Kinetics and mechanism of the refolding of denatured ribonuclease A.
On the basis of two experimental observations, it is established that the refolding mechanism of ribonuclease A (RNase A) is independent of the nature of the denaturant used [urea or guanidine hydrochloride (Gdn.HCl)]. First, by use of a double-jump technique, it is demonstrated that a similar nativelike intermediate exists on the major slow-folding pathway of both urea- and Gdn.HCl-denatured RNase A. Second, from the temperature dependence of the slow-refolding kinetics, it is shown that the activation parameters (both enthalpy and entropy) of the rate-limiting steps, as monitored by tyrosine absorbance and fluorescence, are identical for the refolding of urea- and Gdn.HCl- denatured RNase A. A refolding scheme involving one intermediate on each of the two slow-folding pathways is proposed by adopting the notion that RNase A refolds through a sequential mechanism. However, these two intermediates are formed from their respective unfolded forms (USII and USI) through two different processes of distinct physical origin. The intermediate IN, which is formed from the major slow-folding species USII through a conformational folding step, already possesses many properties of the native protein. In contrast, the intermediate (designated as I') on the minor slow-folding pathway is formed from USI by the isomerization of a proline residue (possibly Pro93) and is still conformationally unfolded. It is shown that such a refolding scheme can account for the known kinetic features of both major and minor slow-refolding pathways of RNase A.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002417:Cattle; D004355:Drug Stability; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008433:Mathematics; D008954:Models, Biological; D010179:Pancreas / Q000201:enzymology; D011487:Protein Conformation; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D006146:Guanidines; D014508:Urea; D012259:Ribonuclease, Pancreatic; D019791:Guanidine