Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits.


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. Study holds ProTherm entries: 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001 Extra Details: additive : EDTA(0.2 mM), tryptophan synthase alpha subunit; thermodynamic stability;,differential scanning calorimetry; accessibility

Submission Details

ID: aMdWddcH4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:16 p.m.

Version: 1

Publication Details
Lim WK;Brouillette C;Hardman JK,Arch. Biochem. Biophys. (1992) Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits. PMID:1727648
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1XCF 2004-11-02 1.8 Crystal structure of P28L/Y173F tryptophan synthase alpha-subunits from Escherichia coli
1WQ5 2005-02-15 2.3 Crystal structure of tryptophan synthase alpha-subunit from Escherichia coli
1V7Y 2005-02-15 2.5 Crystal structure of tryptophan synthase alpha-subunit from Escherichia coli at room temperature
1XC4 2004-11-02 2.8 Crystal structure of wild-type tryptophan synthase alpha-subunits from Escherichia coli

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
97.0 Tryptophan synthase alpha chain B7LS20 TRPA_ESCF3
97.8 Tryptophan synthase alpha chain B7NVN0 TRPA_ECO7I
98.1 Tryptophan synthase alpha chain B1LH32 TRPA_ECOSM
98.5 Tryptophan synthase alpha chain B7N473 TRPA_ECOLU
98.5 Tryptophan synthase alpha chain Q0TIB0 TRPA_ECOL5
98.1 Tryptophan synthase alpha chain B7MU99 TRPA_ECO81
98.9 Tryptophan synthase alpha chain B7UR66 TRPA_ECO27
98.5 Tryptophan synthase alpha chain Q1RCA7 TRPA_ECOUT
98.5 Tryptophan synthase alpha chain A1AAN0 TRPA_ECOK1
98.5 Tryptophan synthase alpha chain B7ML76 TRPA_ECO45
98.9 Tryptophan synthase alpha chain B5YZP0 TRPA_ECO5E
98.9 Tryptophan synthase alpha chain Q8X7B5 TRPA_ECO57
99.3 Tryptophan synthase alpha chain Q32GT0 TRPA_SHIDS
98.9 Tryptophan synthase alpha chain Q3Z108 TRPA_SHISS
99.6 Tryptophan synthase alpha chain Q0T5D6 TRPA_SHIF8
99.6 Tryptophan synthase alpha chain Q31ZV3 TRPA_SHIBS
99.3 Tryptophan synthase alpha chain Q8FHW0 TRPA_ECOL6
99.6 Tryptophan synthase alpha chain B2U0F1 TRPA_SHIB3
99.3 Tryptophan synthase alpha chain A7ZL78 TRPA_ECO24
99.6 Tryptophan synthase alpha chain B7L492 TRPA_ECO55
99.6 Tryptophan synthase alpha chain B6I9X4 TRPA_ECOSE
99.6 Tryptophan synthase alpha chain B7LY16 TRPA_ECO8A
100.0 Tryptophan synthase alpha chain P0A878 TRPA_SHIFL
100.0 Tryptophan synthase alpha chain P0A877 TRPA_ECOLI
100.0 Tryptophan synthase alpha chain B1ITJ5 TRPA_ECOLC
100.0 Tryptophan synthase alpha chain A7ZZJ6 TRPA_ECOHS
100.0 Tryptophan synthase alpha chain B1XBK9 TRPA_ECODH
100.0 Tryptophan synthase alpha chain C4ZTV3 TRPA_ECOBW