NON-PROLYL CIS-TRANS PEPTIDE-BOND ISOMERIZATION AS A RATE-DETERMININGSTEP IN PROTEIN UNFOLDING AND REFOLDING

In wild-type ribonuclease T-1 the peptide bond between Tyr38 and Pro39 is in the cis conformation. When Pro39 is replaced by an alanine this cis conformation is retained, and a non-prolyl cis Tyr38-Ala39 peptid e bond is generated. We employed a stopped-flow double-mixing techniqu e to investigate the kinetics of the cis --> trans isomerization of th is peptide bond in the unfolding and the trans --> cis isomerization i n the refolding of Pro39Ala-ribonuclease T-1. In 6.0 M GdmCl (pH 1.6) and 25 degrees C the protein unfolds rapidly with a time constant of 2 0 ms, followed by Tyr38-Ala39 cis --> trans isomerization. This reacti on shows a time constant of 730 ms and is about 60-fold faster than th e isomerization of the Tyr38-Pro39 bond in the wild-type protein. Unfo lded molecules with the Tyr38-Ala39 bond still in the native-like cis conformation accumulate transiently for a short time after unfolding i s 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 degrees C). Aft er more than three seconds of unfolding virtually all protein molecule s contain an incorrect trans Tyr38-Ala39 bond and refolding is deceler ated approximately 1000-fold, because Tyr38-Ala39 trans --> cis re-iso merization is very slow and, with its time constant of 480 s, determin es the overall rate of refolding. Due to the coupling of the cis-trans equilibrium with protein folding it was possible to measure the kinet ic parameters of the isomerization of a non-prolyl peptide bond in a p rotein. Previously this could not be accomplished, because the trans i somer is strongly preferred for unsubstituted peptide bonds in oligope ptides under virtually all conditions. Our data indicate that the kine tics of Tyr38-Pro39 and of Tyr38-Ala39 isomerization differ predominan tly in the rate of the cis --> trans, rather than of the trans --> cis reaction. The rate of the trans --> cis reaction is, however, measure d during refolding and may be influenced by the formation of ordered p rotein structure.