Structural and Biophysical studies to evaluate the influence of oligomerization on biomolecular function

Abstract

Oligomeric proteins are more common than monomers. This aspect of molecular evolution substantially contributes to functional diversity and the acquisition of features such as cooperativity and regulatory mechanisms. Not surprisingly, oligomeric proteins are seen to play critical roles in cellular homeostasis, regulated biosynthetic cascades, signal transduction and, in some cases, control of gene expression and the cell phenotype. Homo-oligomers illustrate fundamental principles of protein-protein recognition. This thesis describes studies on three model systems to understand specific aspects of protein oligomerization. Dimers are the most prevalent structural state of proteins. Aminotransferases are obligatory dimers for their catalytic activity. In the first study described here, we describe the mechanistic features of this dimeric enzyme that contribute to the multistep catalytic mechanism involving two half reactions. This study was initiated by determining high resolution crystal structures corresponding to the distinct steps in the catalytic mechanism. This study also illustrated the need for flexibility at the dimeric interface as the substrate binding pocket is lined by residues from both monomeric units. The next study involved proteins with higher oligomeric arrangements. Two hexameric proteins- a hormone, Insulin and an unfoldase, ClpX, were used to evaluate the influence of functional and environmental features on oligomeric assembly. In the case of insulin, we could identify an intricate hydrogen bond network at the centre of the hexamer. A natural extension of this finding was to evaluate the role of co-solvents on the oligomeric assembly. Substantial structural perturbations at different ethanol concentrations provided a read-out to evaluate the influence of the solvent in maintaining the integrity of the oligomeric assembly. Insights on how flexibility- essential for enzyme activity- is incorporated in higher order oligomers came from biophysical studies on a hexameric unfoldase Mycobacterium...