The Sequential Unfolding of Ribonuclease A: Detection of a Fast Initial Phase in the Kinetics of Unfolding

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Abstract
Temperature-jump studies have been used to detect a rapid reaction in the thermal unfolding of ribonuclease A (RNase A). The fast reaction occurs over a wide range of pH, and the results of a detailed study at pH 1.3 are reported here. Although its amplitude is small, the reaction is easily measurable over the entire temperature range of thermal unfolding. It occurs in the millisecond time range, and is faster by 3-4 orders of magnitude than the slow unfolding reaction studied previously. Unfolding is measured here by the change in absorbance at 287 nm, which reflects the exposure to solvent of buried tyrosine groups. Since the fast reaction has a detectable amplitude only in the temperature range of unfolding, it apparently detects the presence of intermediate, partly-folded states. Previous equilibrium studies of the unfolding of RNase A in the pH range 1-2 have indicated that it is essentially a 2-state reaction, without detectable intermediates. The existence of a rapid transient phase in the unfolding of RNase A had been predicted previously from a model for this unfolding reaction, based on nucleation-dependent sequential folding. The model served to reconcile kinetic and equilibrium studies of the thermal unfolding reaction of RNase A at neutral pH. Kinetic studies had shown that the slow unfolding reaction, measured at 287 nm, could be represented as a single exponential process, as expected for a 2-state reaction. However, earlier equilibrium measurements, especially the calorimetric studies of Sturtevant and coworkers, had revealed significant deviations from the 2-state behavior at neutral pH. These conflicting observations are explained by the model, which satisfies closely many criteria for a 2-state unfolding, even when appreciable concentrations of partly folded molecules are present. In particular, it predicts that the final, and major, portion of the kinetic reaction will occur as a single process characterized by an exponential time course.