title: Three-state thermodynamic analysis of the denaturation of staphylococcal nuclease mutants.
abstract: Using microcalorimetry, we found an equilibrium intermediate state during the denaturation of the wild-type and five mutant staphylococcal nuclease proteins: V66L, V66W, G88V, D77A, and E75V. The presence of two distinct heat absorption peaks allowed direct measurement of the enthalpy differences between the native, intermediate, and denatured states. Conditions of low pH and high NaCl concentration facilitated observation of the intermediate, or I-state. We propose to consider the nuclease protein as composed of two subdomains, divided along the active-site cleft. The structure of the I-state apparently consists mainly of the folded beta-barrel subdomain, as does that of a nuclease fragment protein [Shortle, D., & Abeygunawardana, C. (1993) Structure 1, 121-134]. The cooperativity of folding of the subdomains is maintained by electrostatic bonds across the active-site cleft. Removal of these bonds by the mutation D77A or E75V results in decooperation of the protein's structure and a three-state mechanism of denaturation at pH 7.0. The origins of differences in the enthalpy change of denaturation and in the m value of guanidinium chloride-induced denaturation with mutant nucleases are discussed in terms of this three-state mechanism.
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
authors.forename: J H
authors.initials: JH
authors.lastname: Carra
authors.forename: E A
authors.initials: EA
authors.lastname: Anderson
authors.forename: P L
authors.initials: PL
authors.lastname: Privalov
mesh_terms: D002151:Calorimetry; D002942:Circular Dichroism; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D017354:Point Mutation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013211:Staphylococcus aureus / Q000201:enzymology; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D008836:Micrococcal Nuclease