A change in the apparent m value reveals a populated intermediate under equilibrium conditions in Escherichia coli ribonuclease HI

Experimental studies of protein stability often rely on the determination o f an "m value", which describes the denaturant dependence of the free energ y change between two states (Delta G = Delta GH(2)O - m[denaturant]). Chang es in the rn value accompanying site specific mutations are usually attribu ted to structural alterations in the native or unfolded ensemble. Here, we provide an example of significant reduction in the in value resulting from a subtle deviation in two-state behavior not detected by traditional method s. The protein that is studied is a variant of Escherchia coli RNase H* in which three residues predicted to be involved in a partially buried salt br idge network were mutated to alanine (R46A, D102A, and B148A). Equilibrium denaturant profiles monitored by both fluorescence and circular dichroism a ppeared to be cooperative, and a two-state analysis yielded a Delta G(UN) O f approximately -3 kcal/mol with an oz value of 1.4 kcal mol(-1) M-1 (vs 2. 3 for RNase H*). Analysis of kinetic refolding experiments suggests that th e system is actually three-state at equilibrium with an appreciable concent ration of an intermediate state under low denaturant concentrations. The st ability of the native state determined from a fit of these kinetic data is -6.7 kcal/mol, suggesting that the stability determined by traditional two- state equilibrium analysis is a gross underestimate. The only hint to this loss of two-state behavior was a decrease in the apparent m value, and the presence of the equilibrium intermediate was only identified by a kinetic a nalysis. Our work serves as a cautionary note; the possibility of a three-s tate system should be closely addressed before interpreting a change in the nz value as a change in the native or unfolded state.