“…Also, the three recombinants (control, D137L, and D137L/G126R) included the mutation C190A to prevent air oxidation. As described previously (16), all of these Tm species were overexpressed in BL21(DE3) according to standard methods, extracted by heating, fractionated by reducing the pH to 4.8, and purified by anion exchange chromatography using a HiTrap QXL column. The C190A mutation mimics the reduced state of cysteine in the Tm molecule, which is typical of living muscle (27), and had no appreciable effect on the tryptic digestion of Tm (9) or on its thermal unfolding and domain structure (12).…”