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Comparison of denaturation by guanidine hydro chloride of the wild type tryptophan synthase ec 4.2.1.20 alpha subunit of escherichia coli and 2 mutant proteins glutamic acid 49 replaced by methionine or glutamine



Comparison of denaturation by guanidine hydro chloride of the wild type tryptophan synthase ec 4.2.1.20 alpha subunit of escherichia coli and 2 mutant proteins glutamic acid 49 replaced by methionine or glutamine



Journal of Biochemistry 85(4): 915-922



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 [EC 4.2.1.20] .alpha.-subunit of E. coli and 2 mutant proteins, trpA33 (Glu49 .fwdarw. Met) and trpA11 (Glu49 .fwdarw. Gln), was compared by CD [circular dichroic] measurements at pH 7.0 and various temperatures. CD spectra of the 2 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 9.7-49.6.degree. C. The free energy change of unfolding in water, .**GRAPHIC**. was evaluated assuming a 3 state denaturation, since the denaturation curves of the 3 proteins suggested the presence of 1 stable intermediate. The values of .**GRAPHIC**. for the trpA33, the wild-type and the trpA11 proteins at 25.8.degree. C and pH 7.0 were 13.4, 8.8 and 6.3 kcal/mol, respectively. The .**GRAPHIC**. 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 3 proteins were correlated with the hydrophobicities of the substituted amino acid residues.

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