Gibbs Free Energy of Folding/Unfolding (ΔG)
Circular Dichroism (CD)
60 mM CH3COO Na, 150 mM NaCl, pH5.4
ΔG values for unfolding of mutant proteins (ΔGu, not reported) were calculated from melting curves using Gibbs-Helmholtz equation,
assuming ΔCp of 624 cal mol-1 deg-1 for all molecules. ΔΔG was calculated at 321 K, the mean of the Tms excluding Gly mutant.
Estimated errors in determining Tm and ΔG are +/- 0.5 and +/- 0.6 kcal/mol, respectively.
See Tm assay for more details.
Tm details: CD measurements were made at a concentration of 10 μM. Melting curves were fitted using Gibbs Helmholtz equation with a fixed ΔCp of 624 cal mol-1 deg-1 assuming a two-state unfolding model. The two-state unfolding model was tested for AASS-53Thr and AASS-53Ala by measuring the enthalpy of unfolding by DSC. Results are ΔHcal = 39.9 kcal mol-1 and 32.8 kcal mol-1; ΔHcal/ΔHvan't Hoff = 0.99 and 0.97 for AASS-53Thr and AASS-53Ala respectively. Calorimetry samples contained ~3 mg/ml protein in 50 mM sodium acetate 150 mM NaCl, pH5.4.
ΔCp for unfolding was determined by combining data from the pH dependence of stability for AASS-53Thr, AASS-53Phe, and AASS-53Val with he stability data obtained at pH 5.4 for each of the guest site mutants (Fig. 1c. The resultant value (ΔCp = 624 +/-62 cal mol-1 deg-1) is in excellent agreement with the previously reported value for WT GB1 (621 +/-71 cal mol-1 deg-1 and these data illustrate that these mutations have little effect on ΔCp, as might be expected for mutations at solvent-exposed positions.