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.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
2P41RR002301-16
Application #
6309185
Study Section
Project Start
2000-04-15
Project End
2005-02-28
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
16
Fiscal Year
2000
Total Cost
$7,533
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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