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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: shamala@physics.iisc.ernet.in or Prof. P.
Balaram, Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560
012, India; e-mail: pb@mbu.iisc.ernet.in
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.
Biopolymers (Peptide Science)
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: deposit@ccdc.cam.ac.uk).
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.
Helical Conformations of Hexapeptides 363
Biopolymers (Peptide Science)
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.
364 Kantharaju et al.
Biopolymers (Peptide Science)
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.
Helical Conformations of Hexapeptides 365
Biopolymers (Peptide Science)
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.
366 Kantharaju et al.
Biopolymers (Peptide Science)
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$NH Regions.
Helical Conformations of Hexapeptides 367
Biopolymers (Peptide Science)
solvent exposure of the residue 3 NH in peptide 1 as com-
pared to peptides 2 and 3 (Figures 4 and 5). To further exam-
ine conformational choices in peptides 1–10, we turn to a
comparison of nuclear Overhauser effects (NOEs) between
peptide backbone protons.
For amino acid residues adopting conformations in the
helical region of /, w space, the distances between the NH
protons of the preceding and succeeding peptide units
(dNNi,i+1) are short (2.8 A)60 resulting in the observation of
relatively intense NOEs. The observation of successive
dNNi,i+1 NOEs along the polypeptide sequence, is diagnostic
of the formation of a helical segment, especially when the
presence of solvent-shielded NH groups is established.
Figure 7 compares the NOEs between backbone protons
(CaH, NH) observed in peptide 2 (Piv-Pro-Pro-Ala-Leu-Aib-
Phe-OMe), peptide 3 (Piv-Pro-Pro-Ala-Aib-Ala-Aib-OMe),
and peptide 4 (Piv-DPro-Ala-Aib-Leu-Aib-Phe-OMe). In pep-
tide 2, which contains the N-terminus diproline segment, suc-
cessive dNNi,i+1 NOEs are observed over the segment residues
3–6. In addition, the CaH$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.
368 Kantharaju et al.
Biopolymers (Peptide Science)
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.
REFERENCES1. DeTar, D. F.; Luthra, N. P. J Am Chem Soc 1977, 99,
1232–1244.
2. MacArthur, M. W.; Thornton, J. M. J Mol Biol 1991, 218, 397–
412.
3. Yaron, A.; Naider, F. CRC Crit Rev Biochem Mol Biol 1993, 28,
31–81.
4. Madison, V. Biopolymers 1977, 16, 2671–2692.
5. Takeuchi, Y.; Marshall, G. R. J Am Chem Soc 1998, 120, 5363–
5372.
6. Chalmers, D. K.; Marshall, G. R. J Am Chem Soc 1995, 117,
5927–5937.
7. Venkatraman, J.; Shankaramma, S. C.; Balaram, P. Chem Rev
2001, 101, 3131–3152.
8. Mahalakshmi, R.; Balaram, P. In D-Amino Acids: A New Fron-
tier in Amino Acid and Protein Research; Konno, R.; Fisher, G.
H.; Brueckner, H.; d’Aniello, A.; Fujii, N.; Homma, H., Eds.;
Nova Science: New York, 2006; pp 415–430.
9. Aravinda, S.; Shamala, N.; Roy, R. S.; Balaram, P. Proc Indian
Acad Sci (Chem Sci) 2003, 115, 373–400.
10. Wuthrich, K.; Grathwohl, C. FEBS Lett 1974, 43, 337–340.
11. Momany, F. A.; McGuire, R. F.; Burgess, A. W.; Scheraga, H. A. J
Phys Chem 1975, 79, 2361–2381.
12. Ramachandran, G. N.; Mitra, A. K. J Mol Biol 1976, 107, 85–92.
FIGURE 8 Partial expansions of the ROESY spectra showing CaH$NH region of peptides 1, 6, 7, and 8.
Helical Conformations of Hexapeptides 369
Biopolymers (Peptide Science)
13. Schulz, G. E.; Schirmer, R. H. Principles of Protein Structure;
Springer: New York, 1979; pp 25–26.
14. Hinderaker, M. P.; Raines, R. T. Protein Sci 2003, 12, 1188–
1194.
15. Richardson, J. S. Adv Protein Chem 1981, 34, 167–339.
16. DeGrado, W. F. Adv Protein Chem 1988, 39, 51–124.
17. Huda’ky, I.; Baldoni, H. A.; Perczel, A. J Mol Struct (Theochem)
2002, 582, 233–249.
18. Chou, P. Y.; Fasman, G. D. J Mol Biol 1977, 115, 135–175.
19. Wilmot, C. M.; Thornton, J. M. J Mol Biol 1988, 203, 221–232.
20. Richardson, J. S.; Richardson, D. C. In Prediction of Protein
Structure and the Principles of Protein Conformation; Fasman,
G. D., Ed.; Plenum: New York, 1989; pp 1–98.
21. Li, S.-C.; Goto, N. K.; Williams, K. A.; Deber, C. M. Proc Natl
Acad Sci USA 1996, 93, 6676–6681.
22. Chan, A. W.; Hutchinson, E. G.; Harris, D.; Thornton, J. M.
Protein Sci 1993, 2, 1574–1590.
23. Barlow, D. J.; Thornton, J. M. J Mol Biol 1988, 201, 610–619.
24. Yun, R. H.; Anderson, A.; Hermans, J. Proteins Struct Funct
Genet 1991, 10, 219–228.
25. Deber, C. M.; Glibowicka, M.; Woolley, G. A. Biopolymers
1990, 29, 149–157.
26. Piela, L.; Nemethy, G.; Scheraga, H. A. Biopolymers 1987, 26,
1587–1600.
27. Sankararamakrishnan, R.; Vishveshwara, S. Biopolymers 1990,
30, 287–298.
28. Sankararamakrishnan, R.; Vishveshwara, S. Int J Peptide Protein
Res 1992, 39, 356–363.
29. Kim, M. K.; Kang, Y. K. Protein Sci 1999, 8, 1492–1499.
30. Presta, L. G.; Rose, G. D. Science 1988, 240, 1632–1641.
31. Richardson, J. S.; Richardson, D. C. Science 1988, 240, 1648–
1652.
32. Aurora, R.; Rose, G. D. Protein Sci 1998, 7, 21–38.
33. Gunasekaran, K.; Nagarajaram, H. A.; Ramakrishnan, C.;
Balaram, P. J Mol Biol 1998, 275, 917–932.
34. Viguera, A. R.; Serrano, L. Protein Sci 1999, 8, 1733–1742.
35. Venkatachalapati, Y. V.; Balaram, P. Nature 1979, 281, 83–84.
36. Kemp, D. S.; Boyd, J. G.; Muendel, C. C. Nature 1991, 352, 451–
454.
37. Kemp, D. S.; Curran, T. P.; Boyd, J. G.; Allen, T. J. J Org Chem
1991, 56, 6683–6697.
38. Kemp, D. S.; Curran, T. P.; Davis, W. M.; Boyd, J. G.; Muendel,
C. J Org Chem 1991, 56, 6672–6682.
39. Job, G. E.; Heitmann, B.; Kennedy, R. J.; Walker, S. M.; Kemp,
D. S. Angew Chem Int Ed 2004, 43, 5649–5651.
40. Heitmann, B.; Job, G. E.; Kennedy, R. J.; Walker, S. M.; Kemp,
D. S. J Am Chem Soc 2005, 127, 1690–1704.
41. Rai, R.; Aravinda, S.; Kanagarajadurai, K.; Raghothama, S.; Sha-
mala, N.; Balaram, P. J Am Chem Soc 2006, 128, 7916–7928.
42. Chatterjee, B.; Saha, I.; Raghothama, S.; Aravinda, S.; Rai, R.;
Shamala, N.; Balaram, P. Chem Eur J 2008, 14, 6192–6204.
43. Mutter, M.; Nefzi, A.; Sato, T.; Sun, X.; Wahl, F.; Wohr, T. Pep-
tide Res 1995, 8, 145–153.
44. Wohr, T.; Wahl, F.; Nefzi, A.; Rohwedder, B.; Saxo, T.; Sun, X.;
Mutter, M. J Am Chem Soc 1996, 118, 9218–9227; and referen-
ces cited therein.
45. Dumy, P.; Keller, M.; Ryan, D. E.; Rohwedder, B.; Wohr, T.; Mut-
ter, M. J Am Chem Soc 1997, 119, 918–925.
46. Keller, M.; Sager, C.; Dumy, P.; Schutkowski, M.; Fischer, G. S.;
Mutter, M. J Am Chem Soc 1998, 120, 2714–2720.
47. Keller, M.; Lehmann, C.; Mutter, M. Tetrahedron 1999, 55, 413–
422.
48. Kantharaju; Raghothama, S.; Raghavender, U. S.; Aravinda, S.;
Shamala, N.; Balaram, P. Biopolymers (Pept Sci) 2009, 92, 405–
416.
49. Lewis, N. J.; Inloes, R. L.; Hes, J. J Med Chem 1978, 21, 1070–
1073.
50. Sheldrick, G. M. SHELXS-97, A Program for Automatic Solu-
tion of Crystal Structures; University of Gottingen: Gottingen,
Germany, 1997.
51. Sheldrick, G. M. SHELXL-97, A Program for the Refinement of
Crystal Structures; University of Gottingen: Gottingen, Ger-
many, 1997.
52. O’Neil, K. T.; DeGrado, W. F. Science 1990, 250, 646–651.
53. Fairman, R.; Anthony-Cahill, S. J.; DeGrado, W. F. J Am Chem
Soc 1992, 114, 5458–5459.
54. Mitchell, J. B. O.; Smith J. Proteins Struct Funct Bioinfor 2003,
50, 563–571.
55. Aravinda, S.; Shamala, N.; Desiraju, S.; Balaram, P. Chem Com-
mun 2003, 2454–2455.
56. Karle, I. L.; Gopi, H. N.; Balaram, P. Proc Natl Acad Sci USA
2003, 100, 13946–13951.
57. Vasudev, P. G.; Ananda, K.; Chatterjee, S.; Aravinda, S.; Sha-
mala, N.; Balaram, P. J Am Chem Soc 2007, 129, 4039–4048.
58. Karle, I. L.; Balaram, P. Biochemistry 1990, 24, 6747–6756.
59. Toniolo, C.; Benedetti, E. Trends Biochem Sci 1991, 16, 350–
353.
60. Wuthrich, K. NMR of Proteins and Nucleic Acids; Wiley: New
York, 1986; pp 127.
370 Kantharaju et al.
Biopolymers (Peptide Science)