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Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits.
Arch Biochem Biophys. 1992 Jan; 292(1):34-41.AB

Abstract

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 degrees 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 degrees C. Eighteen mutant proteins containing alterations in helical and sheet structures had Tm's significantly lower (-1.6 to -7.5 degrees C) than the wild-type Tm (59.5 degrees C). In contrast, 6 mutant alpha subunits with alterations in the regions following beta strands 1 and 3 had increased Tm's (+1.4 to +7.6 degrees 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.

Authors+Show Affiliations

Department of Biological Sciences, University of Alabama, Tuscaloosa 35487.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

1727648

Citation

Lim, W K., et al. "Thermal Stabilities of Mutant Escherichia Coli Tryptophan Synthase Alpha Subunits." Archives of Biochemistry and Biophysics, vol. 292, no. 1, 1992, pp. 34-41.
Lim WK, Brouillette C, Hardman JK. Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits. Arch Biochem Biophys. 1992;292(1):34-41.
Lim, W. K., Brouillette, C., & Hardman, J. K. (1992). Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits. Archives of Biochemistry and Biophysics, 292(1), 34-41.
Lim WK, Brouillette C, Hardman JK. Thermal Stabilities of Mutant Escherichia Coli Tryptophan Synthase Alpha Subunits. Arch Biochem Biophys. 1992;292(1):34-41. PubMed PMID: 1727648.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits. AU - Lim,W K, AU - Brouillette,C, AU - Hardman,J K, PY - 1992/1/1/pubmed PY - 1992/1/1/medline PY - 1992/1/1/entrez SP - 34 EP - 41 JF - Archives of biochemistry and biophysics JO - Arch Biochem Biophys VL - 292 IS - 1 N2 - 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 degrees 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 degrees C. Eighteen mutant proteins containing alterations in helical and sheet structures had Tm's significantly lower (-1.6 to -7.5 degrees C) than the wild-type Tm (59.5 degrees C). In contrast, 6 mutant alpha subunits with alterations in the regions following beta strands 1 and 3 had increased Tm's (+1.4 to +7.6 degrees 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. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/1727648/Thermal_stabilities_of_mutant_Escherichia_coli_tryptophan_synthase_alpha_subunits_ DB - PRIME DP - Unbound Medicine ER -