Kinetics and mechanism of the refolding of denatured ribonuclease A

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Abstract
On the basis of 2 experimental observations, the refolding mechanism of RNase A is independent of the nature of the denaturant used [urea or guanidine hydrochloride (Gdn .cntdot. HCL)]. First, by use of a double-jump technique, a similar nativelike intermediate exists on the major slow-folding pathway of both urea- and Gdn .cntdot. HCl-denatured RNase A. Second, from the temperature dependence of the slow-refolding kinetics, the activation parameters (both enthalpy and entropy) of the rate-limiting steps, as monitored by Tyr absorbance and fluorescence, are identical for the refolding of urea- and Gdn .cntdot. HCl-denatured RNase A. A refolding scheme involving one intermediate on each of the 2 slow-folding pathways is proposed by adopting the notion that RNase A refolds through a sequential mechanism. These 2 intermediates are formed from their respective unfolded forms (USII and USI) through 2 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. The intermediate (designated as I'')- on the minor slow-folding pathway is formed from USI by the isomerization of a Pro residue (possibly Pro93) and is still conformationally unfolded. Such a refolding scheme can account for the known kinetic features of both major and and minor low-refolding pathways of RNase A. Specifically, it can account for the following experimental observations; the merging of the major and minor slow-folding phases into a single one at high concentrations of denaturant; the retention of the biphasic behavior of the slow-folding kinetics at high concentrations of denaturant is added; and the reversal of the relative amplitudes of the 2 phases when detected by Tyr fluorescence as compared to UV absorbance. As an alternative to Pro isomerization, it is also hypothesized that the stereochemical inversion of the chirality of a disulfide bond in the protein molecule might be the underlying molecular basis for the IN .fwdarw. N step.

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