Nonnative isomers of proline-93 and -114 predominate in heat-unfolded ribonuclease A.

The peptide bonds preceding both Pro-93 and Pro-114, which are in the cis conformation in native RNase A, are predominantly in the trans conformation in the heat-unfolded protein. The percentages are estimated to be 60% and 63%, respectively, with a standard deviation of +/- 7% in each quantity. These ratios are close to those found for corresponding sequences in X-Pro-Y peptides. The concentration of the trans proline species was determined from the integrated intensities of resonance peaks of the C alpha H protons of Tyr-92 and Asn-113, which are well resolved in the 1D proton NMR spectrum of heat-unfolded RNase A. The assignments of the resonances were deduced from 2D NOESY and DQF-COSY spectra of unfolded RNase A in D2O. Furthermore, the C alpha H protons of both Tyr-92 and Asn-113 had an intense NOE cross-peak with the C delta H and C delta' H of the respective following prolines. For both Pro-93 and Pro-114, these NOE cross-peaks would arise only if the X-Pro peptide bond were in the trans conformation. It is generally believed that the rate of refolding of RNase A is considerably reduced by nonnative proline isomers, such as trans Pro-93. Two models for folding RNase A, that are consistent with these new results and the work of previous investigators, are presented here.