Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits
Lim, W.K.; Brouillette, C.; Hardman, J.K.
Archives of Biochemistry and Biophysics 292(1): 34-41
ISSN/ISBN: 0003-9861 PMID: 1727648 Accession: 007947467
Random chemical mutagenesis, in vitro, of the 5' portion of the Escherichia coli trpA gene has yielded 66 mutant .alpha. subunits containing single amino acid substitutions at 49 different residue sites within the first 121 residues of the protein; this portion of the .alpha. subunit contains four of the eight .alpha. helices and three of the eight .beta. strands in the protein. Sixty-two of the subunits were examined for their heat stabilities by sensitivity to enzymatic inactivation (52.degree. C for 20 min) in crude extracts and by differential scanning calorimetry (DSC) with 29 purified proteins. The enzymatic activities of mutant .alpha. subunits that contained amino acid substitutions within the .alpha. and .beta. secondary structures were more heat labile than the wild-type .alpha. subunit. Alterations only in three regions, at or immediately C-terminal to the first three .beta. strands, were stability neutral or stability enhancing with respect to enzymatic inactivation. Enzymatic thermal inactivation appears to be correlated with the relative accessibility of the substituted residues; stability-neutral mutations are found at accessible residual sites, stability-enhancing mutations at buried sites. DSC analyses showed a similar pattern of stabilization/destabilization as indicated by inactivation studies. Tm differences from the wild-type .alpha. subunit varied .+-. 7.6.degree. C. eighteen mutant proteins containing alterations in helical and sheet structures had Tm's significantly lower (-1.6 to -7.5.degree. C) than the wild-type Tm (59.5.degree. C). In contrast, 6 mutant .alpha. subunits with alterations in the region following .beta. strands 1 and 3 had increased Tm's (+1.4 to +7.6.degree. C). Because of incomplete thermal reversibilities for many of the mutant .alpha. subunits, most likely due to identifiable aggregated forms in the unfolded state, reliable differences in thermodynamic stability parameters are not possible. The availability of this group of mutant .alpha. subunits which clearly contain structural alterations should prove useful in defining the roles of certain residues or sequences in the unfolding/folding pathway for this protein when examined by urea/guaninidine denaturation kinetic analysis.