title: The pH dependence of staphylococcal nuclease stability is incompatible with a three-state denaturation model.
abstract: Six single substitution mutations, V66F, V66G, V66N, V66Q, V66S, V66T, and V66Y, were made in the background of a highly stable triple mutant (P117G, H124L, and S128A) of staphylococcal nuclease. The thermodynamic stabilities of wild type staphylococcal nuclease, of the stable triple mutant and of its six variants were determined by guanidine hydrochloride denaturation in thirteen different buffers spanning the pH range 4.5 to 10.2. Within experimental error the values of [Formula: see text] and mGuHCl for the various proteins measured over this wide range of pH maintain a constant offset from one another, tracing a series of approximately parallel curves. This data offers an independent means of determining the error of stabilities and slopes determined by guanidine hydrochloride denaturations and shows that previous error estimates are accurate. More importantly, this behavior cannot be reconciled with a three-state denaturation model for staphylococcal nuclease. The large variations in mGuHCl observed in these mutants must therefore arise from other causes.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Spencer
authors.forename: García-Moreno E
authors.initials: GM
authors.lastname: Bertrand
authors.forename: Wesley E
authors.initials: WE
authors.lastname: Stites
mesh_terms: D019943:Amino Acid Substitution; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D055550:Protein Stability; D013210:Staphylococcus / Q000201:enzymology*; D013816:Thermodynamics
chemical_list: D008836:Micrococcal Nuclease; D019791:Guanidine
keywords: Electrostatic effect; Equilibrium denaturation; Equilibrium intermediate; Protein folding; Protein stability; Slope
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