Executive Summary : | There is a dearth of comprehensive knowledge related to the relationship between the amino acid sequence, structure, and dissociation kinetics of various oligomeric states of insulin analogs, the properties that are immensely critical for the biological action of insulin. Not only that, multiple aspects of the interaction of insulin or its analogs with other components of formulation have not been fully understood. This proposal is an attempt to address this Lacuna. Recent studies (Pandyarajan et al. 2016; Hua et al. 2009) have tried to expand the pool of molecular level information on insulin mutation and its effect on stability and absorption kinetics, however complete biophysical analysis and exploration of new peptides to control dissociation kinetics of insulin hexamer is lacking. The study here involves an extensive biophysical analysis of the interaction of various oligomeric states of insulin/analogs and their interaction with protamine or similar peptides. Our group has recently tried to investigate the interaction mechanism of hexamer WT-insulin and protamine (Aggarwal et al. 2022), which are at the heart of insulin formulation. There, isothermal titration calorimetry (ITC) and atomic force microscopy (AFM) studies suggested the condensation of insulin molecules upon protamine binding. The fluorescence and molecular docking implied the role of Tyr residues in the complex formation, further indicating how protamine would penetrate the dimer interface and unsettle hexamer insulin species. In conclusion, we stated that insulin's self-association ability is impaired due to protamine binding. Here, we expect that the objectives of this proposal will deliver knowledge that can be used to build a molecular strategy to augment insulin stability while preserving kinetics. The knowledge gained here may help to guide the development of future analogs for insulin formulations. |