title: A change in the apparent m value reveals a populated intermediate under equilibrium conditions in Escherichia coli ribonuclease HI.
abstract: Experimental studies of protein stability often rely on the determination of an "m value", which describes the denaturant dependence of the free energy change between two states (DeltaG = DeltaG(H2O) - m[denaturant]). Changes in the m value accompanying site specific mutations are usually attributed to structural alterations in the native or unfolded ensemble. Here, we provide an example of significant reduction in the m value resulting from a subtle deviation in two-state behavior not detected by traditional methods. 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 bridge network were mutated to alanine (R46A, D102A, and D148A). Equilibrium denaturant profiles monitored by both fluorescence and circular dichroism appeared to be cooperative, and a two-state analysis yielded a DeltaG(UN) of approximately -3 kcal/mol with an m value of 1.4 kcal mol(-1) M(-1) (vs 2.3 for RNase H). Analysis of kinetic refolding experiments suggests that the system is actually three-state at equilibrium with an appreciable concentration of an intermediate state under low denaturant concentrations. The stability 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 analysis. Our work serves as a cautionary note; the possibility of a three-state system should be closely addressed before interpreting a change in the m value as a change in the native or unfolded state.
authors.affiliation: Department of Molecular and Cell Biology, University of California, 229 Stanley Hall, Berkeley, California 94720, USA.
authors.forename: G
authors.initials: G
authors.lastname: Spudich
authors.forename: S
authors.initials: S
authors.lastname: Marqusee
mesh_terms: D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000138:chemical synthesis; D011489:Protein Denaturation / Q000235:genetics; D017510:Protein Folding; D016914:Ribonuclease H / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea
chemical_list: D001224:Aspartic Acid; D014508:Urea; D001120:Arginine; D016914:Ribonuclease H; C078856:ribonuclease HI