Non-prolyl cis–transPeptide Bond Isomarization as a Rate-determining Step in Protein Unfolding and Refolding

Abstract

In wild-type ribonuclease T 1the peptide bond between Tyr38 and Pro39 is in the cisconformation. When Pro39 is replaced by an alanine this cisconformation is retained, and a non-prolyl cisTyr38–Ala39 peptide bond is generated. We employed a stopped-flow double-mixing technique to investigate the kinetics of the cis→ transisomerization of this peptide bond in the unfolding and the trans→ cisisomerization in the refolding of Pro39Ala-ribonuclease T 1. In 6.0 M GdmCl (pH 1.6) and 25° the protein unfolds rapidly witha time constant of 20 ms, followed by Tyr38–Ala39 cis→ transisomerization. This reaction shows a time constant of 730 ms and is about 60-fold faster than the isomerization of the Tyr38–Pro39 bond in the wild type protein. Unfolded molecules with the Tyr38–Ala39 bond still in the native-like cisconformation accumulate transiently for a short time after unfolding is initiated, and they can refold very rapidly to the native state with a time constant of 290 ms (in 1.0 M GdmCl, pH 4.6, 25°C). After more than three seconds of unfolding virtually all broken molecules contain an incorrect transTyr–Ala39 bond and refolding is decelerated approximately 1000-fold, because Tyr38–ALa39 trans→ cisre–isomerization is very slow and, with its time constant of 480 s, determines the overall rate of refolding. Due to the coupling of the cis–transequilibrium with protein folding it was possible to measure the kinetic parameters of the isomerization of a non-prolyl peptide bond in a protein. Previously this could not be accomplished, because the transisomer is strongly preferred for unsubstituted peptide bonds in oligopeptides under virtually all conditions. Our data indicate that the kinetics of Tyr38–Pro39 and of Tyr38–Ala39 isomerization differ predominantly in the rate of the cis→ trans,rather than of the trans→ cis,reaction. The rate of the trans→ cisreaction is, however, measured during refolding and may be influenced by the formation of ordered protein structure.