Hydrogen exchange and the unfolding pathway of ribonuclease A

Abstract

Recently, when the kinetic unfolding process of ribonuclease A was monitored by hydrogen exchange (T. Kiefhaber and R.L. Baldwin, Proc. Natl. Acad. Sci. USA, 92 (1995) 2657–2661), all peptide hydrogen bonds were found to undergo rapid exchange in a single kinetic step under conditions where unfolding is slow and the intrinsic rate of hydrogen exchange is fast (pH 8.0, 10°C, 4.5 M guanidinium chloride). Comparison with the unfolding rate measured by circular dichroism indicates that hydrogen exchange is caused by the rate-limiting step of unfolding. No evidence was found for partly unfolded intermediates that are formed slowly enough to be observed by EX1 (unfolding-limited) hydrogen exchange. Some peptide NH protons were found to show, in addition to EX1 exchange, faster EX2 exchange that is base-catalyzed. The EX2 exchange is caused by species that equilibrate rapidly with the native protein at the start of the unfolding process. These species might include rapidly formed unfolding intermediates. We show here that any such unfolding intermediates must have large protection factors because the EX2 reactions of ribonuclease A under these unfolding conditions have protection factors ≥ 2500.