Conformational stability of HPr: The histidine-containing phosphocarrier protein from Bacillus subtilis
Corresponding Author
J. Martin Scholtz
Department of Medical Biochemistry & Genetics, Center for Macromolecular Design, Texas A&M University, College Station, Texas 77843–1114
Department of Medical Biochemistry & Genetics, 440 Reynolds Building, Texas A&M University, College Station, Texas 77843–1114Search for more papers by this authorCorresponding Author
J. Martin Scholtz
Department of Medical Biochemistry & Genetics, Center for Macromolecular Design, Texas A&M University, College Station, Texas 77843–1114
Department of Medical Biochemistry & Genetics, 440 Reynolds Building, Texas A&M University, College Station, Texas 77843–1114Search for more papers by this authorAbstract
The conformational stability of the histidine-containing phosphocarrier protein (HPr) from Bacillus subtilis has been determined using a combination of thermal unfolding and solvent denaturation experiments. The urea-induced denaturation of HPr was monitored spectroscopically at fixed temperatures and thermal unfolding was performed in the presence of fixed concentrations of urea. These data were analyzed in several different ways to afford a measure of the cardinal parameters (ΔHg, Tg, ΔSg, and ΔCp) that describe the thermodynamics of folding for HPr. The method of Pace and Laurents (Pace CN, Laurents DV, 1989, Biochemistry 28:2520–2525) was used to estimate ΔCP as was a global analysis of the thermal- and urea-induced unfolding data. Each method used to analyze the data gives a similar value for ΔCp (1,170 Δ 50 cal mol−1 K−1). Despite the high melting temperature for HPr (Tg = 73.5 °C), the maximum stability of the protein, which occurs at 26 °C, is quite modest (AGs = 4.2 kcal mol−1). In the presence of moderate concentrations of urea, HPr exhibits cold denaturation, and thus a complete stability curve for HPr, including a measure of ΔCp, can be achieved using the method of Chen and Schellman (Chen B, Schellman JA, 1989, Biochemistry 28:685–691). A comparison of the different methods for the analysis of solvent denaturation curves is provided and the effects of urea on the thermal stability of this small globular protein are discussed. The methods presented will be of general utility in the characterization of the stability curve for many small proteins.
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