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

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. In an effort to understand the latter paradox, a simple sequential model of protein folding was introduced (II). It can approximate 2-state behavior by several tests, even when sizable concentrations of intermediate, partly-folded molecules are present. A basic prediction of this simple sequential model is the existence of a significant transient phase preceding steady-state kinetics of protein unfolding. If unfolding is highly cooperative, so that the reaction appears to be a 2-state reaction, the amplitude of the transient phase will be small and the separation in time scales between the transient phase and the steady-state phase will be large (e.g., 3-6 orders of magnitude in some hypothetical examples (1I). Nevertheless, temperature-jump studies (11) might detect the transient phases, since the sensitivity of modern instruments is excellent and the time range of observation extends down to a few microseconds.We have found a fast reaction in the thermal unfolding of RNase A over the entire pH range studied (from 1 to 7). Detailed measurements have been made only at pH 1.3, for the following reasons. (a) Unfolding occurs at relatively low temperatures (Fig. 1), which reduces the problem of cavitation (11) in measuring fast effects. (b) The reversibility of unfolding is good at this pH. (c) This is the pH range where an apparent 2-state unfolding of RNase A has been found (6-8).Oscilloscope records of the fast reaction observed at 286 nm are shown in Fig. 2. The reaction occurs in the millisecond time range over the entire temperature range of unfolding (Table 1). It is 3-4 orders of magnitude faster, depending on the temperature, than the slow unfolding reaction studied (11)

Nuclear Magnetic Resonance Study of the Thermal Denaturation of Ribonuclease A: Implications for Multistate Behavior at Low pH

“…The thermal denaturation in particular has been studied by a wide variety of techniques including circular dichroism (5), electron paramagnetic resonance of covalently bound spin labels (6), the action of proteolytic enzymes on RNase A during the course of denaturation (7), thermocalorimetry (8), ultraviolet difference spectroscopy (9), and temperature-jump and pHjump kinetic experiments (10)(11)(12)(13).…”

The Search for Folding Intermediates and the Mechanism of Protein Folding

“…We could look for unfolding intermediates formed between 10 −5 and 10 −1 s. Tian's 1971 T-jump experiments on RNase A unfolding showed that at least one intermediate is present (81). Tian next found a similar result with chymotrypsinogen A (80), which, like RNase A, was considered a paradigm for two-state protein folding.…”

Protein folding: Then and now

“…On these pathways, the protein molecule passes through well-defined partiallystructured intermediate states. Based on this view, numerous experiments and simulations were conducted to test the existence of transient folding intermediates [4,5]. It was expected that the determination of the structures and population of folding intermediates could help elucidate protein folding mechanisms.…”