Recombination of S-peptide with S-protein during folding of ribonuclease S. II. Kinetic characterization of a stable folding intermediate shown by S-protein at pH 1.7

Labhardt, A.M.; Baldwin, R.L.

Journal of Molecular Biology 135(1): 245-254

1979


ISSN/ISBN: 0022-2836
PMID: 43399
DOI: 10.1016/0022-2836(79)90350-4
Accession: 068518784

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Abstract
At pH 1.7 S-peptide dissociates from S-protein but S-protein remains partly folded below 30.degree. 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 RNase S. The 2nd-order combination reaction (k = 0.7 .times. 106 M-1 s-1 at 20.degree. C) can be monitored either by tyrosine absorbance or fluorescence emission; the subsequent 1st-order folding reaction (half-time, 68 ms; 20.degree. C) is monitored by 2'CMP binding. Combination with S-peptide and folding to form native RNase S is considerably slower for both classes of unfolded S-protein. I3 shows a thermal folding transition at pH 1.7: it is completely unfolded above 32.degree. C and reaches a limiting low-temperature value of 65% below 10.degree. C. The 35% S-protein remaining at 10.degree. C is unfolded as judged by its refolding behavior in forming native RNase 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 2 earlier observations. Native RNase A is stable in the temperature range of the S-protein folding transition at pH 1.7, and the binding constant of S-protein for S-peptide falls steadily as the pH is lowered, by more than 4 orders of magnitude between pH 8.3 and pH 2.7, at 0.degree. C. The following explanation is suggested for why folding intermediates are observed easily in the transition of S-protein but not of RNase A. The S-protein transition is shifted to lower temperatures, where folding intermediates should be more stable: consequently, intermediates in the folding of RNase 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.