Guanidine-unfolded state of ribonuclease A contains both fast- and slow-refolding species.

Abstract

The kinetics of the refolding reaction of ribonuclease A from high concentrations of guanidine hydrochloride or urea are biphasic, and show two refolding reactions whose rates differ 450-fold at pH 5.8 and 250. Measurements of cytidine 2'-phosphate binding during refolding, after stopped-flow dilution of guanidine hydrochloride (Gdn-HCl) or urea, show that functional bovine pancreatic ribonuclease A (RNase A; ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) is formed in both the fast and slow phases of the refolding process. We conclude that the guanidine-unfolded state of RNase A is an equilibrium mixture of fast-and slow-refolding species, as was found previously for the heat-unfolded state at low pH. The fraction of the fast-refolding species in guanidine or ureaunfolded RNase A is the same as that in the heat-unfolded protein at pH 2.Previous work has shown that the fast-refolding species disappears as the pH is raised from 3 to 5 for heat-unfolded RNase A. This pH effect is not present in refolding from concentrated GdnHCl solutions: the same proportion of the fast-refolding species is found from pH 2 to pH 6, and also from 2 M to 6 M Gdn-HCI at pH 5.8. We conclude that the same proportion of the fast-refolding species is present at equilibrium whenever the residual structure in unfolded RNase A is reduced to a low level, and that the structural difference between the fast-refolding and slow-refolding species of RNase A lies in the configuration of the random coil polypeptide chain.The observed rates of protein folding reactions are many orders of magnitude faster than predicted from a purely random search of all possible configurations for a random coil polypeptide chain devoid of structuret. A possible explanation for this difference is that an unfolded protein is not a random coil. The elements of residual structure play a crucial role in directing the course of protein folding, and thus considerably restrict the possible pathways (1) There seems to be a contradiction between the two proposals: (a) that a guanidine-unfolded protein behaves as a homogeneous random chain and (b) that this same unfolded state still possesses some elements of residual structure which increase the rate and determine the pathway(s) of folding. However, these proposals are compatible if the residual structure, important for the folding process, is not present as such in the unfolded state, but is rapidly formed after refolding is initiated. For instance, formation of a-helical segments could take place within the dead time of stopped-flow measurements (6) and limit the possible pathways for refolding, if the a-helical segments are stable under the conditions in which refolding is initiated, without a requirement of prior slow steps in refolding to provide a stabilizing environment.Studies of the refolding of heat-unfolded RNase A (7-11) have shown that the heat-unfolded state does not behave as a single species in refolding. Instead, distinct fast-refolding and slow-refolding species of the heat-unfold...

Quantitative analysis of the kinetics of denaturation and renaturation of barstar in the folding transition zone

“…A similar result has been seen with BSCCAA (Schreiber & Fersht, 1993b). Thus, the relative amounts of UF and Us in equilibrium-unfolded barstar or BSCCAA cannot be determined from a l , as it can be in the case of ribonuclease A (Garel & Baldwin, 1973;Garel et al, 1976). A double-jump experiment was therefore devised to determine K2, (Schmid, 1986a).…”

Role of proline isomerization in folding of ribonuclease A at low temperatures

“…In unfolded RNase A there is an equilibrium between fastfolding (UF) and slow-folding (Us) forms (1)(2)(3)(4)(5)(6). The kinetics of the UF Us reaction can be measured by "double-jump" experiments (4,7).…”

CD properties of the fast‐ and slow‐folding forms of unfolded ribonuclease a

“…Hence both species are unfolded to approximately the same extent as assumed by the proline model of generation of slow-folding molecules [5,6] and as expected from the kinetics of RNase A folding [1,7,11].…”

“…This difference was tentatively ascribed to strictly local changes in the environment of one tyrosine residue, coupled to proline isomerization in the unfolded polypeptide chain. However, it could not be excluded definitely that the fluorescence difference between UF and U, was caused by the presence of residual ordered structures in UP in contradiction to the assumption of the proline model and to [ 1,5,7]. Measurements of the circular dichroism in the amide region provide the most useful spectroscopic approach to detect such residual ordered structure.…”