Cured of "stickiness", Poly- l β-hairpin is promoted with ll -to- dd mutation as a protein and a hydrolase mimic

Article abstract of DOI:10.1021/jp1062572

The planar ribbon of the poly-l β-hairpin is modified to a local ～90° bend by mutating a cross-strand pair of residues from ll to dd structure. The bend is furnished aromatic side chains in proximity of acid-base-nucleophile side chains, toward the possib

Full text of DOI:10.1021/jp1062572

J. Phys. Chem. B 2010, 114, 16887–16893
16887
Cured of “Stickiness”, Poly-L ꢀ-Hairpin is Promoted with LL-to-DD Mutation as a Protein
and a Hydrolase Mimic
†
‡
Kirti Patel, Bhupesh Goyal, Anil Kumar, Nand Kishore, and Susheel Durani*
Department of Chemistry, Indian Institute of Technology Bombay, Mumbai-400076, India
ReceiVed: July 7, 2010; ReVised Manuscript ReceiVed: October 25, 2010
The planar ribbon of the poly-L ꢀ-hairpin is modified to a local ∼90° bend by mutating a cross-strand pair
of residues from LL to DD structure. The bend is furnished aromatic side chains in proximity of
acid-base-nucleophile side chains, toward the possibility of catalyzed hydrolysis of an active-site-anchored
substrate. Six sequences permuted in putative catalytic side chains are evaluated for activity and variability
as hydrolase enzymes. Studies using CD, NMR, spectorofluorometry, ITC, and molecular dynamics establish
that the sequences over the bent ꢀ-hairpin are by and large aggregation-free folds soluble to at least millimolar
concentration, and thus remarkably contrasted with “stickiness” of the canonical poly-L ꢀ-hairpin. The
heterochiral fold displays cooperative ordering and affinity for acetylcholine, p-nitrophenylacetate, and
p-nitrophenylphosphate, presumably as the ligands in its aromatic pocket as a bent hairpin. The fold displays
hydrolytic activity against p-nitrophenylacetate and manifests saturation kinetics with respect to substrate
concentration. However, the catalysis power is feeble, which remains unaffected by repositioning
acid-base-nucleophile side chains. Stereochemistry is proven to be critical in the balance between mutually
competitive forces of polypeptide structure involved in guidance of folding or aggregation of the structure.
Residue stereochemistry is confirmed in its value as the alphabet for design of protein folds to desired molecular
shapes.
Introduction
geometry, the canonical ꢀ-hairpin was engineered as a
receptor2
6-29
and was exploited for nanostructure design. The
30
The sequential codes that define proteins over the alphabet
in R-amino acid structures remain poorly understood in their
workings.1 Proteins are approached by joining R-amino acids
canonical structure is in the geometry of its flat ribbon defined
with poly-L stereochemistry. The ribbon may be modified by
bending its geometry without altering the ability in antiparallel
strands to interact mutually, and the approach was advocated
for designing folds to shapes of interest over L- and D-R-amino
-3
4
-7
as defined sequences, but the motivation remains the probing
of the folding principles rather than that of harnessing their
applications. In folding as conformationally well-defined struc-
tures, proteins manifest interactions that are specific for not only
their chemical sequences over R-amino acid residues but also
for their stereochemical sequences. Being always L in residue
stereochemistry, protein polypeptides are stereochemically
invariant sequences poly-L in structure. The stereochemistry
defines protein folds and may determine why the folds are
codable to specific molecular shapes and to specific interaction
11
acids as the stereochemical alphbet. Modification of stereo-
chemistry is also promising for addressing protein-folding
23,31,40
problems.
Stereochemistry determines not only how the
canonical poly-L ꢀ-hairpin folds but also how it may interact
within folded proteins and why it is susceptible to aggregation.
Considering the geometrical role of canonical stereochemistry,
the poly-L ꢀ-hairpin was guided with sequence effects to self-
8
-10
and catalytic specificities as enzymes.
Given that protein
assembly as a C
over peptides of main-chain structure and as a D
tetramer in face-to-face assembly of the C dimer over its side-
2
-symmetric dimer in side-by-side interactions
folds are defined stereochemically and that every sequence
position may be set to L or D stereochemistry, the options were
advocated as the alphabet to have protein shapes designed
2
-symmetric
2
chain structures.32 In the present study, we exploit the stereo-
chemical approach to bend the poly-L ꢀ-hairpin, which is shown,
by suppressing aggregation, to promote a monomolecular fold
as an autonomous protein, which we explore as an enzyme
model. The synthesis and characterization of six peptides
permuted in sequential placements of acid, base, and nucleophile
side chains, as potential chemical catalysts of a proposed
hydrolase model, are described. The sequences are shown to
display on one hand cooperative ordering as proteins and on
the other hand hydrolase activity of an enzyme. Stereochemistry
is proven to be critical in the balance between competitive forces
of polypeptide structure that specifically will direct its folding
or aggregation. The alphabet in L and D structures is further
proven in its value for the design of protein folds to the
molecular shapes of interest.
11
12
13
stereochemically. Illustrating the concept, boat, bracelet,
canoe,14 and pi-cup were reported as the shapes in protein
structure stereospecific for L and D sequences in polypeptides
that were specific in the diastereomer structures.
15
Since it models the ꢀ-sheet motif as an autonomous fold,1
6,17
the canonical poly-L ꢀ-hairpin has allowed a critical test in
protein folding of sequence effects.1
7-20
The studies have
enriched the understanding of protein folding and, by modeling
unfolded structures,2
1-23
have illuminated the aggregation of
2
4,25
unfolded proteins as amyloids.
Given its well-defined
*
Corresponding author. E-mail: sdurani@iitb.ac.in. Phone: +91-22-
2
5767164. Fax: +91-22-25767152.
†
Present address: Department of Chemistry, RNC Arts, JDB Commerce
NSC Science College, Nashik Road-422101, India.
&
‡
Present address: Department of Chemistry, University of Toronto, 80
St. George St., Toronto, ON, M5S 3H6, Canada.
1
0.1021/jp1062572  2010 American Chemical Society
Published on Web 12/02/2010

1
6888 J. Phys. Chem. B, Vol. 114, No. 50, 2010
Patel et al.
Figure 1. Canonical ꢀ-hairpin bent by LL to DD structure mutation.
Panel A: p-nitrophenylacetate docked in the aromatic pocket has its
acetyl moiety in proximity to the acid-base-nucleophile triad. Panel
B: the 10 lowest energy NMR-derived structures modeled with
CYANA.
Results
Design and Synthesis. The peptides 1-6 targeted in the study
are titled according to their variable residues as they read from
C to N terminus. Thus, with Ser (S), His (H), and Asp (D) in
the sequence positions 12, 10, and 7, peptide 1 is named SHD.
The peptides differ in the sequential placements of the potential
catalytic triad of His, Asp, Ser, or Cys (C) residues, as is implied
in the names as given below. All substituted residues are L
Figure 2. Apperance of a coupled exciton of aromatic chromophores
in the CD spectrum.
isomers, except the residues in the sequence positions 3, 8, and
QTOF-ESI MS, which are listed in Table 1 and the spectra
shown in Figure S1 of the Supporting Information. HPLC on
D
1
4, which are D isomers. Pro-Gly in the sequence positions 8
and 9 defines a type II′ ꢀ-turn, which we apply as a ꢀ-hairpin
an RP-C18 column with H O (0.01% TFA)-acetonitrile 0-100%
2
33
nucleator. The nucleated ꢀ-hairpin structure of canonical poly-L
gradient established that the peptides were >95% in apparent
structure is modified to introduce a local bend, as noted in Figure
purity.
1
A, by placing D residues in the sequence positions 3 and 14.
Characterization of Conformation. In CD spectra, the
peptides manifest coupled minima and maxima of ellipticity in,
respectively, 208-211 and 225-240 nm range, as noted in
The aromatic side chains in sequence positions 1, 5, and 16
and the acid-base-nucleophile side chains in sequence posi-
tions 7, 10, and 12 are juxtaposed for the possibility that a
substrate positioned against the aromatic cluster may have an
ester functionality hydrolyzable by acid, base, and nucleophilic
effects of side chains.
Figure 2. The couplets manifest exciton splitting, the hallmark
of interacting aromatic chromophores.1
6,34
Aromatic residues
characterize termini of the peptide, and hence, the peptides are
folded to have the aromatic side chains interacting.
(
(
(
(
(
(
1) SHD:
D
D
D
D
D
D
D
D
Ac-Y
1
1
1
1
1
1
E
E
E
E
E
E
2
2
2
2
2
2
L
L
L
L
L
L
3
3
3
3
3
3
N
N
N
N
N
N
4
4
4
4
4
4
W
W
W
W
W
W
5
5
5
5
5
5
S
S
S
6
6
6
D
7
P
8
G
9
-H10
-H10
-C10
-H10
-H10
-C10
T
11
S
12
A
13
V
14
K
15
F
16-NH
2
2
2
While similar in all peptides, the coupled exciton is unusually
strong in the peptide SHD. Presumably, auxiliary interactions
in this sequence promote stronger interactions of the aromatic
side chains. Trp in position 5 displays quenched fluorescence
relative to N-acetyltryptophanamide (NATA) as a model for
isolated Trp residue. Similar in all peptides, as noted in Figure
S2 of the Supporting Information, the quenching of Trp
fluorescence implies that the residue could be experiencing a
similar chemical environment in all sequences.
2) SHC:
Ac-Y
D
D
D
C
7
P
8
G
9
T
T
11
11
S
S
12
12
A
A
13
13
V
V
14
14
K
K
15
15
F
F
16-NH
16-NH
3) SCH:
Ac-Y
D
H
7
8 9
P G
4) DHS:
Ac-Y
D
D
T
T
T
6
S
7
P
8
G
9
S
11
D
12
A
13
V
14
K
15
F
16-NH
2
5) DHC:
Ac-Y
D
D
6
C
7
P
8
G
9
S
11
D
12
A
13
V
14
K
15
F
16-NH
2
6) DCH:
Ac-Y
D
D
6
H
7
P
8
G
9
S
11
D
12
A
13
V
14
K
15
F
16-NH
2
The peptides were made by manual-solid-phase synthesis
using Fmoc chemistry and displayed the expected ion peaks in
The peptides show a marginal or negligible effect of
concentration on molar ellipticities of CD minima in the 40-100
TABLE 1: Analytical Data Characterizing Structures and Interactions of Hairpin Peptides
SHD SHC SCH DHS
DHC
DCH
MWexp (Da)
MWobs (Da)
1891
1891
1879
1879
1879
1879
1891
1891
1907
1907
1907
1907
Conformational Stability
57.13 ( 0.41
melting temperature (DSC)
56.50 ( 0.65
-20.81 ( 0.23
-6.23
52.67
57.07 ( 1.41
-15.44 ( 0.41
-10.48
67.99
58.52 ( 1.40
-17.40 ( 0.45
-6.84
-
1
∆
G
298 K (kJ M ) (CD)
-15.04 ( 0.45
-9.08
-
1
∆
F
298 K (kJ M ) (MD)
-4.40
-10.90
Ligand Binding
K
∆
∆
d
(µM) (ITC)
423.00 ( 25.00
-18.51 ( 1.09
-16.59 ( 0.33
165.50 ( 15.00
-21.68 ( 1.73
-18.82 ( 0.33
90.21 ( 6.24
-23.06 ( 0.58
-17.04 ( 0.56
102.65 ( 10.99
-22.70 ( 1.09
-17.88 ( 0.07
159.68 ( 7.96
-21.61 ( 0.94
-20.40 ( 0.28
189.08 ( 15.04
-21.23 ( 0.86
-16.42 ( 0.13
G
298 K (kJ M-1) (ITC)
-
1
G (kJ M ) (AutoDock)
Enzyme Activity
K
K
K
M
(µM)
29.84 ( 7.79
0.54 ( 0.04
1.81
242.50 ( 13.79
3.02 ( 0.18
1.24
87.30 ( 8.68
0.53 ( 0.09
0.61
198.56 ( 11.25
0.38 ( 0.05
0.19
54.26 ( 10.35
0.58 ( 0.07
1.07
157.00 ( 15.24
2.93 ( 0.05
1.87
-
1
cat (h
cat/K
)
(M h-1) (104)
-
1
M

Stereochemical Mutation of ꢀ-Hairpin
J. Phys. Chem. B, Vol. 114, No. 50, 2010 16889
TABLE 2: Chemical Shift Assignments of Peptide SHD
residue NH
C
R
H
C
ꢀ
H
C
γ
H
others
Tyr
Glu
8.10 4.65 3.26, 3.17
8.30 4.31 2.03, 1.90 2.30, 2.22
7.46, 6.87
D
Leu 8.12 4.22 1.49
1.51
0.89, 0.85
6.98, 6.73
Asn 8.05 4.44 2.92, 2.80
Trp
Ser
8.37 4.71 2.68, 2.53
8.40 4.42 3.86, 3.80
7.16, 7.43, 7.06, 7.51, 7.18, 10.01
Asp 7.90 4.50 2.91
D
Pro
Gly
His
Thr
Ser
4.31 2.22
8.46 3.83
8.06 4.57 3.22, 3.15
8.36 4.39 4.22
8.21 4.38 3.76, 3.70
8.26 4.38 1.37
1.95
1.17
3.64, 3.56
8.36, 7.16
Figure 3. Schematic representation of NOEs found in the stereochemi-
cally bent ꢀ-hairpin.
Ala
D
Val 8.00 4.05 2.02
0.90
1.17
Lys
Phe
8.51 4.17 1.56
8.16 4.58 3.22, 2.94
1.56, 2.87, 7.51
7.10, 7.33, 7.27
TABLE 3: 3JNHCrH Values of Peptide SHD
residue
3JNHCRH (Hz)
Tyr(1)
Glu(2)
Leu(3)
Asn(4)
Trp(5)
Ser(6)
7.2
7.2
7.2
7.2
7.2
6.4
8.0
6.4
6.4
8.0
6.4
7.2
6.4
8.0
D
Asp(7)
His(10)
Thr(11)
Ser(12)
Ala(13)
D
Val(14)
Lys(15)
Phe(16)
µM concentration regime, as is noted in Figure S3 of the
Supporting Information. In NMR, they have sharpness and
chemical shifts of resonances practically unaffected upon
dilution from 5.0 to 0.5 mM concentration, as is noted in Figure
S4 of the Supporting Information. While showing no evidence
of aggregation on this basis, the peptides were freely soluble to
even higher concentrations.
Portions of TOCSY and NOESY NMR spectra of peptide 1
are shown in Figures S5 and S6 of the Supporting Information.
Chemical shifts assigned with the spectra are given in Table 2.
Figure 4. Temperature dependence of ellipticity is sigmoidal for
several sequences.
ranged, and 44-long-ranged distances calibrated over NOE
volumes, the modeling converged to a bent ꢀ-hairpin fold. The
1
0 best-fit structures superposed in Figure 1B are in mutual
mean global root-mean-square deviation (rmsd) over backbone
atoms, excluding N- and C-terminal residues, 0.27 ( 0.18 Å.
39
The average NMR model, energy minimized with Gromacs,
has the aromatic rings in mutual geometrical modes similar to
C
R
H resonances manifest small and random chemical shift
15
the reported consensus of geometry of the interacting stuctures;
deviations with respect to random-coil values (see Figure S7
of the Supporting Information). Presumably, chemical-shift-
perturbing fields of peptide dipoles are screened by the ready
specifically, Tyr(1) and Phe(16) are in edge-to-tilted (et)
geometry, while Trp(5), Tyr(1), and Phe(16) are in offset-edge
(
oe) geometry, implying the possibility of NH-π and CH-π
3
5 3
access of the small peptide structure to solvent.
JNHCRH values,
interactions.
3
6
another diagnostic of conformation in φ, clearly evidence the
Folding-Unfolding Equilibria. The ꢀ-hairpins were as-
sessed for thermal unfolding with CD. According to the results
in Figure 4, SHD, SCH, and DCH are sigmoidal in the melts;
viz., they are two state in folding-unfolding equilibria. The
other sequences do not find the higher-temperature asymptote
in the melting curves. All equilibria were reversible as g90%
of the CD signal could be recovered on cooling the melts. The
results of differential scanning calorimetry (DSC) experiments
are given in Figure S9 of the Supporting Information. SHD,
SCH, and DCH, sigmoidal in the CD-monitored melts, are
reversible in folding-unfolding transitions, as evidenced by
DSC. The melts in the case of SHC and DHC are irreversible;
possibly the unfolded peptides aggregate. The observed endo-
thermic transitions may in each peptide manifest unfolding.
According to the transition temperature given in Table 1,
replacement of His, Asp, or Ser with Cys in position 7
destabilizes the fold.
ꢀ
J
-sheet conformation. As is noted in Table 3, all observed
3
NHCRH values are 6 Hz or greater, as required for the ꢀ-sheet
37
conformation.
The peptide manifests a rich population of NOEs, implying
close contact between the residues far off in sequence. Specif-
ically, NOEs between Tyr(1)H -Trp(5)H , Tyr(1)H -Trp(5)H ,
Trp(5)H -Phe(16)H , Trp(5)H -Phe(16)H , and Tyr(1)H -
Phe(16)H imply close proximity of aromatic side chains. Long
ε
ꢁ2
ε
δ1
δ1
ε
δ1
ꢁ
δ
ꢁ
range NOEs appear between Trp(5)-Ser(12), Asp(7)-His(10),
D
D
Asp(7)-Ser(12), Leu(3)- Val(14), Glu(2)-Lys(15), and Ser(6)-
Thr(11), which diagnose the ꢀ-hairpin fold, as is illustrated in
Figure 3.
The peptide was taken up for NMR-based conformational
modeling. Using NOE-based interproton distances and the
3
J
NHCRH-suggested range in φ, modeling was undertaken with
38
CYANA-2.1. Constrained with 65-intraresidue, 27-medium-

1
6890 J. Phys. Chem. B, Vol. 114, No. 50, 2010
Patel et al.
Figure 5. Evolution of population in specific folds (conformers
clustered to 0.15 nm rmsd cutoff over CR) during MD.
Computation of Conformational Equilibria. The ideal fold
in each sequence was submitted to molecular dynamics in water
at ambient temperature, and the trajectory was assessed for
evolution of possible alternative conformational folds. The
structures harvested from the molecular dynamics trajectory
were clustered55 to 0.15 nm rmsd cutoff over CR coordinates
as specific folds. Each MD trajectory is noted to evolve to an
asymptote in specific conformational folds, as is noted in Figure
5
. The number of folds defining apparent conformational
equilibrium, ranging from 4 to 10, is sequence specific. The
minimum energy conformer of each ensemble is similar to
the NMR-determined conformation for SHD, as is noted in
Figure 1B. The minimum energy conformer encloses 85-99%
of the equilibrium in different peptides (see Figure S10 of
the Supporting Information). The folding free energies,
calculated by assuming two-state equilibrium, are given in
Table 1. The values are noted to be in reasonable agreement
with the estimates obtained with CD.
Figure 6. Interaction of SHD with p-nitrophenylphosphate (pNPP)
monitored with ITC. Upper panel: heat changes on titrating peptide
(
1.4448 mL of 50 µM solution) with pNPP (10 µL aliquots of 2.5 mM
solution). Lower panel: integrated heat changes in ligand titration, with
the solid line representing the fit to a single binding-site model.
tion of a titratable group in hydrolysis of the substrate.
Activation consequent to binding of substrate, evidenced in the
saturable kinetics, may involve electrophilic activation of the
substrate.
Binding with p-Nitrophenylphosphate. The peptides were
evaluated for affinity with acetylcholine (ACh), p-nitropheny-
lacetate (pNPA), and p-nitrophenylphosphate (pNPP) with
spectrofluorometry and isothermal titration calorimetry (ITC).
On the basis of quenching of Trp fluorescence (Figure S12 of
the Supporting Information), all peptides interact with all of
the ligands. ACh and pNPA are comparable as quenchers, while
pNPP is a stronger quencher, and also it promotes ∼30 nm red
shiftoftheemissionband.Thetitrationsmonitoringpeptide-pNPA
interactions with ITC are in Figure 6 and Figure S13 of the
Supporting Information. The parameters derived on assuming
a 1:1 binding isotherm are given in Table 1. The interactions
Discussion and Conclusions
The protein alphabet of L-R-amino acids remains an open
challenge to know how it directs the specific shapes of protein
structure and the specific interaction and catalysis functions of
enzymes.1
-3,9
Under question are sequence effects that direct
proteins as defined folds of polypeptide structure. Protein folds
are specific for the poly-L diastereomer of polypeptide structure.
A way to probe effects in protein folding of the interactions
over the main-chain and side-chain structures, we envisioned,
-
1
are endothermic, and the enthalpies are in the 16-21 kJ mol
1
1
range. We evaluated the interaction with AutoDock, targeting
the minimum energy fold of the SHD peptide clustered over
aromatic side chains to a rmsd cutoff of 0.15 nm. The central
was to mutate the canonical structures stereochemically. The
mutations, transforming diastereomers, will alter molecular folds,
and thus protein shapes and interactions, and thus may provide
3
9
member, energy minimized with Gromacs, gave the results
summarized in Table 1. The estimates are in reasonable
agreement in the experimental values. According to the results
of simulation, the p-nitrophenyl moiety binds by stacking over
phenylalanine- and tryptophan-ring structures (Figure S14 of
the Supporting Information).
a design approach and approach to investigate folding principles.
Harnessing the inverse algorithm,4
1,42
we have approached
homopolypeptide folds over the L and D alphabet, which we
overlaid with sequences in side chains, to approach the chemical
specificities of interest.4 Illustrating the approach, we have
in a parallel study reported a zinc-based hydrolase, which was
designed first in the fold stereochemically and then in the
3,44
Assessment of Catalyzed Hydrolysis. The results evaluating
catalyzed hydrolysis of pNPA are in Figure S15 of the
Supporting Information. An enzyme-like saturation of hydrolysis
45
11-15
sequence chemically. Testing the systematic approach,
we
targeted in this study the canonical ꢀ-hairpin for the study of
L- to D-mutation effects in its shape and in its interactions as a
protein and possibly as an enzyme.
M
rate occurs with substrate concentration. K and kcat given in
Table 1 are narrowly varied, and kcat/K are comparable. The
M
intended catalytic triad does not affect the rate of substrate
hydrolysis. The results of pH variation summarized in Table
S2 of the Supporting Information do not evidence the participa-
The ꢀ-hairpin evokes interest in the interactions of its
autonomous folding, packing as a protein element, and aggrega-
tion as an independent structure.2
0,23-30
The interactions are in

Stereochemical Mutation of ꢀ-Hairpin
J. Phys. Chem. B, Vol. 114, No. 50, 2010 16891
the canonical structure specific for its poly-L stereochemistry.
The stereochemistry defines the geometry of planar-polypeptide
strands and, over their mutual hydrogen bonds, the pleated
ribbons of the antiparallel-ꢀ-sheet motif. The side chains of
cross-strand hydrogen-bonded and non-hydrogen-bonded resi-
dues project alternately up and down the plane of the natural
poly-L ribbon.4 Given its planarity, the structure can interact
laterally via the array of peptide dipoles and facially via the
array of side chains. The resulting main-chain and side-chain
interactions, along mutually orthogonal planes, possibly provide
in the ꢀ-sheet ribbon the “stickiness” in its participation either
in protein folding or in amyloidogenic self-assembly of unfolded
protein. Exploiting the chemical effects in self-assembly diag-
tBuOH solution as a white powder. Peptide purity was assessed
with HPLC over RP-C18 (10 µM, 10 mm × 250 mm; Merck)
3 2
eluting with CH CN\H O (0.1%TFA) 0-100% gradients.
Mass Spectra. Mass spectra were recorded on a QTOF-ESI
mass spectrometer. Positive ions were detected in linear/
reflectron mode.
6,47
Circular Dichroism. Circular dichroism (CD) was recorded
on a JASCO J-180 CD spectropolarimeter, precalibrated with
d10-camphorsulphonic acid, at 298 K in a 0.2 cm path length
quartz cell with 2 nm bandwidth in far-UV (190-250 nm) or
near-UV (250-350 nm) range. Scanning at 100 nm/min with
1.0 s time constant, in 0.5 nm steps, five scans were averaged
after baseline correction for solvent. Working solutions 20-100
µM in peptide were prepared by optical measurements. The
observations in millidegrees were converted to molar residue
4
8
nosed from protein homodimers, we accomplished poly-L
hairpin sequences that displayed self-assembly along mutual
tetramer.32
ellipticity [θMRW] with a reported relation.
49
orthogonal planes, to define a hemoglobin-like D
The systematic engineering of the canonical ꢀ-hairpin to
2
Unfolding of peptides was monitored by recording the
ellipticity at respective secondary CD bands in the 278-363 K
range in water. The equilibrium constant was calculated as
1
1
specific molecular shapes like bracelet and boat has involved
modifications of the structure in a cross-strand pair of LL to DD
residues, which does not alter the registry of peptide hydrogen
bonds but interchanges the main-chain and side-chain elements,
bending the ribbon locally by about 90°. A single bend was
implemented in this study, while two bends gave, based on the
position along the ribbon, the curved morphology of “bracelet”
and the morphology of “boat” having the ends upturned to
Fu
Keq
)
(1)
1
- Fu
where
1
3,12
enclose an enzyme-like pocket.
The bend was in this study
examined in properties of the polypeptide structure folding as
the protein and interactions as an enzyme. The canonical poly-L
[
Θ]obs - [Θ]F
U
Θ] - [Θ]F
Fu )
(2)
[
ꢀ
-hairpin is notorious for its stickiness, and indeed, it was a
challenge to design the sequences that will not aggregate and
will thus allow the characterization of the structure as an
with [Θ]obs, [Θ]
observed, molar residue ellipticity of the folded state, and molar
residue ellipticity of the unfolded state, respectively.
F
U
, and [Θ] being the molar residue ellipticity
autonomous fold.1
6,17
We reported sequences over the poly-L
hairpin structure that were dimers in the micromolar concentra-
tion regime and tetramers in the millimolar concentration
regime.32 The introduction of a single bend gave in this study
a protein-like fold that is largely monomolecular and is
cooperatively ordered as a protein in millimolar concentrations
and presumably even higher concentrations. The suppression
of stickiness of the canonical ꢀ-hairpin highlights the critical
role poly-L stereochemistry may have in the trade-off in unfolded
proteins between folding as specific structures and aggregating
as nonspecific structures. We exploited the local bend of the
heterochiral hairpin to create a pocket for enzyme-catalyzed
hydrolysis of a suitable substrate. We succeeded in creating a
cooperatively ordered protein-like fold, a binding pocket for a
specific ligand, and rudiments of enzyme catalysis function
against a substrate of interest but not the power of a real enzyme.
∆
G of the unfolding reaction was calculated as
∆
G ) -RT ln Keq
(3)
Spectrofluorometry. Fluorescence was measured on a Perkin-
Elmer LS-55 spectrofluorimeter equipped with standard PMT.
Data were collected at 298 K in a 1 mL cell, with λexcitation as
2
95 nm, λemission in the 300-450 range, and 2.5 nm excitation
and emission slits. A scan rate of 100 nm/min in 1 nm steps
was used. The working concentrations (10-20 µM) of peptide
and NATA (SIGMA chemicals) were calibrated by OD mea-
surements using a molar extinction coefficient of Trp (∼5600
at 280 nm) and Tyr (∼1280 at 280 nm). Binding experiments
were carried out at 15 µM concentration of peptide and 300
µM concentrations of pNPP, pNPA, and acetylcholine.
Experimental Section
1
Nuclear Magnetic Resonance. H NMR was measured with
Materials and Methods. Fmoc-protected amino acids, Rink
Amide AM resin, acetylcholine, p-nitrophenylacetate, and
p-nitrophenylphoshate were purchased from Sigma-Aldrich or
Novabiochem-Merck.
a Bruker 800 MHz spectrometer, equipped with a cryoprobe,
at 298 K. Peptide concentrations of 5 mM were used, and 1D
spectra were recorded also at 10-fold dilution. Solutions were
prepared in 90% H
2 2
O/10% D O with DSS as the internal
Peptide Synthesis. Synthesis was performed manually on
Rink Amide AM resin using standard Fmoc chemistry and
HOBt/DIC as coupling reagents. Each coupling, monitored with
Kaiser and chloranil tests, typically required about 6 h.
Deprotections were carried out with 30% (v/v) piperidine-DMF.
reference. Polypeptide backbone and side-chain assignments
1
1
50
were obtained with 2D H- H TOCSY experiments using a
standard mlevtp pulse sequence for spin lock. A spin lock time
of 90 ms was used to ensure observation of long-range
couplings. The data were collected in 2k time domain points.
The N-terminus was acetylated (-NHCOCH
3
) with Ac
2
O:
512 increments of t
the data being zero-filled to 1k data points in F
transform. Distance constraints were obtained from a 2D H- H
NOESY experiment.51 The data were processed with TOPSPIN
2.0 and 2.1 and analyzed with Computer Aided Resonance
Assignment (CARA) software. Structure calculations were
1
were measured with 48 FIDs per increment,
prior to Fourier
DIPEA:DMF in a 1:2:20 ratio. The cleavage of the final
polypeptide and deprotection of side chains were achieved
together with reagent K (82.5% TFA/5% dry-phenol/5% thio-
anisole/2.5% ethandithiol/5% water). The product precipitated
1
1
1
2
with anhydrous diethyl ether was lyophilized from 1:4H O:

1
6892 J. Phys. Chem. B, Vol. 114, No. 50, 2010
Patel et al.
TABLE 4: Results of Molecular Dynamics Simulation
3
8
performed with CYANA 2.1. Dihedral angle restraints based
on JNHCRΗ values were used, wherever possible, for structure
3
%
population in specific
microstates
refinements. D-Amino acid residues were introduced in the
CYANA library under the guidance of the developer. Structures
were energy minimized using the GROMACS software pack-
age. Structural models were rendered with Pymol, MolMol,
or Viewerlite software.
Isothermal Titration Calorimetry (ITC). The experiments
were performed on a VP-ITC microcalorimeter (Microcal, Inc.)
at 298 K. The sample cell contained peptide in 50 µM
concentration, as determined by optical measurements, and the
reference cell contained water. The 2.5 mM p-nitrophenylphos-
phate (ligand) solution loaded in a 250 µL syringe was titrated
into peptide solution in 10 µL aliquots in 25 steps at 4 min
intervals. The change in enthalpy (∆H) due to dilution was
determined by titrating ligand into solvent as well as solvent
into peptide solution. These backgrounds were subtracted from
no. of
no. of
length of conformers microstates
3
9
peptide simulation sampled
recovered
1
2
3
4
5
a
b
b
b
b
b
SHD
SHC
SCH
DHS
DHC
DCH
100
11
10
14
13
14
20001
11001
10001
14001
13001
14001
10
8
4
5
5
92.52 4.28 2.36 0.51 0.14
85.54 10.28 1.87 1.69 0.33
97.53 1.93 0.45 0.12
98.56 0.88 0.44 0.09 0.03
98.81 0.86 0.25 0.08 0.02
94.08 3.89 1.55 0.34 0.16
6
a
Conformers harvested at the 5 ps interval. b Conformers har-
vested at the 1 ps interval.
dynamics was initialized, and the trajectory was sampled at
intervals reported in the individual cases after allowing an initial
3
ns for equilibration.
∆
H obtained for the corresponding ligand-peptide binding
Conformational clustering to achieve microstates of peptide
experiments, prior to curve fitting. The background-subtracted
data was fitted to a model describing a single binding site using
MicroCal software. The binding enthalpy (∆H), entropy (∆S),
structure to 0.15 nm rmsd cutoff was performed with a reported
procedure.55 The difference Helmholtz free energy (∆FA-B
between specific microstates was calculated from relative
probabilities p and p of finding the system in microstates A
/p , with R as the gas constant,
and p the number of members in
)
d
and dissociation (K ) constant were thus calculated.
A
B
Enzyme ActiWity. The kinetics of hydrolysis was monitored
spectrophotometrically on a Perkin-Elmer spectrophotometer,
fitted with peltier, using p-nitrophenylacetate (pNPA) as a
substrate, by observing the production of p-nitrophenolate anion
at 410 nm. A stock solution of pNPA was prepared in 20 mM
sodium phosphate buffer, pH 7.0, with a few drops of aceto-
nitrile added to solubilize the pNPA. The peptide concentration
in the assays was 24 µM. Hydrolase activity was evaluated in
and B as ∆FA-B ) -RT ln p
B
A
T as the temperature, and p
A
B
microstates A and B.
Molecular Docking. Flexible docking was implemented with
AutoDock 4.0.56 The central members of the top 10 clusters
over aromatic residues of peptide conformers populating the
molecular dynamics trajectory were targeted as the receptor.
Using an rmsd tolerance of 2 Å, structurally distinct confor-
mational clusters of the ligand were ranked in increasing energy.
2
0 mM sodium phosphate buffer, pH 7.0, at 25 °C, by varying
the substrate concentration. The catalyzed rate of pNPA
hydrolysis was measured by an initial slope method, following
the increase in 410 nm absorption of p-nitrophenolate. Errors
in observation were about 5%.
Differential Scanning Calorimetry (DSC). The experiments
were performed on a Mettler-Toledo DSC-822e instrument. The
peptide was dissolved in water in ∼40 mM concentration, and
the solutions were thoroughly degassed prior to data acquisition.
The sample was scanned relative to the empty aluminum pan
as a reference in the temperature range 288-358 K, with a
scanning rate of 5 K/min.
Acknowledgment. We acknowledge DST (09DST028),
Government of India, for financial support, IIT Bombay for the
computing facility “Corona” and C-DAC, Govt. of India, for
the supercomputing facility “BRAF”. We acknowledge Dr.
M. V. Hosur and Dr. Lata Panicker (BARC, Mumbai, India)
for their help with DSC experiments. K.P. and B.G. are
recipients of fellowships from Council of Scientific and
Industrial Research (CSIR) and University Grants Commission
(
UGC), respectively.
Molecular Dynamics. An Intel Pentium PIV Linux server,
Intel Xeon dual CPU 2.4 GHz computational servers, and a
PARAM Padma supercomputer equipped with 248 (Power
Supporting Information Available: Mass, NMR, fluores-
cence, CD, ITC, DSC, molecular dynamics, and kinetics data.
This material is available free of charge via the Internet at http://
pubs.acs.org.
4@1GHz) processors and an aggregate memory of 512 GB were
the hardware platforms used. Molecular dynamics was carried
3
9
out in the GROMACS package (versions 3.1.4, 3.2.1, 3.3.1,
.3.2, and 3.3.3) using the gromos-96 43A1 force field52 in a
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