Effects of solvent viscosity and different guanidine salts on the kinetics of ribonuclease A chain folding

Abstract

The kinetics of folding of the two forms of unfolded ribonuclease A have been measured as a function of solvent viscosity by adding either glycerol or sucrose. The aim is to find out if either reaction is rate limited by segmental motion whose rate depends on external friction. The fast folding reaction (U2 ⇄ N) is known to be the direct folding process, and the slow folding reaction (U1 ⇌ N) is known to be rate limited by an interconversion between two forms (U1 ⇌ U2) which are present after unfolding in strongly denaturing conditions. No dependence on solvent viscosity is found, either for the direct folding reaction or for the interconversion reaction. Each folding reaction has also been tested to see if its rate depends on the concentration of one or more partly folded intermediates, by adding denaturants destabilize any partly folded structures. Different guanidine salts are used as denaturants to vary the denaturing effectiveness of the salt while holding the guanidinium ion concentration constant. The rates both of the direct folding reaction and of the interconversion reaction decrease in relation to the denaturing effectiveness of the salt. However, there is a basic difference between the responses of the fast and slow folding reactions to low concentrations of denaturants. Although each folding reaction produces native protein, there is an 800‐fold decrease in the rate of the fast folding reaction in 1M guanidine thiocyanate and only a 13‐fold decrease in the rate of the slow folding reaction. This is consistent with the fast reaction being the direct folding process and the slow reaction being rate limited by the initial conversion of the slowrefolding to the fast‐refolding form. Both the lack of viscosity dependence and the effects of denaturants indicate that the formation of structure is rate limiting in the direct folding reaction, U2 ⇌ N. The failure to find a viscosity dependence for the interconversion reaction, U1 ⇌ U2, indicates that in this reaction also friction‐limited segmental motion is not the rate‐limiting process. Since the U1 ⇌ U2 interconversion still occurs when the polypeptide chain is completely unfolded, the surprising result is that its rate in refolding conditions depends significantly on a reaction intermediate which is “denatured” by guanidine salts. Copyright © 1978 John Wiley & Sons, Inc.