Insulin is of central importance in human health and provides a general model for studies of protein structure and recognition. How does the hormone bind to the insulin receptor? How does its three-dimensional structure change in the hormone-receptor complex? Of both basic and applied importance, these questions will be investigated by synthetic, biochemical and structural approaches. The following Specific Aims are proposed:
Aim 1 -Toward the Receptor-Active Structure of Insulin: Multidimensional NMR Studies of Paradoxical Analogs. Crystal structures of insulin are now appreciated to represent inactive conformers of the hormone. To deduce the receptor-active structure, we will synthesize active analogs with predicted non-native structures. Of particular interest will be comparison of analogs containing corresponding D and L amino-acid substitutions. Solutions structures will be determined by NMR.
Aim 2 -Mapping the Insulin Receptor by Novel Photo-Crosslinking Analogs. Contact points between insulin and the receptor will be determined by synthesis of A- and B-chain analogs containing a photoactivatable crosslinking amino acid (L-para-azido-Phe). Sites of covalent attachment in the receptor will be mapped by protease digestion and direct micro sequencing.
Aim 3 -Mapping the Insulin-like Growth Factor I (IGF-I) Receptor. Determination of contact points between this growth factor and its receptor will be pursued by the procedures outlined in Aim 2 above, using as parent compound an IGF-I analog consisting of a two-chain molecule embodying the A domain of IGF-I and the B domain of insulin or IGF-I. The feasibility of our Aims are in each case demonstrated by preliminary results. This competing application thus offers the exciting possibility of integrating nonstandard peptide chemistry with biochemistry and biophysics to investigate fundamental aspects of insulin action.
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