Cis-trans isomerization of the proline residue in insulin studied by 13C NMR spectroscopy

Abstract

The natural abundance 13C NMR spectrum of bovine insulin contains two resonances at 49.6 and 48.9 ppm which have a 7:3 intensity ratio at 298 K. Use of the DEPT spectral editing technique shows them to be of CH2 multiplicity. On the basis of their chemical shifts, which are well-resolved from other peaks, they are assigned as the C delta carbon of proline in trans and cis forms respectively. Since insulin contains only a single proline residue, the site of the isomerization can be localized at the peptide bond linking Thr-27 and Pro-28. On heating, the two peaks broaden, coalesce at 308 K, and then sharpen to yield a single peak at higher temperatures. The barrier for this process was calculated to be 64 kJ mol-1 (at the coalesce temperature), which is at the lower end of the range observed for proline isomerization in small peptides. Computer-graphic studies based on the X-ray crystal structure of insulin were used to deduce the structural implications of the cis-trans isomerism in this globular protein.