Zinc-finger hydrolase: Computational selection of a linker and a sequence towards metal activation with a synthetic αββ protein

Article abstract of DOI:10.1016/j.bmc.2010.10.003

The zinc-finger protein is targeted for computational redesign as a hydrolase enzyme. Successful in having zinc activated for hydrolase function, the study validates the stepwise approach to having the protein tuned in main-chain structure stereochemicall

Full text of DOI:10.1016/j.bmc.2010.10.003

Bioorganic & Medicinal Chemistry 18 (2010) 8270–8276
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Zinc-finger hydrolase: Computational selection of a linker and a sequence
towards metal activation with a synthetic
abb protein
⇑
Kirti Patel , Kinshuk Raj Srivastava, Susheel Durani
Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400 076, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The zinc-finger protein is targeted for computational redesign as a hydrolase enzyme. Successful in
having zinc activated for hydrolase function, the study validates the stepwise approach to having the
protein tuned in main-chain structure stereochemically and over side chains chemically. A leucine homo-
Received 23 August 2010
Revised 30 September 2010
Accepted 2 October 2010
Available online 27 October 2010
polypeptide, harboring histidines to tri coordinate zinc and
building blocks of an bb protein, is taken up for modeling, first with CYANA, in a mixed-chirality linker
between the building blocks, and then with IDeAS, in a sequence over side chains. The designed
mixed-chirality polypeptide structure is proven to order as an intended bb fold and capture zinc to acti-
vate its role as a hydrolase catalyst. The design approach to have protein folds defined stereochemically
and receptor and catalysis functions defined chemically is presented, and illustrates - and -amino-
D-amino-acid-nucleated a-helix and b-hairpin
a
Keywords:
Protein design
Zinc-binding sites
Protein stereochemistry
Enzyme-catalysis
Cholinesterase
a
L
D-a
acid structures as the alphabet integrating chemical- and stereochemical-structure variables as its letters.
Ó 2010 Elsevier Ltd. All rights reserved.
p–p interactions
1. Introduction
studies reported the design of a mixed-chirality-peptide hydro-
lase,16 and of a hemoglobin-like tetramer over a canonical b-hair-
Proteins are polypeptide structures defined in the folds, in the
binding sites, and in the catalysis functions with side-chain struc-
tures as the chemical alphabet. The alphabet evokes interest of
application for design of proteins and enzymes, but presents in
the compounded size of conformational- and chemical-search
spaces a formidable challenge. The searches can be undertaken
computationally, and particularly powerful can be the approach
to have the conformational-structure space in main-chain and
chemical-structure space over side chains delinked and explored
independently. Implementing the possibility is the inverse-design
algorithm1–6 which has been applied successfully for the design,
from input of main-chain folds, of new proteins,7 new sensors,8
and new enzymes,9–11 etc. The complementary methods for fold
design are desired, and molecular dynamics12 and Monte Carlo13
procedures are the relevant methods. We have proposed the novel
concept of protein design involving the use of residue stereo-
chemistry as an additional alphabet.14 For the designs, we have
reported relevant computational methods, which may be imple-
mented over preordered secondary structures or unordered poly-
peptides structures as the input.12,14 A complementary method
for design of sequences over the mixed-stereochemistry folds has
also been reported.15 Illustrating the method, we have in parallel
pin structure.17 We now aim the stepwise design of a zinc-finger
hydrolase, first over main-chain and then over side-chain
structures.
Hydrolase enzymes harness acid–base–nucleophile catalysts
from within their polypeptide structures and electrophile catalysts
as external cofactors. Zn is an important protein cofactor contribut-
ing both structural and catalysis roles.18,19 Studies have been
reported where the binding of Zn provides major driving force
for a polypeptide chain to fold.21 Zinc-finger protein,20 having zinc
tetra coordinated with an
abb fold, typifies zinc in the structural
role. Zinc also contributes in enzyme-catalysis functions, and there
are reports of successful modification of zinc-finger structure as a
hydrolase enzyme.22–24 Illustrating
bet, we reported the design of small
L
- and
D-a-amino-acid alpha-
a
bb protein as a binding pock-
et for acetylcholine, calling it pi-cup protein.25 We now report
computational modification of pi-cup-protein structure as a zinc-
based hydrolase. The study illustrates the computational methods
developed in our lab for implementing protein design over L- and
D
-a-amino-acid structures as the combined stereochemical and
chemical alphabet.12,15,16,25,26
2. Results
2.1. Design and synthesis
⇑
Corresponding author. Tel: +91 22 25767164; fax: +91 22 25767152.
E-mail address: sdurani@iitb.ac.in (S. Durani).
We aim to have an
abb fold tri-coordinate zinc, towards metal
Present address: Department of Chemistry, RNC Arts, JDB Commerce & NSC
Science College, Nashik Road, 422 101, India.
activation as a hydrolase catalyst. Figure 1 compares zinc-finger
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.10.003

K. Patel et al. / Bioorg. Med. Chem. 18 (2010) 8270–8276
8271
Figure 1.
abb Folds natural and synthetic. (A) Natural zinc-finger fold. (B) Synthetic pi-cup protein. (C) Zinc-finger fold modified as a potential metallo-hydrolase. (D) Ten
lowest energy NMR models for the synthetic sequence obtained with CYANA
fold,19 pi-cup protein,25 and the synthetic transform we are aiming
the possible use in our design of
the glycines. The glycine in sequence position 12 does not manifest
preference for a specific half of space, and is thus retained in
our final sequence design unchanged.
With the choices for the residues in the intended linker segment
worked out, we took up the design of sequence with IDeAS,15 the
software developed to allow mixed chirality folds to be targeted
L or D residue as replacements for
to achieve as an enzyme. We seek to suitably enlarge
a-helix,
b-hairpin, and linker elements of the pi-cup protein, and incorpo-
rate histidines to tri-coordinate zinc, placing it in the base of an
intended active-site cleft. We implement the design as follows. In
a 21-residue polypeptide, we place in positions 1 and 17 DPro, in
positions 10, 14, and 21 LHis, in positions 11, 12, 13, and 18 Gly,
u,w
L
and in every remaining sequence position Leu. As reported,26 we
with L or D side chains, as required. Keeping specific residues fixed,
27
model an
a
bb fold with NMR-structure solving software CYANA
.
the structure was sequence optimized as the intended protein and
enzyme. Specifically, DPro1 was kept fixed as a nucleator for the
helical fold, DPro17-Gly18 was kept fixed as a nucleator for the
Annealing proteins typically with input of NMR-derived con-
straints, the software can accept design constraints of interest as
well as artificial residues. We have modified CYANA package by
L
L
L
Type-II’ b-hairpin, His10, His14, His21 were kept fixed as the zinc
ligands, and Gly12 was kept fixed as a connector-segment residue,
in accordance with the results of the CYANA-based analysis. Imple-
menting IDeAS, a sequence solution was generated by having the
having
D amino acids incorporated in its library.
We implement fold design by constraining secondary-structure
elements locally and desired tertiary-structure fold globally. The
constraints, imposed in form of distances between atoms or mole-
cular fragments and torsional angles in specific elements of the
structure, are allowed sufficient tolerances for plasticity of confor-
mation and flexibility of atomic packing when implementing sim-
solutions in the sequence position 11 constrained to only
D resi-
dues, and in the sequence positions 2, 3, and 19 constrained only
to aromatic residues. The latter are intended for the possibility of
constraining the folding and ligand binding with aromatic interac-
tions of the selected residues. Allowing in every other sequence po-
sition an unrestricted choice of side chains, the following sequence
generated with IDeAS was selected as the ideal option in respect of
sequence polarity and chemical diversity of the structure.
ulated-annealing cycles of CYANA. Specifically, for the design
objective in this study, we constrained His residues of polypeptide
structure to the geometry suitable for coordination with zinc.18
Constraining intended secondary-structure modules appropriately
to intended tertiary structure, we allowed tri-glycine linker to
sample the possibilities of conformational space compatible with
the targeted aab protein. Multiple runs of CYANA were followed
with analysis of the folds in the conformations over the individual
glycine residues in the desired aab protein. Over 500 annealing
runs, summarized in Figure S2 of the Supplementary data, the
glycines in sequence positions 11 and 13 are noted to sample more
AcDPro1-Tyr2-Trp3-Gln4-Ala5-Ser6-Asp7-Lys8-Arg9-His10-DGlu11
-
Gly12-Asp13-His14-Ser15-Gln16-DPro17-Gly18-Tyr19-Thr20-His21NH2
The sequence describes an
abb fold tri-coordinating zinc, as
noted in Figure 1C. Compared with the zinc-finger20 and pi-cup
proteins,25 the designed protein is extended in the linker between
the a-helix and b-hairpin elements, and harbors a pocket towards
eventual tune-up for a desired binding ligand. The peptide was
frequently either the left or the right half of
u,w space, suggesting
synthesized by manual solid-phase chemistry. Ion peak in

8272
K. Patel et al. / Bioorg. Med. Chem. 18 (2010) 8270–8276
correspondence of the expected molecular mass is found to appear
in MALDI–MS, as is noted in Figure S1 of the Supplementary data.
HPLC over RP-C18 column with H2O (0.01% TFA)–acetonitrile
0–100% gradient established that the peptide was >90% pure.
1H NMR spectrum recorded in 90:10 H2O/D2O mixture at pH 6.7
is well dispersed in chemical shifts, and is unaffected by dilution of
the peptide from 3 mM and 0.3 mM concentration, as is noted in
Figure S4 of the Supplementary data. Apparently, the peptide is
free of aggregation in the concentration regime of NMR experiment
as well. Chemical shift assignments were achieved with TOCSY29
and NOESY.30 Portions of 2D spectra are shown in Figures S5–S7
of the Supplementary data. Chemical shift assignments are given
2.2. Characterization of conformation
A coupled minimum and maximum of ellipticity appears at 212
and 228 nm in CD spectrum, as is noted in Figure S3A. The bands
characterize a coupled exciton, the hallmark of aromatic chro-
mophores in mutual interaction.28 The Tyr in sequence position
19 may be interacting with the Tyr in sequence position 2 or the
Trp in sequence position 3, implying that the polypeptide has its
termini in close proximity, even when it is not ligated with zinc.
in Table 1. Deviations of CaH shifts from random-coil values, called
CSI, is a useful diagnostic of secondary structure.31,32 The CSIs in
our peptide, noted in Figure 2, are in accordance with the expecta-
tion for its
a-helical fold but not for its b-hairpin fold. Arising lo-
cally from peptide-dipolar fields, CSIs are known to diminish in
proportion of the access a peptide has to solvent.33 Hence, small
in size, the peptide may not conform to the CSIs derived from pro-
b-Hairpin and a-helix elements may be ordered, but the character-
3
istic minima of CD, expected in the range of ꢀ208–235 nm, cannot
be observed, possibly due to the strength of the coupled exciton. As
evidence for a chirally perturbed aromatic group, the peptide dis-
plays a CD band in 260–350 nm range, as is noted in Figure S3B
of the Supplementary data. The peptide is in its molar ellipticity
teins. JNHC values, as could be read off from 1D spectrum, are
a
H
3
listed in Table 2. With JNHC H 6 3.2 Hz, Tyr(2), Ser(6), Asp(7),
a
Lys(8), and His(10) evidently are in
a-helical conformation, while
3
with JNHC H P 8 Hz, His(14), Ser(15), and Gln(16) evidently are
a
in b-sheet conformation.30
concentration invariant in the 20–100
l
M regime, as is noted in
Rich population of NOEs appeared in NOESY, implying short
contacts between several residues possibly far off in sequence. As
noted in Figures 3 and 4 and Figure S7 of the Supplementary data,
NOEs appear between the main-chain protons of Gln(16)-Tyr(19),
Ser(15)-Thr(20), and His(14)-His(21), in support of b-hairpin
fold, of Gln(4)-Asp(7), Gln(4)-Lys(8), Ser(6)-His(10), and Ala(5)-
Figure S3A of the Supplementary data. Presumably, the peptide is
a monomolecular fold aggregation free in the concentration regime
examined.
Table 1
Proton chemical shifts
Arg(9), in support of
a-helical fold, and between the side-chain
protons of Tyr(2)-Tyr(19), Trp(3)-Tyr(19), Gln(16)-Trp(3), and
Residue
NH
C
aH
CbH
C
c
H
Others
Gln(16)-Arg(9), in support of abb fold of the protein.
Pro-1
Tyr-2
Trp-3
4.29
4.63
4.17
2.07, 1.86
3.05, 2.97
3.26, 3.21
1.70, 1.51
3.525
7.49, 6.84
7.22, 10.11, 7.54,
7.21, 7.46, 7.11
7.44, 6.81
Modeling with CYANA 2.1,27 using 128 NOE-calibrated distance
8.01
8.07
restraints (76 intra residue, 31 medium, and 21 long range) and
3
11 JNHC H-calibrated
u
restraints, led to convergence of the pep-
a
tide as an
abb fold. The 10 best-fit conformers, superposed in
Gln-4
Ala-5
Ser-6
Asp-7
Lys-8
8.37
8.18
8.53
8.13
8.43
7.95
8.07
8.44
8.40
7.82
8.12
8.22
8.08
4.17
4.19
4.50
4.49
4.24
4.15
4.57
4.67
3.88
4.51
4.63
4.45
4.17
4.38
3.83
4.60
4.30
4.62
1.79, 1.70
1.38
3.83
2.77, 2.69
1.74
1.95, 1.79
2.94, 2.74
2.07, 1.91
1.66, 1.41
Figure S1D of the Supplementary data, are in mutual global root-
mean-square deviation (RMSD) over backbone atoms, excluding
N- and C-terminal residues, of 0.26 0.27 Å. The structures are
with side chains of Tyr(2), Trp(3), and Tyr(19) in close enough
1.54
2.11, 2.18
1.69, 3.10, 7.35
3.24, 3.20, 7.39, 6.76
8.02, 7.51
Arg-9
proximity for NH-p, CH-p, and p–p interactions.
His-10
Glu-11
Gly-12
Asp-13
His-14
Ser-15
Gln-16
Pro-17
Gly-18
Tyr-19
Thr-20
His-21
2.33, 2.32
2.3. Binding with Zn2+
2.75, 2.64
3.21
3.87
1.96, 1.80
2.26, 1.98
7.57, 7.20
Evaluation of Zn2+ binding was undertaken with MALDI-MS,
NMR, CD, and Isothermal Titration Calorimetry (ITC). In MALDI–
MS recorded in the presence of 10-fold excess of Zn(ClO4)2Á6H2O,
shown in Figure S8 of the Supplementary data, ion peak appears
in correspondence of Zn2+-peptide complex in stoichiometry of
2.20, 2.14
1.86
7.05, 6.75
3.75, 3.67
8.29
7.97
8.13
8.32
3.01, 2.94
4.09
3.21, 3.08
6.94, 6.73
7.16, 6.82
1.10
1:1. In NMR,
a slight perturbation occurs on addition of
Zn(ClO4)2Á6H2O in 5 mol excess, but only in NH region, as is noted
0.3
0.1
-0.1
-0.3
-0.5
-0.7
P
Y
W
Q
A
S
D
K
R
H
E
G
D
H
S
Q
P
G
Y
T
H
Figure 2. Deviations of observed CaH chemical shifts (DdHa) from mean random-coil values.

K. Patel et al. / Bioorg. Med. Chem. 18 (2010) 8270–8276
8273
Table 2
3JNHC values
aH
Residue
3JNHC (Hz)
aH
Tyr(2)
Ser(6)
Asp(7)
Lys(8)
His(10)
Asp(13)
His(14)
Ser(15)
Gln(16)
Tyr(19)
His(21)
3.20
2.80
2.80
2.40
2.40
7.20
8.00
8.00
8.00
7.20
7.20
Figure 3. Near neighbor backbone NOEs of abb fold.
Figure 4. Schematic representations of long range NOEs of abb fold.

8274
K. Patel et al. / Bioorg. Med. Chem. 18 (2010) 8270–8276
Figure 5. (A) Concentration-dependent perturbation of exciton-coupled CD band of peptide with Zn2+, (inset) the dependence of ellipticity at 212 nm on concentration of
zinc. (B) Microcalorimetric evaluation of peptide–Zn2+ interaction. Upper panel: measured heat change on adding aliquots of Zn(ClO4)2 into peptide solution. Lower panel:
integrated heat change in each step with solid line in correspondence of single-site binding model.
in Figure S9 of the Supplementary data. Presumably, the peptide is
preordered to the zinc-binding conformation. Titration with Zn2+
leads to perturbation of exciton-coupled CD band of the peptide,
affecting its ellipticity at 212 nm. Monitoring zinc interaction with
be monitored with ITC as well. According to the results given in
Figure 5B, the interaction is saturable and exothermic. Parameters
derived from the titration, by assuming 1:1 isotherm, are given in
Table 3. Kd of 716 55 lM, and correspondingly DG° of
CD and fitting the data to 1:1 isotherm, gave Kd as 874 360
lM
À17.9 kJ molÀ1 are in agreement with the results of CD
and correspondingly
D
G° as À16.5 kJ molÀ1. The interaction could
experiment.
Table 3
2.4. Evaluation of catalysis
Thermodynamic parameters for peptide–Zn2+ interaction derived with CD and ITC
D
H° (kJ/mol)
D
S° (J/mol)
Kd
(
l
M)
DG° (kJ/mol)
The peptide was evaluated as a hydrolase against p-nitrophenyl-
acetate (pNPA) as the acetylcholine surrogate. Hydrolysis of the
substrate with only zinc, with only peptide, and with Zn–peptide
ITC
CD
À50.87 1.80
À26.41
716.40 55.55
873.97 359.69
À17.93 0.04
À16.51 1.12
—
—
Figure 6. (A) UV monitored rate of hydrolysis of pNPA in buffer showing effects of added Zn(ClO4)2, free peptide, and Zn–peptide complex. (B) Rate of hydrolysis of pNPA with
Zn–peptide complex (1 M peptide + 5
M Zn2+) as a function of substrate concentration.
l
l

K. Patel et al. / Bioorg. Med. Chem. 18 (2010) 8270–8276
8275
Table 4
Initial rate (
tune the determinants of its affinity for zinc and its further devel-
opment as an enzyme.
m
) for hydrolysis of pNPA
(10À9 MsÀ1
2.30 0.03
a
Catalyst
m
)
m/mcontr
Buffer
Zn
Peptide
1.00
2.5
4.99
4. Experimental section
4.1. Materials
5.75 0.03
11.49 0.21
103.45 0.25
Zn–peptide
44.98
a
Rate of hydrolysis of pNPA by buffer was taken as mcontr
.
p-Nitrophenylacetate and Zn(ClO4)2Á6H2O were from Sigma–
Aldrich. Fmoc-protected amino acids, reagents for solid-phase pep-
tide synthesis, Rink-Amide AM resin, DMF, methanol, diethylether,
dichloromethane, were from Sigma–Aldrich or Novabiochem-
Merck.
complex is plotted in Figure 6 A and the derived rate constants are
summarized in Table 4. Small rate enhancement occurs with only
zinc and with only the peptide, while a ꢀ45-fold rate enhancement
occurs with the zinc–peptide complex. The results in Figure 6B
show that the hydrolysis rate does not saturate with the substrate
concentration. Michaelis–Menten model is ruled out and the hydro-
lysis with zinc–peptide complex may involve collisional mecha-
nism rather than a specific complexation between the substrate
and the enzyme.
4.2. Peptide synthesis
Synthesis was performed on Rink Amide AM resin using stan-
dard Fmoc chemistry and HOBt/DIC as coupling reagents. Each
coupling, monitored with Kaiser and chloranil tests, typically re-
quired about 6 h. Deprotections were carried out with 30% (v/v)
piperidine-DMF. N-Terminus was acetylated (–NHCOCH3) with
Ac2O/DIPEA/DMF in 1:2:20 ratio. The cleavage of the final polypep-
tide and deprotection of side chains were achieved together with
reagent K (82.5% TFA/5% dry-phenol/5% thioanisole/2.5% ethan-
dithiol/5% water). The product precipitated with anhydrous diethyl
ether was lyophilized from 1:4 H2O/tBuOH solution as a white
powder. Peptide purity was assessed with HPLC over RP-C18
3. Discussion and conclusions
The protein alphabet of
a-amino-acid structures, by now
clarified to some extent in its operational physics, has begun to
yield in the efforts to have it applied.1–6 Algorithms developed
for its application12,13 have been validated with the successful
design of proteins,2,15 receptors,20 sensors,8 and enzymes.9–11 The
algorithms work by delinking the search space of the structure in
sequences over side chains from that of conformational folds over
main-chain. The development motivates the exploration of fold de-
sign with the recruitment of possible newer variables of structure.
We have proposed and explored residue chirality as a variable for
fold design,14 and have reported the computational methods suit-
(10
l
M, 10 mm  250 mm; Merck) eluting with CH3CN/H2O (0.1%
TFA) 0–100% gradients.
4.3. Mass spectra
Mass spectra were recorded in MALDI-TOF (Matrix Assisted
Laser Desorption Ionization-Time of Flight) mode or on AXIMA-
CFR Kratos instrument.
able for sampling and exploring mixed-L,D-polypeptide structures
in their diversity of folds,12 for having the folds evaluated in fitness
to be designable as proteins,34 and for having main-chain folds
optimized for possible desired application over the chemical alpha-
bet in side chains.15 To test the methods,15,25 the present study
aimed the computational reprogramming of zinc-finger protein
as a hydrolase enzyme. While protein design field has witnessed
improvements in the force fields,35,36 the search size of conforma-
tional structures and sequence over side chains remains prohibi-
tive and the reason protein-structure prediction from input of
sequence remains a challenge largely usolved.37 The design field
has advanced by having the sequence design and the conforma-
tional design delinked.3–5 Leveraging the delinking, we have
reported independent methods for design first over the stereo-
4.4. Circular dichroism
Circular dichroism (CD) was recorded on JASCO J-180 CD spec-
tropolarimeter at 298 K in 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 1 nm
steps, five scans were averaged after baseline correction for sol-
vent. Working solutions 20–100 lM in peptide were prepared by
optical measurements and of Zn(ClO4)2Á6H2O gravimetrically. The
observations in millidegrees were converted to molar residue ellip-
ticity [hMRW] with a reported relation.38
4.5. Nuclear magnetic resonance
chemical alphabet in
L and D structure and then over the chemical
alphabet in side chains.14
1H NMR was on Bruker 800 MHz spectrometer, equipped with
cryoprobe, at 298 K. Peptide concentrations of 3 mM were used,
and 1D spectra were recorded also at 10-fold dilution. Solutions
were prepared in 90% H2O/10% D2O with DSS as the internal refer-
ence at pH 6.7 in presence and absence of Zn. Polypeptide back-
bone and side-chain assignments were obtained with 2D 1H–1H
TOCSY28 experiments using standard mlevtp pulse sequence for
spin lock. Spin lock time of 80 ms was used to ensure observation
of long-range couplings. The data were collected in 2 k time do-
main points. 512 increments of t1 were measured with 48 FIDs
per increment, the data being zero-filled to 1 k data points in F1
prior to Fourier transform. Distance constraints were obtained
from a 2D 1H–1H NOESY experiment29 with mixing time of
300 ms. The data were processed with TOPSPIN 2.0 and 2.1 and
The catalysis function in metal-hydrolases involves the side
chains in a multitude of roles, including the coordination with me-
tal ion and its activation as a hydrolase. Here we have approached
the activation of zinc in a possible zinc-finger-like
abb protein. The
design of a linker to join -amino-acid nucleated secondary struc-
D
26
tures was implemented with CYANA
.
Several software have been
developed for inverse optimizing folds,2 and we have developed
IDeAS to have side chains applied to hetero-chiral folds in the chi-
rality of interest.15 Applying first CYANA and then IDeAS, an
abb pro-
tein was achieved as a Zn-based hydrolase in this study. The
coordination and activation of zinc as a hydrolase catalyst was
achieved successfully but not the binding of the substrate and its
enzyme-like hydrolysis. Design of a metal-free hydrolase, illustrat-
ing rational approach to the modeling of folds over the alphabet in
L
and
D
structures, is reported elsewhere.16 Future iterations of the
analyzed with Computer Aided Resonance Assignment
(CARA)
software. Structure calculations were performed with CYANA 2.1.27
structure accomplished in the present study will aim to find and

8276
K. Patel et al. / Bioorg. Med. Chem. 18 (2010) 8270–8276
Dihedral angle restraints based on 3JNHC Y values were used, wher-
Supplementary data
a
ever possible, for structure refinements.
D-Amino acid residues
were introduced in CYANA library under the guidance of the devel-
oper. Structures were energy minimized using GROMACS software
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.10.003.
package.39 Structural models were rendered with PYMOL
VIEWERLITE softwares.
, MOLMOL or
References and notes
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D
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Acknowledgments
We acknowledge DST (09DST028), Government of India, for
financial support. We acknowledge Professor Nand Kishore for
their help with ITC experiments. K.R.S. is recipient of fellowships
from Council of Scientific and Industrial Research (CSIR). We
acknowledge TIFR, Mumbai for the help with NMR experiments.