Conformational stability of HPr: The histidine‐containing phosphocarrier protein from Bacillus subtilis

Abstract

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 (AH,, Tg, AS,, and AC,) 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 AC, 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 AC, (1,170 * 50 cal mol" K"). Despite the high melting temperature for HPr (T, = 73.5 "C), the maximum stability of the protein, which occurs at 26 "C, is quite modest (AGs = 4.2 kcal mol"). In the presence of moderate concentrations of urea, HPr exhibits cold denaturation, and thus a complete stability curve for HPr, including a measure of AC,, 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 giobular protein are discussed. The methods presented will be of general utility in the characterization of the stability curve for many small proteins. Keywords: cold denaturation; conformational stability; protein foldingA complete thermodynamic description of the stability of globular proteins is crucial to our understanding of the energetics of protein structure, folding, and function. Here we describe the complete stability curve, the variation of the Gibbs energy of folding with temperature, for histidine-containing phosphocarrier protein (HPr) using a combination of thermal-and ureainduced unfolding experiments and spectroscopic probes for the folding transition. The data have been analyzed in several ways to afford measures of the cardinal thermodynamic parameters (AH,, AS,, T,, etc., evaluated at the temperature where A G = 0; see Becktel & Schellman, 1987) necessary for a description of the conformational stability of HPr. Included in the analysis is the assumption that AC, is temperature independent and that the denatured forms of HPr at high temperature or in the presence of high concentrations of urea are thermodynamically inReprint requests to: .I.

Determining the Conformational Stability of a Protein from Urea and Thermal Unfolding Curves

“…Therefore, before starting either of these protocols with a new protein, the CD signal should be measured as a function of time near the midpoint of the transition to determine the time required to reach equilibrium. For urea‐induced unfolding, in cases where equilibrium is quickly reached, one may choose to use titration methods instead of preparing individual solutions (Scholtz, 1995).…”