The biosynthesis of insulin proceeds through a precursor protein, proinsulin. This larger protein (86 residues) folds to form three native disulfide bonds, which are retained in the proteolytic 2-chain fragment, insulin. Unfortunately, proinsulin is refractory to crystallization under all conditions successfully applied to insulin itself. The absence of structural information has blocked efforts to understand the role of the C-peptide and the targets for cleavage by the converting enzymes. We therefore propose to determine the solution structure of human proinsulin. Preliminary results strongly suggest that this can be accomplished using the Zn-coordinated hexamer at 55xC and pH 7. The thermal stability and structural rigidity of the hexamer provide high-resolution spectra to be obtained under these conditions. Because of the limited resolution in spin systems at the junctions between the connecting peptide and the A- and B-domains (sites of biological recognition by converting enzymes) and because isotopic labeling is impeded by inefficient expression in E. coli, we seek to obtain homonuclear data at the highest field strength.
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