Observation of intermediates in the folding of ribonuclease A at low temperature using proton nuclear magnetic resonance.

The refolding of ribonuclease A (RNase A) has been investigated in aqueous methanol cryosolvents in the 0 to -20 degrees C range. When a thermally unfolded sample was brought under renaturing conditions (e.g., -16 degrees C, 35% methanol, pH 2.8), the refolding, as monitored by the absorbance change at 286 nm (which reflects the degree of solvent exposure of Tyr), was triphasic and took approximately 1 h for completion. The 360-MHz proton nuclear magnetic resonance (NMR) spectrum of the native enzyme in either 35% or 50% aqueous methanol is very similar to that in aqueous solution. When the refolding of RNase A was monitored in the subzero temperature range with the signals of the His C2 protons, new resonances rapidly appeared, in addition to those from native protein. The new resonances are attributed to a partially folded intermediate state that has a relatively compact structure. Time-dependent changes were observed in the areas of the resonances from both native and partially folded species. The rates of peak area reduction for the intermediate state were the same as those for the increase in area of the native resonances, and similar to those for the second phase observed in the absorbance experiments. The results are consistent with the slow-refolding form of RNase A consisting of a least two distinct populations. A model for the folding of RNase A is proposed.