Ensemble modulation as an origin of denaturant-independent hydrogen exchange in proteins

Native state hydrogen exchange (HX) has become a powerful tool for the anal ysis of conformational states that exist under native conditions. However, the interpretation of HX data in terms of conformational fluctuations is st ill controversial. In particular, it has been shown that many residues disp lay exchange behavior that is independent of denaturant concentration. It h as been postulated that this lack of denaturant dependence results from loc al fluctuations that do not expose appreciable amounts of buried surface ar ea. Here, we use a general thermodynamic description of HX to explore the d ifferent possibilities for this behavior. We find that the denaturant depen dence seen in HX experiments under native conditions is not a de facto indi cation of the amount of surface area exposure required for exchange. Instea d, this behavior results from the relatively homogenous character of the co nformational ensemble that exists under native conditions and the non-speci fic nature of denaturant effects. Furthermore, a comparison of the HX behav ior from a stabilized mutant of Staphylococcal nuclease (SNase) with that p redicted for the wild-type SNase from the COREX algorithm suggests that den aturant-independent exchange of many residues is consistent with significan t (similar to 10 %) surface area exposure for this protein. (C) 2000 Academ ic Press.