HYDROGEN-EXCHANGE AND THE UNFOLDING PATHWAY OF RIBONUCLEASE-A

Recently, when the kinetic unfolding process of ribonuclease A was mon itored by hydrogen exchange (T. Kiefhaber and R.L. Baldwin, Proc. Natl . Acad, Sci. USA, 92 (1995) 2657-2651), all peptide hydrogen bonds wer e found to undergo rapid exchange in a single kinetic step under condi tions where unfolding is slow and the intrinsic rate of hydrogen excha nge is fast (pH 8.0, 10 degrees C, 4.5 M guanidinium chloride). Compar ison with the unfolding rate measured by circular dichroism indicates that hydrogen exchange is caused by the rate-limiting step of unfoldin g. No evidence was found for partly unfolded intermediates that are fo rmed slowly enough to be observed by EX1 (unfolding-limited) hydrogen exchange, Some peptide NH protons were found to show, in addition to E X1 exchange, faster EX2 exchange that is base-catalyzed, The EX2 excha nge is caused by species that equilibrate rapidly with the native prot ein at the start of the unfolding process. These species might include rapidly formed unfolding intermediates. We show here that any such un folding intermediates must have large protection factors because the E X2 reactions of ribonuclease A under these unfolding conditions have p rotection factors greater than or equal to 2500.