PH dependence of stability of the wild-type tryptophan synthase α-subunit and two mutant proteins (Glu49 → Met or Gln)

Abstract

In order to elucidate the role of individual amino acid residues in the electrostatic interaction affecting the conformational stability of proteins, the pH dependence of stability of tryptophan synthase α-subunit of the wild-type and two mutant proteins, trpA33 (Glu49 → Met) and trpA11 (Glu49 → Gln), has been compared by means of circular dichroism measurements in the absence and presence of guanidine hydrochloride. The denaturation of the three proteins is discussed, assuming the existence of one stable intermediate. In the first denaturation step, i.e. the transition from the native to the intermediate state, the midpoint of the conformational transition in the acid region was pH 3.9 and pH 5.1 for the wild type and the trpA11 proteins, respectively; whereas, in the alkaline region the midpoint was pH 10.9 and pH 11.6 for the wild-type and the trpA11 proteins, respectively. The trpA33 protein belonged to the stronger group in stability in both the acid and alkaline regions. In the acid region in the presence of 0.4 m-guanidine hydrochloride, the trpA11 protein was more labile than the wild-type and trpA33 proteins; whereas, in the alkaline region in 0.8 m-guanidine hydrochloride, the order of stability among the three proteins was the trpA33, trpA11 and wild-type proteins at pH values between 7.5 and 9.8. In the second denaturation step, i.e. the transition from the intermediate to the completely denatured state, the order of stability among the three proteins was the trpA33, wild-type and trpA11 proteins. These results showed that a negatively charged group at position 49 served as a destabilizing factor in the alkaline region, and that the hydrophobicity of the residue at position 49 contributed to the stabilization of the conformation of the protein.