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 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.
Spudich
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Department of Molecular and Cell Biology, University of California, 229 Stanley Hall, Berkeley, California 94720, USA.
Marqusee
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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
D001224:Aspartic Acid; D014508:Urea; D001120:Arginine; D016914:Ribonuclease H; C078856:ribonuclease HI