A quantitative treatment of the kinetics of the folding transition of ribonuclease A

Paul J Hagerman, Robert L. Baldwin

Research output: Contribution to journalArticlepeer-review

163 Scopus citations


New experimental data and a quantitative theoretical treatment are given for the kinetics of the thermal folding transition of ribonuclease A at pH 3.0. A three-species mechanism is used as a starting point for the analysis: U1 (slow) ⇌ U2 (fast) ⇌ N, where U1 and U2 are two forms of the unfolded enzyme with markedly different rates of refolding and N is the native enzyme. This mechanism is based on certain facts established in previous studies of refolding. The kinetics of unfolding and of refolding show two phases, a fast phase and a slow phase, over a range of temperatures extending above the transition midpoint, Tm. The three-species mechanism can be used in this range. At higher temperatures a new, much faster, kinetic phase is also observed, corresponding to the transient formation of a new intermediate (I). Although the general solution for a four-species mechanism is complex, it is not difficult to extend the three-species analysis for the special case found here, in which the fast reaction (I ⇌ N) is well separated from the other two reactions. At temperatures below the transition zone the slow phase of refolding becomes kinetically complex. No attempt has been made to extend the analysis to include this effect. The basic test of the three-state analysis is the prediction as a function of temperature of α2, the relative amplitude of the fast phase, both for unfolding and refolding. At temperatures above Tm, for which the three-state analysis must be extended to include the new intermediate I, a corresponding quantity α2(cor) is predicted and compared with measured values. Data used in the three-state prediction are values of τ2 and τ1, the time constants of the fast and slow kinetic phases, plus a single value of α2 measured when τ2 and τ1 are well separated. The observed and predicted values of α2 agree within experimental error. The analysis predicts correctly that, for these experiments, α2 should have the same value in unfolding as in refolding in the same final conditions. The analysis also predicts satisfactorily the equilibrium transition curve from kinetic data alone. Four striking properties of the kinetics are explained or correlated by the analysis: (a) the drop in α2 to a minimum near Tm as well as the delayed rise in α2 above Tm; (b) the vanishing of α1 above the transition zone; (c) the sharp drop in TI inside the transition zone followed by a partial leveling off outside this zone; and (d) the passage of τ2 through a maximum near Tm. Through a comparison of observed and predicted values of α2, the analysis also rules out the alternative three-species mechanism U1 (slow) ⇌ N (fast) ⇌ U2. Finally, the temperature dependence of the amplitude for the fast reaction (I ⇌ N) is discussed: the behavior of I is like that of U2, and I may be an unfolded species populated at equilibrium. If so, I accounts for only 2% of the total unfolded enzyme and would not be detected in refolding experiments below Tm. Possible molecular interpretations of the U1 ⇌ U2 ⇌ I ⇌ N mechanism are discussed briefly.

Original languageEnglish (US)
Pages (from-to)1462-1473
Number of pages12
Issue number7
StatePublished - 1976
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


Dive into the research topics of 'A quantitative treatment of the kinetics of the folding transition of ribonuclease A'. Together they form a unique fingerprint.

Cite this