Chain length effects on helix-hairpin distribution in short peptides with Aib-DAla and Aib-Aib segments

Article abstract of DOI:10.1002/bip.21613

The Aib-DAla dipeptide segment has a tendency to form both type-I'/III' and type-I/III β-turns. The occurrence of prime turns facilitates the formation of β-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 α β-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 = ^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 β-hairpin conformations in CD₃OH. The β-turn conformations (type-I'/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 α-turn conformation, stabilized by three intramolecular hydrogen bonds (two 4→1 and one 5→1). The Aib-^DAla segment adopts a type-I' β-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.

Full text of DOI:10.1002/bip.21613

Chain Length Effects on Helix-Hairpin Distribution in Short Peptides
D
Chain Length Effects on Helix-Hairpin Distribution in Short Peptides with
with Aib- Ala and Aib-Aib Segments
Aib- Ala and Aib-Aib Segments
D
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
(type-I0/III) stabilized by intramolecular 4?1 hydrogen
D
bonds are observed for the peptide Boc-Aib- Ala-NHMe
ABSTRACT:
D
The Aib- Ala dipeptide segment has a tendency to form
(4) and Boc-Aib-Aib-NHMe (8) in crystals. The
both type-I0/III0 and type-I/III b-turns. The occurrence of
tetrapeptide Boc-Val-Aib-Aib-Leu-NHMe (7) adopts an
prime turns facilitates the formation of b-hairpin
incipient 310-helical conformation stabilized by three
conformations, while type-I/III turns can nucleate helix
D
4?1 hydrogen bonds. The peptide Boc-Val-Aib- Ala-
D
formation. The octapeptide Boc-Leu-Phe-Val-Aib- Ala-
Leu-NHMe (3) adopts a novel a-turn conformation,
Leu-Phe-Val-OMe (1) has been previously shown to form
stabilized by three intramolecular hydrogen bonds (two
a b-hairpin in the crystalline state and in solution. The
D
4?1 and one 5?1). The Aib- Ala segment adopts a
effects of sequence truncation have been examined using
type-I0 b-turn conformation. The observation of an NOE
the model peptides Boc-Phe-Val-Aib-Xxx-Leu-Phe-
between Val (1) NH$HNCH3 (5) in CD3OH suggests,
that the solid state conformation is maintained in
NHMe (2, 6), Boc-Val-Aib-Xxx-Leu-NHMe (3, 7), and
D
Boc-Aib-Xxx-NHMe (4, 8), where Xxx ¼ Ala, Aib. For
peptides with central Aib-Aib segments, Boc-Phe-Val-
#
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; nuclear
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
Overhauser effects; a-turns
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
D
hexapeptide Boc-Phe-Val-Aib- Ala-Leu-Phe-Val-NHMe
(2) favors helical conformations in CDCl3 and b-hairpin
conformations in CD3OH. The b-turn conformations
INTRODUCTION
ydrogen 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
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
C
VV
2011 Wiley Periodicals, Inc.
bonded ring, is the one of the best characterized
H
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PeptideScience Volume 96 / Number 6

Chain Length Effects on Helix-Hairpin Distribution
745
Table I Designed Peptide Sequences
No.
1
Peptide Sequence
Observed Conformations
References
D
Boc-Leu-Phe-Val-Aib- Ala-Leu-Phe-Val-OMe (1)
NMR: Helix in CDCl3; b-hairpin in CD3OH and DMSO-d6
X-ray: b-hairpin, Type I0 b-turn
NMR: Helix in CDCl3; Helix + b-hairpin in CD3OH
NMR: Helix in CDCl3; b-hairpin in CD3OH
X-ray: a-turn
X-ray: Type-I0 b-turn
NMR: Helix in CDCl3; Helix + b-hairpin in
CD3OH and DMSO-d6
25
21
D
Boc-Phe-Val-Aib- Ala-Leu-Phe-NHMe (2)
D
Boc-Val-Aib- Ala-Leu-NHMe (3)
2
3
Present study
Present study
D
Boc-Aib- Ala-NHMe (4)
4
5
Present study
25
Boc-Leu-Phe-Val-Aib-Aib-Leu-Phe-Val-OMe (5)
6
7
Boc-Phe-Val-Aib-Aib-Leu-Phe-NHMe (6)
Boc-Val-Aib-Aib-Leu-NHMe (7)
NMR: Helix in CDCl3 and CD3OH
NMR: Helix in CDCl3 and CD3OH
X-ray: 310-helix
Present study
Present study
8
9
Boc-Aib-Aib-NHMe (8)
Boc-Leu-Val-Val-Aib-Gly-Leu-Val-Val-OMe (9)
X-ray: Type-III b-turn
Present study
26
NMR: Helix in CDCl3 and CD3CN; b-hairpin in
CD3OH and DMSO-d6
NMR: b-hairpin, Type-II0 b-turn
L
Boc-Leu-Val-Val- Pro- Pro-Leu-Val-Val-OMe (10)
D
10
11
25
25
L
Boc-Leu-Phe-Val- Pro- Pro-Leu-Phe-Val-OMe (11) NMR: b-hairpin, Type-II0 b-turn
D
X-ray: b-hairpin, Type-II0 b-turn
X-ray: b-hairpin, Type-II0 b-turn
NMR: Helix in CDCl3; b-hairpin in CD3OH
NMR: b-hairpin in CDCl3 and CD3OH
NMR: Helix in CDCl3; b-hairpin in CD3OH
NMR: b-hairpin in CDCl3, CD3OH and DMSO-d6
X-ray: b-hairpin, Type-I’ b-turn
D
Boc-Leu-Val-Val- Pro-Aib-Leu-Val-Val-OMe (12)
D
Boc-Leu-Val-Val-Aib- Ala-Leu-Val-Val-OMe (13)
D
Boc-Leu-Val-Val-Aib- Val-Leu-Val-Val-OMe (14)
12
13
14
15
16
25
25
25
25
25
22
25
Boc-Leu-Phe-Val-Aib-Gly-Leu-Phe-Val-OMe (15)
D
Boc-Leu-Val-Val-Aib- Pro-Leu-Val-Val-OMe (16)
17
Boc-Leu-Phe-Val-Aib-Ala-Leu-Phe-Val-OMe (17)
NMR: Helix in CDCl3 and DMSO-d6
elements in polypeptide structures.3–6 This conformational permitted crystallographic characterization of the b-hairpin
D
feature is determined by the backbone torsion angles (/, w) conformation in the octapeptide, Boc-Leu-Val-Val-Aib- Pro-
of two residues (i and i + 1) in peptides.7,8 The type I/III b- Leu-Val-Val-OMe.22 The achiral dipeptide segment Aib-Gly
turn, which is formed when both residues i and i + 1 lie in has also been used to stabilize b-hairpin folding, with several
the right handed helical (aR) region of Ramachandran space, model sequences investigated by isotope-edited IR spectros-
is the conformational element which, when repeated, leads to copy.23,24 An earlier study from this laboratory addressed the
the formation of a 310-helical segment in a polypeptide conformation directing effect of the Xxx residue in the octa-
chain. For example, the repetitive type-III consecutive b-turn peptide Boc-Leu-Phe-Val-Aib-Xxx-Leu-Phe-Val-OMe. For
structure corresponds to a single turn of a 310-helix.9–11 The Xxx ¼ Ala, the b-hairpin conformation appears strongly
D
type-I0/III0 (prime) turn is formed when residues i/i + 1 favored in hydrogen bonding solvents like CD3OH and
adopt left handed helical (aL) conformations in which both DMSO-d6, whereas in poorly interacting solvents like CDCl3,
/ and w have positive values. This prime turn nucleates b- NMR evidence favors a mixture of a-helical and b-hairpin
hairpin formation when placed centrally in a polypeptide conformations. For Xxx ¼ Aib, helical conformations are
segment consisting of L-Residues.12,13 Conformationally con- exclusively observed in CDCl3, while in CD3OH and DMSO-
strained residues have been developed as a strategy to con- d6 the NMR data supports a mixed population of a-helices
struct obligatory turn segments of both type-I/III and type- and b-hairpins.25 Evidence for similar conformational equili-
I0/III0 categories. In particular, Aib-LXxx segments may be bria was also obtained for the octapeptide Boc-Leu-Val-Val-
used to preferentially populate local type-I/III conforma- Aib-Gly-Leu-Val-Val-OMe26 (see Table I). For an eight resi-
0
D
tions, while Aib- Xxx segments show a preference for type-I / due protected peptide, formation of a 310-helix results in six
III0 b-turn structures.14 The formation of right handed 310/a- intramolecular (C10) hydrogen bonds, while an a-helix yields
helices in peptides containing Aib-LXxx sequence has been five intramolecular (C13) hydrogen bonds. b-Hairpin results
widely demonstrated.15–20 The b-hairpin structure of an oc- in four cross-strand hydrogen bonds. Thus, in solvents like
0
D
tapeptide containing a central Aib- Ala type-I b-turn has CDCl3 which do not compete for backbone hydrogen bond-
been characterized in crystals by X-ray diffraction.21 More ing sites, helix formation is likely to be favored, while b-hair-
recently, the obligatory type-I b-turn template, Aib- Pro has pins may be promoted in solvents like CD3OH, which can
0
D
Biopolymers (Peptide Science)

746
Rajagopal et al.
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
FIGURE 1 Schematic representation of the solvent dependent
conformational equilibrium between an a-helix (left) and a
b-hairpin (right).
Da); 830.1 [M + Na]+; 846.1 [M + K]+; 7, 514.3 [M + H]+ (Mcal
¼
513 Da); 536.2 [M + Na]+; 8, 302.2 [M + H]+ (Mcal ¼ 301 Da);
324.2 [M + Na]+.
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-
NMR Spectroscopy
Experiments 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.
D
Val-Aib-Xxx-Leu-Phe-Val-OMe, Xxx ¼ Ala (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-
D
D
Phe-Val-Aib- Ala-Leu-Phe-NHMe (2), Boc-Val-Aib- Ala-
Leu-NHMe (3), Boc-Phe-Val-Aib-Aib-Leu-Phe-NHMe (6),
and Boc-Val-Aib-Aib-Leu-NHMe (7). In addition, we report
X-Ray Diffraction
Crystals 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
D
the crystal structure of turn segments, Boc-Aib- Ala-NHMe
(4), Boc-Aib-Aib-NHMe (8) and the truncated tetrapeptide
D
models Boc-Val-Aib- Ala-Leu-NHMe (3) and Boc-Val-Aib-
Aib-Leu-NHMe (7).
MATERIALS AND METHODS
Peptide Synthesis
Peptides 1 and 5 were synthesized as previously reported.22 Peptides diffraction parameters for peptides 3, 4, 7, and 8 are summarized in
2, 3, 4, 6, 7, and 8 were prepared by standard solution phase meth- Table II. The crystallographic coordinates for the structures are de-
ods. The t-butyloxycarbonyl (Boc) and methyl ester were used for posited at the Cambridge Crystallographic Data Centre with deposi-
N- and C-terminal protection, respectively. Peptide bond formation tion numbers CCDC 801464 (3) 801463, (4) 801466 (7), and
was achieved by using N,N0-dicyclohexylcarbodiimide (DCC) and 801465 (8). These data can be obtained free of charge via
1-hydroxybenzotriazole (HOBt). The conversion of C-terminal www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge
Biopolymers (Peptide Science)

Chain Length Effects on Helix-Hairpin Distribution
747
Table II Crystal and Diffraction Parameters of Peptides 3, 4, 7, and 8
Peptide 3
Peptide 4
Peptide 7
Peptide 8
Empirical formula
Crystal habit
C24H45N5O6 ꢀ H2O
Clear
C13H25N3O4 ꢀ 0.33H2O
Clear
C25H47N5O6
Clear
C14H27N3O4 ꢀ H2O
Clear
Crystal size (mm)
Crystallizing solvent
Space group
0.31 3 0.25 3 0.13
Methanol/water
C2
0.26 3 0.1 3 0.04
Methanol/water
P212121
0.66 3 0.34 3 0.14
Methanol/water
P21
0.6 3 0.25 3 0.04
Methanol/water
P21
Cell parameters
˚
a (A)
˚
17.674(1)
11.508(8)
16.693(1)
90, 108.1(1), 90
3228.2(4)
4/1
10.526(2)
21.781(4)
22.489(4)
90, 90, 90
5155.8(2)
12/3
10.244(8)
11.164(8)
13.954(1)
90, 104.3(1), 90
1546.4(2)
2/1
8.653(1)
12.517(2)
8.861(1)
90, 94.6(2), 90
956.7(2)
2/1
b (A)
˚
c (A)
a, b, c (deg)
3
˚
Volume (A )
Z/Z’
Cocrystallized solvent
Molecular weight
Density (g/cm3)(cal)
F (000)
1 water
515.65
1.06
1 water
292.69
1.131
None
513.6
1 water
319.4
1.109
1.103
560
1120
MoKa
2.14 to 25.0
1904
MoKa
2.04 to 27.1
348
MoKa
2.82 to 26.2
Radiation
h range (o)
Scan type
MoKa
2.1 to 26.8
x
15504
x
20866
x
11697
x
6690
Measured reflections
Unique reflections
Observed reflections [|F|>4r(F)]
Final R/wR2 (%)
Goodness of fit (S)
2995
2672
6264
1900
3216
2988
1857
1688
6.73/18.1
1.11
8.58/14.6
0.93
3.53/9.93
1.07
4.12/10.8
0.968
3
˚
Dqmax/Dqmin (e/A )
No. of restraints/parameters
Data to parameter ratio
0.69/ꢁ0.17
1/325
8.2:1
0.22/ꢁ0.27
0/550
3.5:1
0.12/ꢁ0.13
1/325
9.2:1
0.17/ꢁ0.14
1/267
6.3:1
D
Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ,
UK; fax: (+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk).
OMe (5). The central Aib- Ala 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,
RESULTS
NMR Studies in Solution
The nature of the peptide conformations in solution was the type-I0 b-turn is expected to promote b-hairpin confor-
probed using backbone nuclear Overhauser effects (NOEs) mations. The chemical shift values for backbone NH and
to establish local residue conformations and by solvent de- CaH protons in CDCl3 and CD3OH are summarized in Table
pendence of amide NH chemical shifts, for differentiating III for peptides 2, 3, 6, and 7. Corresponding values for the
D
between internally hydrogen bonded and solvent exposed parent peptides Boc-Leu-Phe-Val-Aib- Ala-Leu-Phe-Val-
NH groups. NMR data were collected in two different solvent OMe (1) and Boc-Leu-Phe-Val-Aib-Aib-Leu-Phe-Val-OMe
systems CDCl3 and CD3OH. In the case of the aprotic solvent (5) are also listed for comparison.
CDCl3, conformations which maximize intramolecular
Figures 2 and 3 provide a comparison of key backbone
hydrogen bonding were expected to be favored, while NOEs obtained for the hexapeptides 2 and 6, in CD3OH sol-
CD3OH is anticipated to promote conformers which have a utions. In b-hairpin structures cross-strand (non-sequential)
greater number of solvent exposed backbone NH groups.
NOEs may be anticipated, while a-helices are characterized
The NMR parameters obtained for the truncated tetra by a succession of sequential NiH$Ni+1H NOEs. Figure 2a
D
and hexapeptides 2, 3, 6, and 7 are compared with those of reveals that in the hexapeptide Boc-Phe-Val-Aib- Ala-Leu-
D
the parent octapeptide Boc-Leu-Phe-Val-Aib- Ala-Leu-Phe- Phe-NHMe (2) a relatively intense NOE is observed between
Val-OMe (1) and Boc-Leu-Phe-Val-Aib-Aib-Leu-Phe-Val- Val (2) NH and Leu (5) NH. Furthermore, an appreciable
Biopolymers (Peptide Science)

748
Rajagopal et al.
Biopolymers (Peptide Science)

Chain Length Effects on Helix-Hairpin Distribution
749
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.
NOE is also observed between Phe (1) CaH and Phe (6) terminus with Phe 6 adopting local conformation in the
CaH (Figure 2b). The absence of the sequential Leu (5) NH helical region, with consequent loss of the hydrogen bond,
$Phe (6) NH NOE suggests that continuous helical confor- Boc (CO) ꢀ ꢀ ꢀ NHCH3. Interestingly, a completely different
mations are not significantly populated. These results sug- pattern of NOEs is seen for peptide 2 in CDCl3 solution.
gest that despite truncation of the N- and C- terminal The cross-strand Val (2) NH$Leu (5) NH NOE is absent
strands, the b-hairpin conformation, nucleated by the cen- and a succession of sequential NiH$Ni+1H NOEs are
D
trally positioned Aib- Ala segment, is still appreciably popu- observed, suggesting that a continuous helical conformation
D
D
lated. The observed Aib (3) NH$ Ala (4) NH and Ala (4) predominates in CDCl3. Thus, in peptide 2 the competing
NH$Leu (5) NH NOEs are compatible with type-I0 b-turn effects of the intrapeptide and peptide-solvent interactions,
D
at the Aib- Ala segment. The Phe (6) NH$NHCH3 (7) determine the nature of conformations that are populated
NOE may be rationalized by occurrence of fraying at C- in solution.
Biopolymers (Peptide Science)

750
Rajagopal et al.
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-
D
cated tetrapeptide models Boc-Val-Aib- Ala-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
D
3 (Aib- Ala) 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 (ꢂ 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). The
data 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 obtained
in CDCl3-DMSO-d6 mixtures clearly establish that Aib (3),
Leu (4), and NHCH3 (5) groups are solvent shielded (Dd ꢃ
0.21 ppm), while Val (1) NH and Aib (2) NH groups exhibit
FIGURE 3 Partial 500 MHz ROESY spectra (NH$NH NOEs) of
peptide 6 (a) CD3OH, (b) CDCl3.
The behavior of the related hexapeptide, Boc-Phe-Val-
Aib-Aib-Leu-Phe-NHMe (6) contrasts sharply with that
observed for peptide 2. Figure 3 shows the partial ROESY
spectra of peptide 6 in CDCl3 and CD3OH. In this case, only
sequential NiH$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
Biopolymers (Peptide Science)

Chain Length Effects on Helix-Hairpin Distribution
751
D
FIGURE 4 (a) Partial 700 MHz ROESY Spectra of Boc-Val-Aib- Ala-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.
D
large Dd values (ꢂ1.25 ppm) characteristic of solvent obtained for peptide 7. Peptide 3 (Aib- Ala) appears to have
exposed groups. These observations strongly suggest the pre- a significant population of hairpin conformations in CDCl3;
ponderance of states in which three intramolecular hydrogen an inference drawn from the low Dd value for Val (1) NH.
bonds are present, consistent with a 310-helical conforma- The observed pattern of Dd values is consistent with mixed
tion. Temperature coefficient (dd/dT) values in CD3OH are populations of 310 helical and hairpin structures. The large
indeed lower for the Aib (3) NH, Leu (4) NH, and NHCH3 dd/dT values for obtained Val (1) NH in CD3OH is sugges-
(5) groups, although the distinction between the various NH tive of fraying of the hairpin in the more strongly interacting
groups is less pronounced. This is suggestive of a shift of con- solvent.
former populations, with a greater proportion of nonhelical
In the hexapeptide Boc-Phe-Val-Aib-Aib-Leu-Phe-NHMe
structures in CD3OH, a solvent which may be expected to (6), the Dd values are consistent with continuous 310-helical
compete for backbone hydrogen bonding sites. Replacement conformations with five intramolecularly hydrogen bonded
of residue 3 in peptide 7 (Boc-Val-Aib-Aib-Leu-NHMe) with NH groups. Indeed, only Phe (1) NH and Val (2) NH groups
D
D
Ala yields peptide 3 (Boc-Val-Aib- Ala-Leu-NHMe). Inspec- have Dd values (ꢂ1.1 ppm) characteristic of exposed NH
tion of Table III reveals that Aib (2) NH shows a very high groups. In CD3OH, differentiation of NH groups is not
Dd value (1.92 ppm) indicative of exposure to solvent. The obtained from dd/dT values. In the case of peptide 2 (Boc-
D
other four NH groups show much lower solvent dependent Phe-Val-Aib- Ala-Leu-Phe-NHMe), Dd values for Phe (1)
chemical shifts, with Val (1) NH yielding the lowest Dd value NH are low (0.58 ppm), suggesting that hairpin conformers
of 0.23 ppm. These results are in sharp contrast to those in which the N-terminus NH group is hydrogen bonded are
Biopolymers (Peptide Science)

752
Rajagopal et al.
Table IV Torsion Angles (deg)
Residues
/
w
x
v1
v2
Peptide 3
Val(1)
Aib(2)
ꢁ54.9 137.2
175.6 61.5, ꢁ60.8
18.9 ꢁ175.8
1.0 ꢁ171.2
59.0
81.3
D
Ala(3)
Leu(4) ꢁ111.9 ꢁ36.1 ꢁ178.4 ꢁ60.3
Peptide 4
ꢁ62.8, 174.0
Molecule-A
Aib
54.4
80.3
38.3
174.9
D
Ala
Molecule-B
Aib
7.6 ꢁ178.8
59.4
95.7
34.4
ꢁ5.7
177.5
177.0
D
Ala
Molecule-C
Aib
56.1
78.0
34.3
6.2
176.4
179.8
D
Ala
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
D
FIGURE 5 (a) Molecular conformation of Boc-Aib- Ala-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-
The estimated standard deviation & 0.58, 1.28, 0.28, and 0.38 for Peptides
3, 5, 7, and 8, respectively.
D
Aib- Ala-NHMe (4, molecule C) with the corresponding enan-
tiomeric conformation of Boc-Aib-Aib-NHMe (8).
by comparing the parameters obtained for the parent octa-
D
peptide 1 (Aib- Ala) 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.
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
Molecular Conformation in Crystals
Diffraction quality, single crystals were obtained for the
D
dipeptides Boc-Aib- Ala-NHMe (4), Boc-Aib-Aib-NHMe
D
(8) and tetrapeptides Boc-Val-Aib- Ala-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-
D
marized in Tables IV and V. Boc-Aib- Ala-NHMe (4) crystal-
D
FIGURE 6 Molecular conformation of (a) Boc-Val-Aib- Ala-Leu-
lized with three independent molecules in the orthorhombic
NHMe (3) and (b) Boc-Val-Aib-Aib-Leu-NHMe (7) in crystals.
cell. All three molecules adopt b-turn conformations,
Biopolymers (Peptide Science)

Chain Length Effects on Helix-Hairpin Distribution
753
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)
N(2)
O1w
O1w
O(4)a
O1wb
O(2)
3.010
2.874
2.872
2.776
2.164
2.055
144.0
140.8
167.8
158.8
O(3)c
Intramolecular
4?1
4?1
5?1
Peptide 4
N(3)
N(4)
O(0)
O(1)
O(1)
3.000
2.862
3.055
2.265
2.031
2.221
144.1
127.6
131.7
140.5
132.3
135.8
143.5
162.1
163.3
N(1M)
Intermolecular
N(11)
N(12)
N(21)
N(22)
N(31)
N(32)
O1w
O(21)d
O(12)e
O(31)f
O(11)
O1w
3.008
2.885
2.996
2.869
3.014
3.316
3.006
2.869
2.197
2.058
2.210
2.092
2.168
2.654
139.1
147.7
140.3
149.0
145.4
146.8
131.1
153.6
157.2
161.0
151.8
149.9
167.6
134.8
O(22)
O(22)
O(32)g
129.1
133.5
O1w
Intramolecular
4?1
4?1
4?1
Peptide 7
N(11M)
N(21M)
N(31M)
O(02)
O(04)
O(06)
2.952
3.008
2.913
2.144
2.187
2.077
120.3
115.1
121.2
127.1
119.3
125.6
156.2
159.7
163.8
Intermolecular
N(2)
O(4)d
2.923
2.180
144.0
153.2
144.4
Intramolecular
4?1
4?1
4?1
Peptide 8
N(3)
N(4)
O(0)
O(1)
O(2)
3.098
2.973
2.977
2.313
2.146
2.157
121.0
129.9
121.3
127.0
134.3
127.0
152.0
161.1
159.1
N(1M)
Intermolecular
N(1)
N(2)
O1w
O1w
O(1)h
O1w
O(1)h
O(2)i
3.091
2.938
2.889
2.832
2.306
2.089
2.179
1.997
152.2
153.7
168.4
170.0
155.6
173.1
134.6
128.7
139.8
130.6
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, ꢁzꢁ1.
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 –xꢁ1, y ꢁ 1/2, ꢁz.
i Symmetry related by ꢁx, y ꢁ 1/2, ꢁz.
D
stabilized by a single intramolecular 4?1 hydrogen bond was based on the presence of the Ala residue. It should be
Boc C ¼ O ꢀ ꢀ ꢀ HNMe. The torsion angles presented in Table noted that the diffraction data would also be consistent with
IV correspond to a type-I0 b-turn conformation. This choice an enantiomer structure. Boc-Aib-Aib-NHMe (8) crystallizes
Biopolymers (Peptide Science)

754
Rajagopal et al.
30
D
L
D
FIGURE 7 Examples of a-turns observed in peptide crystals (a) Piv- Pro- Pro- Ala-NHMe , (b)
31
D
Dnp-Val-Aib-Gly-Leu-pNA , and (c) Boc-Val-Aib- Ala-Leu-NHMe (3). Only backbone atoms of
the a-turn are shown for clarity. The torsion angles and hydrogen bond lengths are shown.
22
D
in the chiral, monoclinic space group P21, despite the ab- Leu-Val-Val-Aib- Pro-Leu-Val-Val-OMe. In both crystal-
sence of chirality in the sequence. The conformational angles line octapeptides, the central segment nucleating the b-hair-
presented in Table IV correspond to the Type-III conforma- pin adopts a type-I0 b-turn conformation.
tion. This achiral sequence would be expected to exist in so-
The conformational angles (/, w) for the tetrapeptide seg-
D
lution as an equimolar mixture of the energetically degener- ments reveal that Boc-Val-Aib- Ala-Leu-NHMe (3) differs
ate type-III/III0 b-turn conformations. While crystallization from the corresponding segment in b-hairpins only in the
in an achiral, centric space group containing both enantio- torsion angles Val (1) / and Leu (4) w. The former is altered
meric conformations, might have been anticipated, in the by 608, while the latter is changed by *1608. These two
present case the less frequently observed trapping of a single, changes result in the transformation of the b-hairpin struc-
enantiomeric conformation in a chiral space group has been ture to the crystallographically observed conformation, in
obtained. A 4?1 hydrogen bond, Boc C ¼ O ꢀ ꢀ ꢀ HNMe, is which Val (1) CO forms bifurcated hydrogen bonds with Leu
observed in the dipeptide Boc-Aib-Aib-NHMe (8).
(4) NH (C10, 4 ?1) and NHMe (C13, 5?1) The observed
The tetrapeptide Boc-Val-Aib-Aib-Leu-NHMe (7) adopts C13 hydrogen bond in peptide 3 constitutes an interesting
the expected 310-helical conformation stabilized by three suc- example of a nonhelical a-turn, previously characterized as
cessive 4?1 hydrogen bonds. All four residues adopt /, w isolated conformational features in protein structures.30–32
values lying in right handed (aR) region of conformational Such C13, 5?1 hydrogen bonds are observed relatively infre-
space. Boc-Val-Aib- Ala-Leu-NHMe (3) adopts a dramati- quently in the crystal structures of short peptides.33,34 Two
D
cally different conformation (see Figure 6). The observed examples are illustrated in Figure 7.
conformation is stabilized by three intramolecular hydrogen
All examples illustrated in Figure 7 correspond to distinct
bonds: Boc C ¼ O ꢀ ꢀ ꢀ H-N Ala (3) (4?1) Val (1) C ¼ a-turn families. Peptide 3 may be classified as a type-I0 b-turn
D
O ꢀ ꢀ ꢀ HN Leu (4) (4 ?1) and Val (1) C ¼ O ꢀ ꢀ ꢀ HNMe (5) followed by a distorted aR (bridge) conformation. In the pre-
L
D
D
(5?1). Inspection of the backbone torsion angles in Table IV viously reported peptide Piv- Pro- Pro- Ala-NHMe, a dis-
reveals that the Aib (2) and Ala (3) segments adopt the torted type-II0 b-turn conformation is followed by a distorted
D
type-I0 b-turn conformation, while the Val (1) residue lies in aL residue. In this case, the C13 hydrogen bond is significantly
the polyproline (PII, semi extended) region of conforma- shorter than the C10 hydrogen bond.33 The structure of tetra-
tional space. Leu (4) lies in the bridge region of the Rama- peptide Dnp-Val-Aib-Gly-Leu-Pna constitutes an example of
chandran map (/ ¼ ꢁ1128, w ¼ 308). The tetrapeptide, a type-II0 b-turn conformation followed by an aL residue.34
Boc-Val-Aib- Xxx-Leu-NHMe segment has been character- This results in a consecutive type-II0-III b-turn structure,
D
ized in two independent octapeptide b-hairpin structures, which has been widely observed in short peptides containing
Boc-Leu-Phe-Val-Aib- Ala-Leu-Phe-Val-OMe (1)21 and Boc- the constrained a,a-dialkylated amino acids.11,35–38 In this
D
Biopolymers (Peptide Science)

Chain Length Effects on Helix-Hairpin Distribution
755
˚
case, the C13 hydrogen bond (3.3 A) is appreciably longer lated in solution. The results of this study may be used
˚
than the two C10 hydrogen bonds (2.98 A and 3.13 A).
˚
to design suitable sequences for directly monitoring helix-
hairpin equilibria in solution.
DISCUSSION
Insertion of Aib-XXxx segments into peptide sequences pro-
motes the formation of folded b-turns, which in turn can nu-
S. Aravinda thanks the Department of Science and Technology for
the award of SERC FAST Track Young Scientist Fellowship.
X
cleate further secondary structure formation. When Xxx is
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X
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ˇ
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