Biennial Report 2005-2007 - Saha Institute of Nuclear Physics

Biophysical Sciences 207basis of stability of the proteases has been explained from the high resolution X-ray structures. Wehave determined the substrate specificity of each protease using spectroscopic techniques with arange of chromogenic peptide substrates. The complexes of ervatamins with a substrate analoginhibitor have been crystallized and X-ray structures have been determined to further explain thesubstrate specificity from a structural point of view.Raka Ghosh, Sampa Biswas, Chandana Chakrabarti, JK DattaguptaC& MB6.1.2.4 Cloning and expression of Papain to improve thermostability for industrialapplicationPapain-like plant cysteine proteases form a well-characterized enzyme having various industrial andpharmacological applications. Papain is the archetype enzyme of this family having high proteolyticactivity suitable for industrial applications. Structure-based protein engineering studies have beeninitiated to increase the stability of the enzyme, another important factor responsible for moreuseful application of any enzyme used in industry. The papain precursor has been cloned andexpressed in a bacterial system. The recombinant protein has been refolded from inclusion bodiesand purified to homogeneity.Debi Choudhury, Sumana Roy, Sampa Biswas, JK Dattagupta, Chandana ChakrabartiC& MB6.1.2.5 Spacer Asn determines the fate of Kunitz (STI) inhibitors, as revealed bystructural and biochemical studies on WCI mutantsNatural serine protease inhibitors, in their own right, bind tightly to their target proteases like asubstrate, yet their rate of hydrolysis is very slow. We have demonstrated the role of a conservedspacer Asn, projecting from the scaffold of the inhibitor towards the binding loop, that resisthydrolysis of the inhibitor. We prepared five mutants of WCI and the results of the structural,kinetic and limited proteolysis studies on these mutants showed that the side chain of spacer Asnfits snugly in the reactive site loop cavity and its ND2 atom forms hydrogen bonds with P2 and P1carbonyl O at either side of the scissile bond holding the cleaved products together and favoringreligation. Through a database analysis, we identified such spacer asparagines in 5 other familiesof serine protease inhibitors with similar side chain disposition and on that basis we have proposeda mechanism of retaining the inhibitory property for most of the serine protease inhibitors.Jhimli Dasgupta, Susmita Khamrui, Jiban K Dattagupta, Udayaditya SenC&MB6.1.2.6 Single mutation at P1 of a chymotrypsin inhibitor changes it to a trypsininhibitor: X-ray structural (2.15 Å) and biochemical basisTo explore the change in specificity, caused by the mutations at P1 site of WCI, we mutated its P1residue (Leu to Arg) and solved the crystal structure at 2.15Åresolution. Structural, biochemical,phylogenetic and docking studies showed that P1 mutation converted WCI to a strong trypsininhibitor. The reactive site loop conformation of the mutant deviated from WCI and adopted a