Structucture function realtionship of winged bean basic agglutin (WBA I): carbohydrate binding, Domain structure and amino acid sequesnce analyses

Abstract

The thesis deals mainly with the following aspects of Winged bean (Psophocarpus tetragonolobus) basic agglutinin (WBA I):

i) Thermodynamic studies on the binding of WBA I with various blood group A and B reactive sugars using fluorescence spectroscopy to elucidate the topography of the combining site of WBA I, the forces involved in their interaction, and the role of the fucosyl residues of these antigens in stabilization of the complex with WBA I.

ii) Extrinsic fluorescence thermodynamic analysis of the binding of 4-methylumbelliferyl glycosides of galactose (Gal) and N-acetylgalactosamine (GalNAc) to WBA I to probe the role of bulky substituents at the anomeric and at C-2 carbon of galactose.

iii) Stopped-flow spectrofluorimetric studies to study the effect of bulky substituents at the anomeric and at C-2 carbon of galactose on the mechanism of binding of ligands to WBA I.

iv) Titration calorimetric studies to determine the binding behavior of galactosides to WBA I and direct determination of the stoichiometries and the thermal effects involved therein.

v) Differential scanning calorimetry to study the domain structure of WBA I, its stability, and interactions within the protein and with different galactosides at the denaturation temperature.

vi) Complete amino acid sequence determination of WBA I and description of its structural features.