NSF Award
9004670
We have been studying the structural features of insulin analogs in solution as part of project to engineer proteins with altered biologic and physical properties. A striking finding has been the degree of flexibility observed for insulin in solution. Most notably, the binding potency of several analogs correlates with overall molecular flexibility. Selected mutations and bridging structures can lock conformational substructures that are not productive for receptor recognition. Moreover, mutations that increase molecular flexibility appear to reverse these effects. Therefore, we have identified a system for relating protein structures in crystals, in solution (NMR) and at the receptor surface. It appears that changes in molecular structure are necessary accompaniments to high affinity receptor recognition. In the current proposal I have outlined three complimentary approaches for defining the structural requirements of insulin action. First, site-site interactions between insulin and the insulin receptor will be positively identified. In this fashion, comparative binding surfaces of insulin and the insulin receptor will be constructed, using the three- dimensional crystal structure of insulin as a template. Second, solution structures of selected series of analogs are being analyzed by NMR and CD spectroscopy. Specific structural features will be correlated with receptor binding potency. Finally, increasing concentrations of truncated receptor homologs will be added to solutions of insulin and selected analogs; the effects on insulin structure will be characterized. In combination these studies should identify the structural features in solution that are necessary for receptor binding and the changes in structure that accompany binding. Furthermore, these studies will help to provide a more general foundation for studying relationships between protein crystal structures, solution structures, and changes in structure that occur during complex formation.
