Insulin regulates metabolic homeostasis in vertebrates by binding to the insulin receptor and thereby triggering a signal-transduction pathway. How insulin binds is an outstanding problem in structural molecular biology. This application describes 2D and multi-dimensional heteronuclear NMR studies of (i) native and genetically altered insulins and (ii) extracellular domains of the insulin receptor. These studies probe in a logical series of steps relationships between structure and function and are likely to have broad implications for the general analysis of protein structure, dynamics, and folding. The results will have long-term application to rational design of insulin agonists in clinical medicine. Particular emphasis will be placed on the combined use of genetic engineering and 15N and 13C isotopic labeling. Heteronuclear 1H-detected 2D, 3D, and 4D-NMR spectroscopy of uniformly and selectively labeled proteins will be used to study conformation and dynamics by means of NOEs, J-coupling constants, 15N and 13C spin-lattice (T1) relaxation times, and amide proton exchange rates. Experimental parameters will be used as restraints to define three-dimensional structures in solution and to compare observed NMR parameters with those predicted by molecular-dynamics simulations. The design of this application is stimulated in part by the extraordinary observation that previous crystal structures of insulin dimers and hexamers depict inactive conformers (Derewenda et al., J. Mol. Biol. 256, 1406-1412 (1991). The proposed NMR studies will proceed in three steps. Short-term. The relationship between X-ray and solution structures will be evaluated by comparative NMR studies of engineered dimers and monomers (the functional species). Medium-term. Correlation of structure and function will be evaluated by comparative study of analogues containing mutations in the receptor-binding surface that confer enhanced or decreased receptor- affinity. Long-term. Structural domains of the insulin receptor alpha- subunit will be characterized as NMR models of the receptor-hormone complex.
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