Calorimetric study of tryptophan synthase α‐subunit and two mutant proteins

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Abstract
Heat-denaturation of tryptophan synthase α-subunit from E. coli and two mutant proteins (Glu 49 ± Gln or Ser; called Gln 49 or Ser 49, respectively) has been studied by the scanning microcalorimetric method at various pH, in an attempt to elucidate the role of individual amino acid residues in the conformational stability of a protein. The partial specific heat capacity in the native state at 20°, Cp20, has been found to be (0.43 ± 0.02) cal ± K-1 ± g-1, the unfolding heat capacity change, ΔdCp, (0.10 ± 0.01) cal ± K-1 ± g-1, and the unfolding enthalpy value extrapolated to 110°, Δdh110, (9.3 ± 0.5) cal ± g-1 for the three proteins. The value of Cp20 was larger than those found for fully compact protein and that of Δdh110 was smaller. Unfolding Gibbs energy, ΔdG at 25° for Wild-type, Gln 49, and Ser 49 were 5.8, 8.4, and 7.1 kcal ± mol-1 at pH 9.3, respectively. Unfolding enthalpy, ΔdH, of the three proteins seemed to be the same and equal to (23.2 ± 1.2) kcal ± mol-1 at 25°. As a consequence of the same value of ΔdH and the different value in ΔdG, substantial differences in unfolding entropy, ΔdS, were found for the three proteins. The values of ΔdG for the three proteins at 25° coincided with those from equilibrium methods of denaturation by guanidine hydrochloride.