Thermal unfolding of staphylococcal nuclease and several mutant forms thereof studied by differential scanning calorimetry

Abstract

The effects of eight mutations on the thermodynamics of the reversible thermal unfolding of staphylococcal nuclease have been determined over a range of pH and protein concentration by means of differential scanning calorimetry. Variation of the protein concentration was included in our study because we found a significant dependence of the thermodynamics of protein unfolding on concentration. Values for the change in the standard free energy of unfolding, AAG:, produced by the mutations in the pH range 5.0-7.0 varied from 1.9 kcal mol" (apparent stabilization) for H124L to -2.8 kcal mol" (apparent destabilization) for L25A. As has been observed in numerous other cases, there is no correlation in magnitude or sign between AAG: and the corresponding values for AM,, and TAAS:, the latter quantities being in most cases much larger in magnitude than AAG:. This fact emphasizes the difficulty in attempting to correlate the thermodynamic changes with structural changes observed by X-ray crystallography.Keywords: differential scanning calorimetry; mutations; staphylococcal nuclease; thermal unfoldingThe thermal denaturation of staphylococcal nuclease (SNase) and several of its mutant forms has been studied by Shortle et al. (1988) using fluorescence emission to monitor the thermally induced conformational changes. The data were subjected to van't Hoff analysis to obtain the thermodynamic parameters for the denaturations. The denaturations were observed over a range of pH, which led to a variation in the mid-temperature of denaturation of approximately 30 "C and permitted evaluation of the permanent change in heat capacity accompanying the denaturations.We have undertaken a reinvestigation of the thermal unfolding of five of these mutants-V66L, G79S, G88V, V66L + G88V, and V66L + G79S + G88V-using differential scanning calorimetry (DSC). We have also included in our study H124L, a naturally occurring form obtained from the V8 strain of Staphylococcus aureus, and the mutants L25A and A90S. It was also necessary to do a careful study of the wild-type (WT) enzyme for comparison

Thermodynamics of staphylococcal nuclease denaturation. I. The acid‐denatured state

“…In the range of 1-3 mg/mL, we do not observe a significant protein concentration dependence of the T, or AHfi*/AHCa' ratio (Table 2). At higher protein concentrations, however, aggregation may be extensive, as was found by Tanaka et al (1993) (see Discussion). Aggregation of denatured proteins is also generally highly salt dependent far from the isoelectric point, which for SNase is pH 9.62 (Heins et al, 1967).…”

“…Indeed, rationalization of a greater enthalpy change for a less stable mutant would be very difficult without this consideration. A plot of AH versus Td for the mutant protein L25A (Tanaka et al, 1993) looks similar to that for V66A (Fig. 3), also giving greater AH values than wild type at the same Td.…”

“…The reasons for this most likely involve experimental procedures and accuracy of instrumentation. In the calorimetric study of Tanaka et al (1993), the ratios of the fitted 2-state and calorimetric enthalpies were found to be significantly greater than 1, and a protein concentration dependence of Td was found. The authors interpreted this as evidence of partial aggregation or oligomerization in the denatured state.…”

“…The studies of Griko et al (1988) and Shortle et al (1988), which also used relatively low protein concentrations, found that the ratios of these enthalpies were close to 1. Two previous studies (Calderon et al, 1985;Tanaka et al, 1993) did not take into account light scattering in determination of protein concentrations, which may produce an underestimation of the calorimetric enthalpy and an overestimation of the ratio of enthalpies. Extensive aggregation of the denatured state at high protein concentrations might also preclude observation of the effects of pH on enthalpy and ACp reported here.…”

“…Staphylococcal nuclease (SNase) is a widely studied model system for protein folding and has previously been the subject of calorimetric investigation (Calderon et al, 1985;Griko et al, 1988;Shortle et al, 1988;Tanaka et al, 1993). As part of a project to study the effects of mutations on the heat capacities, enthalpies, and entropies of proteins, we herein reexamine the thermodynamics of SNase denaturation using differential scanning calorimetry.…”