By use of intrinsic fluorescence to determine the apparent equilibrium constant Kapp as a function of temperature, the midpoint temperature Tm and apparent enthalpy change delta Happ on reversible thermal denaturation have been determined over a range of pH values for wild-type staphylococcal nuclease and six mutant forms. For wild-type nuclease at pH 7.0, a Tm of 53.3 +/- 0.2 degrees C and a delta Happ of 86.8 +/- 1.4 kcal/mol were obtained, in reasonable agreement with values determined calorimetrically, 52.8 degrees C and 96 +/- 2 kcal/mol. The heat capacity change on denaturation delta Cp was estimated at 1.8 kcal/(mol K) versus the calorimetric value of 2.2 kcal/(mol K). When values of delta Happ and delta Sapp for a series of mutant nucleases that exhibit markedly altered denaturation behavior with guanidine hydrochloride and urea were compared at the same temperature, compensating changes in enthalpy and entropy were observed that greatly reduce the overall effect of the mutations on the free energy of denaturation. In addition, a correlation was found between the estimated delta Cp for the mutant proteins and the d(delta Gapp)/dC for guanidine hydrochloride denaturation. It is proposed that both the enthalpy/entropy compensation and this correlation between two seemingly unrelated denaturation parameters are consequences of large changes in the solvation of the denatured state that result from the mutant amino acid substitutions. Study holds ProTherm entries: 2427, 2428, 2429, 2430, 2431, 2432, 2433 Extra Details: staphylococcal nuclease; heat capacity change; enthalpy; entropy;,free energy
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:18 p.m.
|Number of data points||20|
|Proteins||Thermonuclease ; Thermonuclease|
|Assays/Quantities/Protocols||Experimental Assay: dCp ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ|