.99

5.40

4.25

1.62

(7.99)

(4.68)

(�9.42)

Phe(1)

(6.91)

(4.36)

(�6.05)

Val(3)

6.99

3.81

0.31

Val(2)

6.60

3.84

1.12

Val(1)

4.91

3.63

1.45

(7.72)

(3.92)

(�9.98)

(7.84)

(3.89)

(�5.37)

(6.81)

(3.65)

(�8.15)

Aib(4)

7.30

—0.34

Aib(3)

7.60

—0.25

Aib(2)

6.46

—1.25

(7.96)

(�)

(�9.17)

(8.12)

(�)

(�6.97)

(8.26)

(�)

(�7.72)

Aib(5)

7.16

—0.38

Aib(4)

7.19

—0.23

Aib(3)

7.38

—0.13

(7.75)

(�)

(�6.87)

(7.65)

(�)

(�5.87)

(7.84)

(�)

(�6.85)

Leu(6)

7.54

4.16

0.05

Leu(5)

7.63

4.01

0.11

Leu(4)

7.41

4.39

0.21

(7.78)

(4.18)

(�4.12)

(7.85)

(4.22)

(�5.97)

(7.89)

(4.24)

(�3.71)

Phe(7)

7.67

4.73

0.04

Phe(6)

7.54

4.70

0.06

NH(5)

7.30

—0.11

(8.02)

(4.61)

(�6.82)

(7.84)

(4.54)

(�4.17)

(7.64)

(�)

(�5.54)

Val(8)

7.23

4.44

0.05

NH(7)

7.17

—0.32

(7.68)

(4.28)

(�5.34)

(7.47)

(�)

(�12.0)

aThereported

chem

icalshiftvalues

areafteradditionof1.1%

ofDMSO-d

6to

separateoverlappingresonance

from

thediagonalpeak.

b,Dd¼

(d28.3%

ofDMSO-d

6/CDCl 3-d

CDCl 3).

cdd/dTvalues

(CD3OH)forpeptide1and5arefrom

Ref.1.

dDd¼

(d11.5%

ofDMSO-d

6/CDCl 3-d

CDCl 3).

eExperim

entperform

edat283K.

Biopolymers (Peptide Science)

748 Rajagopal et al.

NOE is also observed between Phe (1) CaH and Phe (6)

CaH (Figure 2b). The absence of the sequential Leu (5) NH

$Phe (6) NH NOE suggests that continuous helical confor-

mations are not significantly populated. These results sug-

gest that despite truncation of the N- and C- terminal

strands, the b-hairpin conformation, nucleated by the cen-

trally positioned Aib-DAla segment, is still appreciably popu-

lated. The observed Aib (3) NH$DAla (4) NH and DAla (4)

NH$Leu (5) NH NOEs are compatible with type-I0 b-turnat the Aib-DAla segment. The Phe (6) NH$NHCH3 (7)

NOE may be rationalized by occurrence of fraying at C-

terminus with Phe 6 adopting local conformation in the

helical region, with consequent loss of the hydrogen bond,

Boc (CO) � � � NHCH3. Interestingly, a completely different

pattern of NOEs is seen for peptide 2 in CDCl3 solution.

The cross-strand Val (2) NH$Leu (5) NH NOE is absent

and a succession of sequential NiH$Ni+1H NOEs are

observed, suggesting that a continuous helical conformation

predominates in CDCl3. Thus, in peptide 2 the competing

effects of the intrapeptide and peptide-solvent interactions,

determine the nature of conformations that are populated

in solution.

FIGURE 2 Partial 500 MHz ROESY spectra of peptide 2 in CD3OH (a) NH$NH region. (b)

Long range NOE Phe(1)CaH $ Phe(6)CaH. (c) NH$NH region of peptide 2 in CDCl3. (d) Sche-

matic representation of the proposed b-hairpin structure of peptide 2. The expected hydrogen bonds

are shown by broken lines. Key conformation sensitive NOEs are shown by double edged arrows.

Chain Length Effects on Helix-Hairpin Distribution 749

Biopolymers (Peptide Science)

The behavior of the related hexapeptide, Boc-Phe-Val-

Aib-Aib-Leu-Phe-NHMe (6) contrasts sharply with that

observed for peptide 2. Figure 3 shows the partial ROESY

spectra of peptide 6 in CDCl3 and CD3OH. In this case, only

sequential NiH$Ni + 1H NOEs are observed in both sol-

vents, consistent with the anticipated continuous helical con-

formation. In CD3OH, the presence of relatively intense se-

quential NOEs over the segment residues 3–6 also favors

helix formation. The low intensity of the Phe (1) NH$Val

(2) NH NOE and the absence of the Val (2) NH$Aib (3)

NH NOE suggests unfolding of the helix at the N-terminus.

The absence of the Phe (1) CaH$Phe (6) CaH NOE argues

against the population of folded hairpin conformation,

which were considered for peptide 2 (Figure 2d). It should be

noted that for peptide 6 in CD3OH, Val (2) NH and Leu (5)

NH protons have very similar chemical shifts, precluding a

definitive comment on the presence or absence of these

NOEs.

Figure 4 compares the partial ROESY spectra of the trun-

cated tetrapeptide models Boc-Val-Aib-DAla-Leu-NHMe (3)

and Boc-Val-Aib-Aib-Leu-NHMe (7). In both cases, succes-

sive NiH$Ni + 1H NOEs are observed, supportive of a short

stretch of 310-helical conformation, stabilized by three intra-

molecular hydrogen bonds. A similar pattern of NOEs is also

observed in CD3OH solution. For both tetrapeptides 3 and

7, the absence of the Val (1) NH$Leu (4) NH NOEs sug-

gests that b-hairpin like conformations are not significantly

populated. Inspection of the ROESY spectra of tetrapeptides

3 (Aib-DAla) in CD3OH revealed a weak NOE between Val

(1) NH$NHMe (5) at 300 K. Cooling of the sample to 283

K enhanced the Val (1) NH$NHMe (5) NOE (Figure 4a).

The anticipated distance (� 3.5 A) between these two pro-

tons in both helical and b-hairpin conformation lies outside

the range expected to result in NOEs. The nature of the con-

formation that gives rise to the unexpected NOE is discussed

subsequently, in relation to the conformation observed in the

solid state by X-ray diffraction, where an interproton dis-

tance of 3.3 A is observed (Figure 4a).

The number of solvent shielded (intramolecularly hydro-

gen bonded NH groups) in significantly populated confor-

mations in peptides 1 to 8 may be estimated from the tem-

perature coefficients of NH chemical shifts in CD3OH (dd/dT) and the solvent dependent chemical shift upon titrating

CDCl3 solutions with small amount of DMSO-d6 (Dd). Thedata are summarized in Table III. Ideally, internally hydrogen

bonded groups in short peptides exhibit significantly lower

dd/dT and Dd values than their solvent exposed counterparts.

Conformational heterogeneity often results in intermediate

values, which do not permit a clear delineation between

exposed and hydrogen bonded NH groups. For NH groups

which are internally hydrogen bonded in a large population

of conformational states, assignment of hydrogen bonded

groups is straight forward. On the other hand, situations

where an NH group is buried in one conformation and

exposed in another can lead to intermediate values, which

are hard to interpret. In the present situation, the equilibria

present in solution are likely to involve short helices, hair-

pins, and other partially unfolded structures. In the tetrapep-

tide Boc-Val-Aib-Aib-Leu-NHMe (7), the Dd values obtainedin CDCl3-DMSO-d6 mixtures clearly establish that Aib (3),

Leu (4), and NHCH3 (5) groups are solvent shielded (Dd �0.21 ppm), while Val (1) NH and Aib (2) NH groups exhibit

FIGURE 3 Partial 500 MHz ROESY spectra (NH$NH NOEs) of

peptide 6 (a) CD3OH, (b) CDCl3.

750 Rajagopal et al.

Biopolymers (Peptide Science)

large Dd values (�1.25 ppm) characteristic of solvent

exposed groups. These observations strongly suggest the pre-

ponderance of states in which three intramolecular hydrogen

bonds are present, consistent with a 310-helical conforma-

tion. Temperature coefficient (dd/dT) values in CD3OH are

indeed lower for the Aib (3) NH, Leu (4) NH, and NHCH3

(5) groups, although the distinction between the various NH

groups is less pronounced. This is suggestive of a shift of con-

former populations, with a greater proportion of nonhelical

structures in CD3OH, a solvent which may be expected to

compete for backbone hydrogen bonding sites. Replacement

of residue 3 in peptide 7 (Boc-Val-Aib-Aib-Leu-NHMe) withDAla yields peptide 3 (Boc-Val-Aib-DAla-Leu-NHMe). Inspec-

tion of Table III reveals that Aib (2) NH shows a very high

Dd value (1.92 ppm) indicative of exposure to solvent. The

other four NH groups show much lower solvent dependent

chemical shifts, with Val (1) NH yielding the lowest Dd value

of 0.23 ppm. These results are in sharp contrast to those

obtained for peptide 7. Peptide 3 (Aib-DAla) appears to have

a significant population of hairpin conformations in CDCl3;

an inference drawn from the low Dd value for Val (1) NH.

The observed pattern of Dd values is consistent with mixed

populations of 310 helical and hairpin structures. The large

dd/dT values for obtained Val (1) NH in CD3OH is sugges-

tive of fraying of the hairpin in the more strongly interacting

solvent.

In the hexapeptide Boc-Phe-Val-Aib-Aib-Leu-Phe-NHMe

(6), the Dd values are consistent with continuous 310-helical

conformations with five intramolecularly hydrogen bonded

NH groups. Indeed, only Phe (1) NH and Val (2) NH groups

have Dd values (�1.1 ppm) characteristic of exposed NH

groups. In CD3OH, differentiation of NH groups is not

obtained from dd/dT values. In the case of peptide 2 (Boc-

Phe-Val-Aib-DAla-Leu-Phe-NHMe), Dd values for Phe (1)

NH are low (0.58 ppm), suggesting that hairpin conformers

in which the N-terminus NH group is hydrogen bonded are

FIGURE 4 (a) Partial 700 MHz ROESY Spectra of Boc-Val-Aib-DAla-Leu-NHMe (3) in CD3OH,

illustrating NH$NH regions at 283 K. The Val (1) NH$HNCH3 (5) 1/5 NOE is circled. A view of

the crystallographically determined conformation indicating the relevant interproton distance is

shown. (b) Partial 500 MHz ROESY spectra of peptide 3 in CDCl3, illustrating the NH$NH region,

at 300 K. (c and d) Partial 500 MHz ROESY spectra of peptide 7 NH$NH region at 300 K (c)

CD3OH and (d) CDCl3.

Chain Length Effects on Helix-Hairpin Distribution 751

Biopolymers (Peptide Science)

indeed significantly populated. As in the case of peptide 6, a

clear differentiation between backbone NH groups is not im-

mediately apparent from the temperature coefficient in

CD3OH. The effect of sequence truncation may be assessed

by comparing the parameters obtained for the parent octa-

peptide 1 (Aib-DAla) and 5 (Aib-Aib) (Table III). Lengthen-

ing the peptide chain appears to stabilize hairpin formation

in CD3OH solution. It should be noted that for the hexapep-

tide methylamide sequences five internal hydrogen bonds are

anticipated in 310-helical conformations, while hairpins con-

tain three cross-strand hydrogen bonds. For the tetrapeptide

sequence 3 and 7, the hairpin conformations accommodate

two hydrogen bonds, whereas helical structures can result in

three hydrogen bonds. The N-terminus Val (1) NH group is

hydrogen bonded internally in hairpins and is solvent

exposed in helices.

Molecular Conformation in Crystals

Diffraction quality, single crystals were obtained for the

dipeptides Boc-Aib-DAla-NHMe (4), Boc-Aib-Aib-NHMe

(8) and tetrapeptides Boc-Val-Aib-DAla-Leu-NHMe (3) and

Boc-Val-Aib-Aib-Leu-NHMe (7). Figures 5 and 6 show a

view of the molecular conformations in crystals. The back-

bone torsion angles and hydrogen bond parameters are sum-

marized in Tables IV and V. Boc-Aib-DAla-NHMe (4) crystal-

lized with three independent molecules in the orthorhombic

cell. All three molecules adopt b-turn conformations,

FIGURE 5 (a) Molecular conformation of Boc-Aib-DAla-NHMe

(4) in crystals. Only one molecule in the asymmetric unit is shown.

(b) Molecular conformation of peptide Boc-Aib-Aib-NHMe (8) in

crystals. (c) Superposition of the three independent molecules in

the asymmetric unit of peptide 4. (d) Superposition of Boc-

Aib-DAla-NHMe (4, molecule C) with the corresponding enan-

tiomeric conformation of Boc-Aib-Aib-NHMe (8).

FIGURE 6 Molecular conformation of (a) Boc-Val-Aib-DAla-Leu-

NHMe (3) and (b) Boc-Val-Aib-Aib-Leu-NHMe (7) in crystals.

Table IV Torsion Angles (deg)

Residues / w x v1 v2

Peptide 3

Val(1) �54.9 137.2 175.6 61.5, �60.8

Aib(2) 59.0 18.9 �175.8DAla(3) 81.3 1.0 �171.2

Leu(4) �111.9 �36.1 �178.4 �60.3 �62.8, 174.0

Peptide 4

Molecule-A

Aib 54.4 38.3 174.9DAla 80.3 7.6 �178.8

Molecule-B

Aib 59.4 34.4 177.5DAla 95.7 �5.7 177.0

Molecule-C

Aib 56.1 34.3 176.4DAla 78.0 6.2 179.8

Peptide 7

Val(1) �52.0 �39.3 �173.9 �60.3, 174.8

Aib(2) �53.1 �33.6 �175.5

Aib(3) �56.2 �27.6 �178.6

Leu(4) �69.0 �21.5 �177.4 �61.1 �63.6, 171.7

Peptide 8

Aib(1) �53.6 �38.3 �175.6

Aib(2) �64.5 �19.5 �174.4

The estimated standard deviation & 0.58, 1.28, 0.28, and 0.38 for Peptides3, 5, 7, and 8, respectively.

752 Rajagopal et al.

Biopolymers (Peptide Science)

stabilized by a single intramolecular 4?1 hydrogen bond

Boc C ¼ O � � �HNMe. The torsion angles presented in Table

IV correspond to a type-I0 b-turn conformation. This choice

was based on the presence of the DAla residue. It should be

noted that the diffraction data would also be consistent with

an enantiomer structure. Boc-Aib-Aib-NHMe (8) crystallizes

Table V Hydrogen Bonds

Type Donor (D) Acceptor (A) D � � �A (A) H � � �A (A) C¼O � � �H (deg) C¼O � � �D (deg) DH � � �A (deg)

Peptide 3

Intermolecular

N(1) O(4)a 3.010 2.164 144.0 140.8 167.8

N(2) O1wb 2.874 2.055 158.8

O1w O(2) 2.872

O1w O(3)c 2.776

Intramolecular

4?1 N(3) O(0) 3.000 2.265 144.1 140.5 143.5

4?1 N(4) O(1) 2.862 2.031 127.6 132.3 162.1

5?1 N(1M) O(1) 3.055 2.221 131.7 135.8 163.3

Peptide 4

Intermolecular

N(11) O(21)d 3.008 2.197 139.1 145.4 157.2

N(12) O(12)e 2.885 2.058 147.7 146.8 161.0

N(21) O(31)f 2.996 2.210 140.3 131.1 151.8

N(22) O(11) 2.869 2.092 149.0 153.6 149.9

N(31) O1w 3.014 2.168 167.6

N(32) O(22) 3.316 2.654 129.1 133.5 134.8

O1w O(22) 3.006

O1w O(32)g 2.869

Intramolecular

4?1 N(11M) O(02) 2.952 2.144 120.3 127.1 156.2

4?1 N(21M) O(04) 3.008 2.187 115.1 119.3 159.7

4?1 N(31M) O(06) 2.913 2.077 121.2 125.6 163.8

Peptide 7

Intermolecular

N(2) O(4)d 2.923 2.180 144.0 153.2 144.4

Intramolecular

4?1 N(3) O(0) 3.098 2.313 121.0 127.0 152.0

4?1 N(4) O(1) 2.973 2.146 129.9 134.3 161.1

4?1 N(1M) O(2) 2.977 2.157 121.3 127.0 159.1

Peptide 8

Intermolecular

N(1) O(1)h 3.091 2.306 152.2 153.7 168.4

N(2) O1w 2.938 2.089 170.0

O1w O(1)h 2.889 2.179 134.6 139.8 155.6

O1w O(2)i 2.832 1.997 128.7 130.6 173.1

Intramolecular

4?1 N(1M) O(0) 2.985 2.229 123.7 125.0 169.1

The estimated standard deviation in bond length and bond angles & 0.006 A, 0.58; 0.01 A, 0.78; 0.003 A, 0.28 and 0.004 A, 0.28 for peptides 3, 4, 7, and 8,

respectively.a Symmetry related by �x � 1/2, y + 1/2, �z�1.b symmetry related by �x � 1/2, y + 1/2, �z.c Symmetry related by �x, y, �z.d symmetry related by x + 1, y, z.e Symmetry related by x + 1/2, �y � 1/2, �z.f Symmetry related by �x + 3/2, �y, z + 1/2.g Symmetry related by �1 + x, y, z.h Symmetry related by –x�1, y � 1/2, �z.i Symmetry related by �x, y � 1/2, �z.

Chain Length Effects on Helix-Hairpin Distribution 753

Biopolymers (Peptide Science)

in the chiral, monoclinic space group P21, despite the ab-

sence of chirality in the sequence. The conformational angles

presented in Table IV correspond to the Type-III conforma-

tion. This achiral sequence would be expected to exist in so-

lution as an equimolar mixture of the energetically degener-

ate type-III/III0 b-turn conformations. While crystallization

in an achiral, centric space group containing both enantio-

meric conformations, might have been anticipated, in the

present case the less frequently observed trapping of a single,

enantiomeric conformation in a chiral space group has been

obtained. A 4?1 hydrogen bond, Boc C ¼ O � � �HNMe, is

observed in the dipeptide Boc-Aib-Aib-NHMe (8).

The tetrapeptide Boc-Val-Aib-Aib-Leu-NHMe (7) adopts

the expected 310-helical conformation stabilized by three suc-

cessive 4?1 hydrogen bonds. All four residues adopt /, wvalues lying in right handed (aR) region of conformational

space. Boc-Val-Aib-DAla-Leu-NHMe (3) adopts a dramati-

cally different conformation (see Figure 6). The observed

conformation is stabilized by three intramolecular hydrogen

bonds: Boc C ¼ O � � �H-N DAla (3) (4?1) Val (1) C ¼O � � �HN Leu (4) (4 ?1) and Val (1) C ¼ O � � �HNMe (5)

(5?1). Inspection of the backbone torsion angles in Table IV

reveals that the Aib (2) and DAla (3) segments adopt the

type-I0 b-turn conformation, while the Val (1) residue lies in

the polyproline (PII, semi extended) region of conforma-

tional space. Leu (4) lies in the bridge region of the Rama-

chandran map (/ ¼ �1128, w ¼ 308). The tetrapeptide,

Boc-Val-Aib-DXxx-Leu-NHMe segment has been character-

ized in two independent octapeptide b-hairpin structures,

Boc-Leu-Phe-Val-Aib-DAla-Leu-Phe-Val-OMe (1)21 and Boc-

Leu-Val-Val-Aib-DPro-Leu-Val-Val-OMe.22 In both crystal-

line octapeptides, the central segment nucleating the b-hair-pin adopts a type-I0 b-turn conformation.

The conformational angles (/, w) for the tetrapeptide seg-ments reveal that Boc-Val-Aib-DAla-Leu-NHMe (3) differs

from the corresponding segment in b-hairpins only in the

torsion angles Val (1) / and Leu (4) w. The former is altered

by 608, while the latter is changed by *1608. These two

changes result in the transformation of the b-hairpin struc-

ture to the crystallographically observed conformation, in

which Val (1) CO forms bifurcated hydrogen bonds with Leu

(4) NH (C10, 4 ?1) and NHMe (C13, 5?1) The observed

C13 hydrogen bond in peptide 3 constitutes an interesting

example of a nonhelical a-turn, previously characterized as

isolated conformational features in protein structures.30–32

Such C13, 5?1 hydrogen bonds are observed relatively infre-

quently in the crystal structures of short peptides.33,34 Two

examples are illustrated in Figure 7.

All examples illustrated in Figure 7 correspond to distinct

a-turn families. Peptide 3 may be classified as a type-I0 b-turnfollowed by a distorted aR (bridge) conformation. In the pre-

viously reported peptide Piv-DPro-LPro-DAla-NHMe, a dis-

torted type-II0 b-turn conformation is followed by a distorted

aL residue. In this case, the C13 hydrogen bond is significantly

shorter than the C10 hydrogen bond.33 The structure of tetra-

peptide Dnp-Val-Aib-Gly-Leu-Pna constitutes an example of

a type-II0 b-turn conformation followed by an aL residue.34

This results in a consecutive type-II0-III b-turn structure,

which has been widely observed in short peptides containing

the constrained a,a-dialkylated amino acids.11,35–38 In this

FIGURE 7 Examples of a-turns observed in peptide crystals (a) Piv-DPro-LPro-DAla-NHMe30, (b)

Dnp-Val-Aib-Gly-Leu-pNA31, and (c) Boc-Val-Aib-DAla-Leu-NHMe (3). Only backbone atoms of

the a-turn are shown for clarity. The torsion angles and hydrogen bond lengths are shown.

754 Rajagopal et al.

Biopolymers (Peptide Science)

case, the C13 hydrogen bond (3.3 A) is appreciably longer

than the two C10 hydrogen bonds (2.98 A and 3.13 A).

DISCUSSIONInsertion of Aib-XXxx segments into peptide sequences pro-

motes the formation of folded b-turns, which in turn can nu-

cleate further secondary structure formation. When XXxx is

an L-residue type-III b-turns are preferentially formed,

resulting in nucleation of 310-helical structures. In contrast,

when Xxx is a D-residue type-I0 b-turn formation may be

preferred, resulting in nucleation of b-hairpin structures.

The intrinsic preference of D-residues to favour aL (/*+608, w *+308) conformations result in the bias for type-

I0/III0 turns. It should be noted, however that D-residues with

aliphatic side chains (DAla, DVal, and DLeu) have also been

accommodated into right handed (aR) oligopeptide helices,

as demonstrated by several crystal structure determinations.

Indeed, there is a relatively small energy penalty to be paid

for accommodating D-residues in right handed helical

(aR)conformation.39–42

The purpose of this study was to examine the consequen-

ces of truncating the b-hairpin octapeptide, Boc-Leu-Phe-

Val-Aib-DAla-Leu-Phe-NHMe (1). The corresponding trun-

cated peptides containing the Aib-Aib segment were also

studied for comparison. These results demonstrate clearly

that chain length truncation in the case of Aib-DAla peptides

leads to strongly solvent dependent conformational equili-

bria. In poorly interacting solvents like CDCl3, b-hairpins aredisfavored and helical conformations, which posses a greater

number of intramolecular hydrogen bonds seem to be pre-

ferred in the hexapeptide Boc-Phe-Val-Aib-DAla-Leu-Phe-

NHMe (2). In CD3OH, which can form hydrogen bonds to

donor and acceptor groups which face outwards b-hairpinsare significantly populated. These observations suggests a

finely tuned interplay between the energetics of the two types

of b-turns formed by the Aib-XXxx, Aib-DXxx segments, the

number of intramolecular backbone hydrogen bonds in dif-

ferent conformation and peptide solvation.

Crystal structures establishes the anticipated b-turn con-

formation in the dipeptides Boc-Aib-DAla-NHMe (4) and

Boc-Aib-Aib-NHMe (8) and the short 310 helix in the tetra-

peptide Boc-Val-Aib-Aib-Leu-NHMe (7). Interestingly, the

conformation determined in crystals for the tetrapeptide

Boc-Val-Aib-DAla-Leu-NHMe (3) reveals a type-I0 b-turn at

the Aib-DAla segment. Examination of the folded conforma-

tion reveals that this can be derived from a potential b-hair-pin conformation by backbone distortion at the Leu (4) resi-

due. Notably, the observed NOE between Val (1) NH and

NHCH3 (5) protons in CD3OH solution suggests that the

crystal state conformation may indeed be significantly popu-

lated in solution. The results of this study may be used

to design suitable sequences for directly monitoring helix-

hairpin equilibria in solution.

S. Aravinda thanks the Department of Science and Technology for

the award of SERC FAST Track Young Scientist Fellowship.

REFERENCES1. Baker, E. N.; Hubbard, R. E. Prog Biophys Mol Biol 1984, 44,

97–179.

2. Sticke, D. F.; Presta, L. G.; Dill, K. A.; Rose, G. D. J Mol Biol

1992, 226, 1143–1159.

3. Rose, G. D.; Gierasch, L. M.; Smith, J. A. Adv Protein Chem

1985, 37, 1–109.

4. Smith, J. A.; Pease, L. G. CRC Crit Rev Biochem 1980, 8, 315–399.

5. Richardson, J. S. Adv Protein Chem 1981, 34, 167–339.

6. Venkatachalam, C. M. Biopolymers 1968, 6, 1425–1436.

7. Sibanda, B. L.; Blundell, T. L.; Thornton, J. M. J Mol Biol 1989,

206, 759–777.

8. Ramachandran, G. N.; Sasisekharan, V. Adv Protein Chem

1968, 23, 283–438.

9. Shamala, N.; Nagaraj, R.; Balaram, P. Biochem Biophys Res

Commun 1977, 79, 292–298.

10. Nagaraj, R.; Shamala, N.; Balaram, P. J Am Chem Soc 1979, 101,

16–20.

11. Prasad, B. V. V.; Balaram, P. CRC Crit Rev Biochem 1984, 16,

307–347.

12. Sibanda, B. L.; Thornton, J. M. Nature 1985, 316, 170–174.

13. Gunasekaran, K.; Ramakrishnan, C.; Balaram, P. Protein Eng

1997, 10, 1131–1141.

14. Venkatraman, J.; Shankaramma, S. C.; Balaram, P. Chem Rev

2001, 101, 3131–3152.

15. Toniolo, C.; Benedetti, E. Trends Biochem Sci 1991, 16, 350–353.

16. Toniolo, C.; Benedetti, E. Macromolecules 1991, 24, 4004–4009.

17. Karle, I. L.; Balaram, P. Biochemistry 1990, 29, 6747–6756.

18. Kaul, R.; Balaram, P. Bioorg Med Chem 1999, 7, 105–117.

19. Toniolo, C.; Crisma, M.; Formaggio, F.; Peggion, C.; Broxterman,

Q. B.; Kaptein, B. Biopolymers (Pept Sci) 2004, 76, 162–176.

20. Crisma, M.; Formaggio, F.; Moretto, A.; Toniolo, C. Biopoly-

mers (Pept Sci) 2006, 84, 3–12.

21. Aravinda, S.; Shamala, N.; Rai, R.; Gopi, H. N.; Balaram,

P. Angew Chem Int Ed 2002, 41, 3863–3865.

22. Raghavender, U. S.; Aravinda, S.; Rai, R.; Shamala, N.; Balaram,

P. Org Biomol Chem 2010, 8, 3133–3135.

23. Huang, R.; Setnicka, V.; Etienne, M. A.; Kim, J.; Kubelka, J.;

Hammer, R. P.; Keiderling, T. A. J Am Chem Soc 2007, 129,

13592–13603.

24. Wu, L.; McElheny, D.; Takekiyo, T.; Keiderling, T. A. Biochemis-

try 2010, 49, 4705–4714.

25. Rai, R.; Raghothama, S.; Sridharan, R.; Balaram, P. Biopolymers

(Pept Sci) 2007, 88, 350–361.

26. Awasthi, S. K.; Shankaramma, S. C.; Raghothama, S.; Balaram,

P. Biopolymers 2001, 58, 465–476.

27. Kubasik, M.; Blom, A. ChemBioChem 2005, 6, 1187–1190.

28. Waters, M. L. Biopolymers (Pept Sci) 2004, 76, 435–445.

29. Sheldrick, G. M. Acta Crystallogr 2008, A64, 112–122.

30. Nataraj, D. V.; Srinivasan, N.; Sowdhamini, R.; Ramakrishnan,

C. Curr Sci 1995, 69, 434–447.

Chain Length Effects on Helix-Hairpin Distribution 755

Biopolymers (Peptide Science)

31. Ramakrishnan, C.; Nataraj, D. V. J Pept Sci 1997, 4, 239–

252.

32. Pavone, V.; Gaeta, G.; Lombardi, A.; Nastri, F.; Maglio, O.;

Isernia, C.Saviano, M. Biopolymers, 1996, 38, 705–721.

33. Chatterjee, B.; Saha, I.; Raghothama, S.; Aravinda, S.; Rai, R.;

Shamala, N.; Balaram, P. Chem Eur J 2008, 14, 6192–6204.

34. Yamada, T.; Nakao, M.; Miyazawa, T.; Kuwata, S.; Sugiura, M.;

In, Y.; Ishida, T. Biopolymers 1993, 33, 813–822.

35. Raghavender, U. S.; Aravinda, S.; Shamala, N.; Kantharaju; Rai,

R.; Balaram, P. J Am Chem Soc 2009, 131, 15130–15132.

36. Raghavender, U. S.; Kantharaju; Aravinda, S.; Shamala, N.;

Balaram, P. J Am Chem Soc 2010, 132, 1075–1086.

37. Kantharaju; Raghothama, S.; Aravinda, S.; Shamala, N.;

Balaram, P. Biopolymers (Pept Sci) 2010, 94, 360–370.

38. Crisma, M.; Valle, G.; Formaggio, F.; Toniolo, C. Z Kristallogr

1998, 213, 599–604.

39. Aravinda, S.; Shamala, N.; Desiraju, S.; Balaram, P. Chem Com-

mun 2002, 2454–2455.

40. Karle, I. L.; Gopi H. N.; Balaram, P. Proc Natl Acad Sci USA

2003, 100, 13946–13951.

41. Aravinda, S.; Shamala, N.; Bandyopadhyay, A.; Balaram, P. J Am

Chem Soc 2003, 125, 15065–15075.

42. Fairman, R.; Anthony-Cahill, S. J.; DeGrado, W. F. J Am Chem

Soc 1992, 114, 5459–5460.

756 Rajagopal et al.

Biopolymers (Peptide Science)