Tests of the simple model of Lin and Brandts for the folding kinetics of ribonuclease A

Abstract

Brandts recently proposed a simple model for the folding kinetics of RNase A in which folding intermediates are not detectable. The basic assumption of the simple model for the major unfolded species, which is produced by a slow isomerization (the "X .dblarw. Y reaction" according to Lin and Brandts) after unfolding, was tested. The simple model assumes that in refolding the slow Y .fwdarw. X reaction must occur before any folding can take place. The Y .fwdarw. X reaction was measured during folding. Tyrosine-detected folding occurs before the Y .fwdarw. X reaction; the difference in rate between the Y .fwdarw. X reaction and folding monitored by tyrosine absorbance becomes large when the stabilizing salt 0.56 M (NH4)2SO4 is added. The simple model predicts that the kinetic properties of the X .dblarw. Y reaction in unfolded RNase are the same as those of tyrosine-detected folding. However, the kinetics of the X .fwdarw. Y reaction in unfolded RNase are apparently independent of urea concentration, whereas the rate of tyrosine-detected folding decreases almost 100-fold between 0.3-5 M urea, as reported by Linn and Brandts. The kinetic properties of the X .dblarw. Y reaction in unfolded RNase are apparently characteristic of proline isomerization.