HYDROGEN-EXCHANGE IN THERMALLY DENATURED RIBONUCLEASE-A

Hydrogen exchange has been used to test for the presence of nonrandom structure in thermally denatured ribonuclease A (RNase A). Quenched-flow methods and 2D H-1 NMR spectroscopy were used to measure exchange rates for 36 backbone amide protons (NHs) at 65-degrees-C and at pH* (uncorrected pH measured in D2O) values ranging from 1.5 to 3.8. The results show that exchange is approximately that predicted for a disordered polypeptide [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158]; we thus are unable to detect any stable hydrogen-bonded structure in thermally denatured RNase A. Two observations suggest, however, that the predicted rates should be viewed with some caution. First, we discovered that one of the approximations made by Molday et al. (1972), that exchange for valine NHs is similar to that for alanine NHs, had to be modified; the exchange rates for valine NHs are about 4-fold slower. Second, the pH minima for exchange tend to fall at lower pH values than predicted, by as much as 0.45 pH units. These results are in accord with those of Roder and co-workers for bovine pancreatic trypsin inhibitor [see Table I in Roder, H., Wagner, G., & Wuthrich, K. (1985) Biochemistry 24, 7407-7411]. The origin of the disagreement between predicted and observed pH minima is unknown but may be the high net positive charge on these proteins at low pH. In common with some other thermally unfolded proteins, heat-denatured ribonuclease A shows a significant circular dichroism spectrum in the far-ultraviolet region [Labhardt, A. M. (1982) J. Mol. Biol. 157, 331-355]. This spectrum is reduced to a low level by adding guanidine hydrochloride. The nature of the residual structure responsible for this spectrum is not known. Our results show that it is not stable helix formation by the three alpha-helices of native ribonuclease A, which would give measurable protection against amide proton exchange.