Melting Temperature (Tm)
Circular Dichroism (CD)
60 mM CH3COO Na, 150 mM NaCl, pH5.4
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.