Enthalpy of Unfolding (ΔH)
Circular Dichroism (CD)
50 mM phosphate
Thermal denaturation profiles for all protein variants were used to determine thermodynamic parameters for the unfolding transition. After fitting both the lower and upper baselines for the denaturation curve, the program ThermoDynaCD  was used to determine the Tm and ∆Hm for each mutant.
See Tm pH 7.2 assay for details.
Tm pH7.2 details: Purification and circular dichroism of mutant proteins
Protein variants were generated by primers that encoded specific amino acid substitutions into the synthetic β1 gene on a pET11a∆ vector, and protein was expressed as described previously . Protein purification was achieved by ion-exchange chromatography on Q-Sepharose  and followed by gel-filtration chromatography on Hi-Load Superdex 75 (Pharmacia) in 50 mM sodium acetate buffer, pH 5.2, the buffer in which all CD spectra were acquired, except for the charged mutants. The charged mutants were studied at pH 7.2 in 50 mM phosphate buffer. Far-UV CD spectra were acquired from 20 μM protein samples in a cuvette of 0.1 cm path length. We used 50 μM protein samples in a 0.2 cm path length cuvette for thermal denaturation studies sampling at 1°C increments with a 2 min equilibration and a 1 min averaging time on an Aviv 62DS CD spectrometer (Aviv Instruments).