Structure and Interactions of Lectins
The facility was used in the structure analysis of a complex of peanut
lectin with C-lactose. The study showed that C- and O – lactose have identical
conformation when bound to the lectin, as indeed when they are free. Another
major effort involved the structural analysis of the dimeric mannose binding
lectin from garlic. It has type II b -prism fold, similar to that observed
in tetrameric snowdrop lectin. However, the oligosacharide specificity
of the two lectins is different, providing an example of how carbohydrate
specificity can be generated by oligomerization. In another effort, the
facility was extensively used in a detailed analysis and theoretical study
of the variability in quaternary association of legume lectins.
Hydration, plasticity and action of proteins
The facility continues to be used in studies employing water-mediated
transformations, in particular in the X-ray analysis of orthorhombic lysozyme
grown at basic pH and its low humidity variant. Comparison with structures
of crystals grown at acidic pH does not indicate any systematic pH-dependent
variations in molecular geometry. The changes in the molecule during the
transformation to the low humidity form are more pronounced in the C-terminal
Structural analysis of E-coli uracil DNA glycosylase
In a new initiative, in which the facility was used, the X-ray analysis
of a complex of E.coli uracil DNA glycosylase with a proteinaceous
inhibitor has been carried out. The structure, the first UDG form a prokaryotic
source to be X-ray analyzed, provides a framework for examining UDG’s from
bacteria, including pathogenic ones.
Three-dimensional structure of physalis mottle virus
The crystal structure of physalis Mottle Virus belonging to the Tymovirus
group of plant viruses has been solved at 3.8Å resolution. The structure
determination was based on initial phasing using the known structure of
TYMV. The electron density maps were convincing at the level of backbone
detail, and at the level of side chain interpretation. The interactive
computer graphics facility was used for visualizing and building the molecular
model of the virus particle.
Structural studies on a Xylanase
The facility was used in the structure analysis at 1.8Å resolution
of a thermostable xylanase isolated from Thermoascus Aurantiacus.
The structure solved by molecular replacement method has the overall (a
/b )8 fold, i.e., the TIM barrel fold. The aminoacid sequence was determined
from the electron density map aided by multiple alignment of related xylanase
sequences. The sequence thus obtained provides a correction to the sequence
reported earlier based on biochemical methods. The active site consists
of two glutamates located at the C-terminus end of b -barrel. The residues
Cys 255 and Cys 261 are linked by a disulfide bond. Three salt bridges
have been identified which together with the disulfide bond might be contributing
to the thermal stability of the enzyme which retains its activity even
at 70° C.
Rational Design of temperature sensitive mutants of globular protein
Temperature sensitive (Ts) mutants of a protein are an extremely powerful
tool for studying protein function in vivo and in cell culture. We have
devised a method to predict those residues in a protein sequence that,
when appropriately mutated, are most likely to give rise to a Ts phenotype.
Since substitutions of buried hydrophobic residues often result in significant
destabilization of the protein, our method predicts those residues in the
sequence that are likely to be buried in the protein structure. We also
indicate a set of amino acid substitutions that should be made to generate
a Ts mutant of the protein.
Residue Depth: A novel parameter for the analysis of protein structure
Accessible surface area is a parameter that is widely used in analyses
of protein structure and stability. However accessible surface area does
not distinguish between atoms just below the protein surface and those
in the core of the protein. In order to differentiate between such buried
residues, we describe a computational procedure for calculating the depth
of a residue from the protein surface. Residue depth correlates significantly
better than accessibility with effects of mutations on protein stability
and on Protein-protein interactions.
Crystallographic studies of the denaturation of Rnase S
Although chemical denaturants are widely used to characterize protein
folding and stability, there is relatively little detailed structural information
on protein in the presence of denaturants. In an attempt to view the onset
of urea denaturation in ribonuclease we have collected x-ray diffraction
data on ribonuclease S crystals soaked in 0.0, 1.5, 2.0, 3.0 and 5.0 Molar
(M) urea. At concentrations above 2M urea, crystals were stabilized by
glutaraldehyde cross-linking. We have also collected data on ribonuclease
S crystals at low pH in an attempt to study the onset on pH denaturation.
The resolution of the datasets ranges from 1.9 to 3.0 Å.
Genome analysis of thermophilic protein sequences
We have analyzed complete genomic sequences from several thermophilic
and mesophilic organism in order to find out if there are any sequence
based rules that describe protein thermostability. Preliminary analysis
indicates compositions of thermophiles and mesophiles are
not significantly different. However there appear to be differences in
the distribution of charged residues, especially in helical regions of
Structural studies on plasmodium falciparam triosephosphate isomerase
Cavity creating mutation, Y74C, at the dimeric interface of pfTIM was
designed to link the subunits through disulfide bridge. Disulfide bonds
were formed between mutated position and native C13. It was found by spectroscopic
studies that mutation decreased the stability of the protein. However,
DTNB treatment restored the stability. Crystal structure of this mutant
was determined to understand the molecular basis of the decreased and restored
stability. Electron density analysis using the graphics program O, interestingly,
showed that one of the two symmetry related disulfide bonds was reduced.
Critical analysis of the effects of mutation revealed the molecular basis
for the spectroscopic observation.
Crystal structure of pfTIM with its transition state analogue phosphoglycolic
acid was determined to a resolution of 3.0Å. Analysis of protein-ligand
interactions were being done using the state-of-the-art packages available
at the Interactive Graphics facility.
Structural studies on calcium binding protein from
To elucidate the mechanism of calcium signaling and the role of calcium
and CaBP in host-parasite relationships, structural studies on CaBP from
entamoeba histolytica was pursued. Amino acid sequence analysis revealed
the presence of four calcium binding sites. However, the sequence identity
of the CaBP with other CaBP’s is fairly low. Moreover the ion-binding parameters
of the CaBP was also different when compared to other CaBP. Crystals structure
was determined in order to understand molecular details of calcium binding.
The crystals belong to the space group P6122 and diffracted to a medium
resolution of 3.5Å. Initial analysis of electron density maps using
the program O showed good densities for most part of the side chain atoms.
Further analysis and refinement of the structure is in progress.
Structural studies on glycolytic enzymes from Mycobacterium tuberculosis
Glycolysis is an ubiquitous metabolic pathway involving ten key enzymes.
In mycobacterium tuberculosis, apart from being the main source
of energy, the pathway also leads to the formation of intermediates in
the synthesis of its major protective fatty acid coat. As a first step
in exploring the usefulness of these enzymes in design of antitubercular
drugs, their three dimensional structures are being studied by molecular
modelling techniques using the Interactive Graphics facility.
Analysis of the structure, function relationship of dopamine agonists
Several drugs in clinical use for psychiatric disorders act as either
dopamine agonists of antagonists, but their exact mode of action is not
known. The atomic structure of the dopamine receptor, therefore is being
studied which will enable the analysis of binding of various drugs to this
receptor, which in turn will help in understanding the molecular basis
for the action of these drugs. Modelling studies are being carried out
using the Interactive Graphics facility.
Visualization of RNA-protein complex and its modelling
The silicon graphics workstation at the interactive Graphics facility
is used extensively by our group for visualization of molecular models
of proteins and protein-ligand complexes. The BIOSYM software, INSIGHT
is used for this purpose. Insight was used as visual verification tool
for our RNA-Protein modeled complexes as well as for pictorially representing
several snap-shots from molecular dynamics simulation. Small minimization
and molecular dynamics routines using the Discover software were also made
Design of new organic porous solids
A few supramolecular synthons were synthesized and their self-organization
behavior were studied in crystalline state. To understand the role of various
non-covalent interactions acting in these systems. For this purpose, the
state-of-the-art packages in the Interactive Graphics facility have been
used extensively for visualizing and building the molecular structures
of the assembly.
Energy conformation of nucleosides and nucleotides
INSIGHT/DISCOVER package was used for the non-bonded energy calculation
to find the potential energies for various possible conformers in modified
nucleosides and nucleotides. Geometric calculations were also carried out
to study the weak interactions such as X-H…pi (X = C, N and O) and pi…pi
bonds which play significant role in deciding the conformation and packing
of aromatic molecules. Moreover mono, stereo illustrations and the molecular
packing diagrams were generated using the BIOSYM package at the Interactive
C-H...O interactions in Tetraoxa--Peristylane
Facility (Frodo & Bobscript graphics Packages) at the Interactive
Graphics has been used to generate the packing diagrams of the organic
compound Tetraoxa--Peristylane. In addition, to study the novel architecture
of the C-H…O interactions, the packages developed in the Interactive Graphics
facility have also been used.
Packing of a novel metal cluster in the crystal lattice
Using Interactive Graphics user facility, we were able to see the packing
arrangement of a chiral metal cluster in the unit cell. The molecule shows
an interesting helical pattern which is stabilized by inter and intra molecular
hydrogen bonding in the crystal lattice. By rotating the molecule in different
directions, the three dimensional view of the molecule is visualized in
depth which helped in understanding the conformation.