COMPARISON OF DENATURATION BY GUANIDINE-HYDROCHLORIDE OF THE WILD-TYPE TRYPTOPHAN SYNTHASE ALPHA-SUBUNIT OF ESCHERICHIA-COLI AND 2 MUTANT PROTEINS (GLU-49-] MET OR GLN)

In order to elucidate the roles of individual amino acid residues in the conformational stability of proteins, the denaturation by guanidine hydrochloride of the wild-type tryptophan synthase α-subunit of Escherichia coli and two mutant proteins, trpA33 (Glu49→Met) and trpA11 (Glu49→Gln), has been compared by means of CD measurements at pH 7.0 and various temperatures. CD spectra of the two mutant proteins were similar to that of the wild-type protein. The trpA33 and the trpA11 proteins were more and less resistant, respectively, to guanidine hydrochloride than the wild-type protein at 9.7 to 49.6°C. The free energy change of unfolding in water, δGH2ond, was evaluated assuming a three state denaturation, since the denaturation curves of the three proteins suggested the presence of one stable intermediate. The values of δGH2ond for the trpA33, the wild-type, and the trpA11 proteins at 25.8°C and pH 7.0 were 13.4, 8.8, and 6.3 kcal/mol, respectively. The δGH2ond of the trpA11 protein was almost independent of temperature, though that of the trpA33 protein was remarkably dependent on temperature. The conformational stabilities of the three proteins were correlated with the hydrophobicities of the substituted amino acid residues. © 1979 By The Journal Of Biochemistry.