Conformational Stability of Muscle Acylphosphatase: The Role of Temperature, Denaturant Concentration, and pH

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Abstract
The conformational stability (ΔG) of muscle acylphosphatase, a small α/β globular protein, has been determined as a function of temperature, urea concentration, and pH. A combination of thermally induced and urea-induced unfolding, monitored by far-UV circular dichroism, was used to define the conformational stability over a wide range of temperature. Through analysis of all these data, the heat capacity change upon unfolding (ΔCp) could be estimated, allowing the determination of the temperature dependence of the main thermodynamic functions (ΔG, ΔH, ΔS). Thermal unfolding in the presence of urea made it possible to extend such thermodynamic analysis to examine these parameters as a function of urea concentration. The results indicate that acylphosphatase is a relatively unstable protein with a ΔG(H2O) of 22 ± 1 kJ mol-1 at pH 7 and 25 °C. The midpoints of both thermal and chemical denaturation are also relatively low. Urea denaturation curves over the pH range 2−12 have allowed the pH dependence of ΔG to be determined and indicate that the maximum stability of the protein occurs near pH 5.5. While the dependence of ΔG on urea (the m value) does not vary with temperature, a significant increase has been found at low pH values, suggesting that the overall dimensions of the unfolded state are significantly affected by the number of charges within the polypeptide chain. The comparison of these data with those from other small proteins indicates that the pattern of conformational stability is defined by individual sequences and not by the overall structural fold.