CONFORMATIONS OF INSULIN &ITS RECEPTOR COMPLEXES

The relationship of structure and bioactivity in insulin is complex. Determination of a solution conformation may yield insight into its activity, and establish methods for studying insulin's interaction with its natural receptors, and rational design of new drugs for diabetes mellitus. It is proposed to study the conformations of several derivatives of differing activity, synthetic analogs similar to known mutant insulins in humans, and new analogs designed to test possible structure/activity relations. The major approach will be application of Nuclear Magnetic Resonance (NMR) techniques to insulin analogs specifically designed and synthesized for those studies. We shall assign spectra and detect protein secondary structure by a combination of conventional and two-dimensional homo- and heteronuclear techniques. Distance geometry calculations will be used to produce structural information on those portions of the analogs which are conformationally static, from nuclear Overhauser measurements (nOe). Comparative conformational analyses of the analogs will test the possible relation of the solution structure to bioactivity. Molecular dynamics will be used to model flexible regions of the hormone. With the long term view of extending these correlations to direct measurements of the hormone/receptor complex, we shall apply new methods of NMR, for connectivity and nOe measurements, which use detection of protons, and secondary irradiation of 13C and 15N at labeled sites in insulin analogs. The objectives of these methods, are the selection of proton homonuclear connectivity and nOe data for atoms spincoupled to 13C or 15N, and the indirect measurement of 13C and 15N NMR values in, hormone-receptor complexes. We shall attempt to identify fragments of insulin receptor suitable for complex formation and study such complexes. The conformational information derived will be used to design possible analogs which might test the role of flexibility and orientation of various parts of insulin. These analogs will be synthesized, assayed, and conformationally characterized, by NMR and as time permits, directly in complexes with receptor fragments.