Peptide hormones regulate metabolic pathways through specific hormone- receptor interactions at the cell surface.
The aim of this grant application is to invetigate the biophysical basis of hormone structure, folding and interactions in solution. Heteronuclear 2D and 3D-NMR techniques will be applied to insulin as a model system; comparative studies of wild-type and mutant insulins associated with diabetes mellitus will enable structure-function relationships to be defined. This proposal represents the first high-resolution structural study of the insulin monomer in solution, which is the physiologically active form. Although protein aggregation has limited previous one-dimensional NMR studies, new buffer conditions described in Section C make possible high-quality two-dimensional NMR analysis (Preliminary Results). Under these conditions insulin binds to the WGA-purified insulin receptor, demonstrating the biological relevance of the proposed measurements; additional control experiments ae provided by comparative NMR studies of genetically engineered monomeric insulins in aqueous solution. The interdisciplinary approach proposed in this grant application--combining NMR, genetic and theoretical techniques--will be of general utility in the study fo macromolecules of biological interest. High resolution 1 H, 13 C, 15 N-NMR spectroscopy will be used to study the conformation and dynamics of human insulin in solution. Mutant insulins bearing single amino-acid substitutions will be studied to correlate structural perturbations with receptor binding and to make rigorous assignment of NMR resonances. Mutations identified in patients with diabetes mellitus will be examined to determine the structural origins of their impaired function. Comparative studies of insulin and proinsulin will be undertaken to explore prohormone folding and elements of nascent protein structure. Analysis of folding mutants bearing systematic Cys Alpha E A la (or Ser) substitutions will be used to test models of protein structure and dynamics. Particular emphasis will be placed on the use of 15 N and 13 C labels, which provide intrinsic nonperturbing probes for protein structure and dynamics. Such labels may be observed indirectly through proton coherence and enable 2D-NMR spectra to be edited. Experimentally determined interproton distances will be used as restraints in molecular dynamics modeling to define elements of three-dimensional structure in solution. It is expected that this interdisciplinary strategy will lead to a deeper understanding of insulin structure, folding and function.
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