RECOMBINATION OF S-PEPTIDE WITH S-PROTEIN DURING FOLDING OF RIBONUCLEASE-S .2. KINETIC CHARACTERIZATION OF A STABLE FOLDING INTERMEDIATE SHOWN BY S-PROTEIN AT PH 1.7

At pH 1.7 S-peptide dissociates from S-protein but S-protein remains partly folded below 30 °C. A folded form of S-protein, labeled I3, is detected and measured by its ability to combine rapidly with S-peptide at pH 6.8 and then to form native ribonuclease S. The second-order combination reaction (k = 0.7 × 106 m-1 s-1 at 20 °C) can be monitored either by tyrosine absorbance or fluorescence emission; the subsequent first-order folding reaction (half-time, 68 ms; 20 °C) is monitored by 2′CMP † † See footnote to the summary of the preceding paper (Labhardt & Baldwin, 1979). binding. Combination with S-peptide and folding to form native RNAase S is considerably slower for both classes of unfolded S-protein (see preceding paper). I3 shows a thermal folding transition at pH 1.7: it is completely unfolded above 32 °C and reaches a limiting low-temperature value of 65% below 10 °C. The 35% S-protein remaining at 10 °C is unfolded as judged by its refolding behavior in forming native RNAase S at pH 6.8. The folding transition of S-protein at pH 1.7 is a broad, multi-state transition. This is shown both by the large fraction of unfolded S-protein remaining at low temperatures and by the large differences between the folding transition curves monitored by I3 and by tyrosine absorbance. The fact that S-protein remains partly folded after dissociation of S-peptide at pH 1.7 but not at pH 6.8 may be explained by two earlier observations. (1) Native RNAase A is stable in the temperature range of the S-protein folding transition at pH 1.7, and (2) the binding constant of S-protein for S-peptide falls steadily as the pH is lowered, by more than four orders of magnitude between pH 8.3 and pH 2.7, at 0 °C. The following explanation is suggested for why folding intermediates are observed easily in the transition of S-protein but not of RNAase A. The S-protein transition is shifted to lower temperatures, where folding intermediates should be more stable: consequently, intermediates in the folding of RNAase A which do not involve the S-peptide moiety and which are populated to almost detectable levels can be observed at the lower temperatures of the S-protein transition. © 1979.