Interactive Graphics Facility

• 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 region.

• Structural analysis of E-coli uracil DNA glycosylase (EcUDG)

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 and stability

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 the protein.

• 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 entamoebahistolytica

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 and antagonists

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 use of.

• 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 Graphics facility.

• C-H...O interactions in Tetraoxa-[4]-Peristylane

Facility (Frodo & Bobscript graphics Packages) at the Interactive Graphics has been used to generate the packing diagrams of the organic compound Tetraoxa-[4]-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.