Unfolding properties of tryptophan-containing alpha-subunits of the Escherichia coli tryptophan synthase.


Abstract

The urea-induced unfolding of the Escherichia coli tryptophan synthase alpha-subunit is examined via fluorescence measurements with tryptophan-containing alpha-subunit mutants, constructed by in vitro mutagenesis. Early unfolding studies with urea and guanidine suggested that the wild type protein unfolded in a two-step process with a stable intermediate composed of a native alpha-1 folding unit (residues 1-188) and a completely unfolded alpha-2 folding unit (residues 189-268). Recently, more detailed spectroscopic and calorimetric data from the Matthews and Yutani groups indicate that such a structure for the intermediates seems unlikely. Previously, we described the introduction of Trp residues as unfolding reporter groups separately into each of the folding domains and showed that these proteins are wild type enzymatically and in their stability to urea. The unfolding behavior of these alpha-subunits, monitored by fluorescence intensity changes at the discrete emission lambda max for each, in both equilibrium and kinetic experiments, suggest that: (a) both folding units commence unfolding simultaneously (near 2 M urea); (b) the larger alpha-1 unit unfolds in a multistep process, initially yielding a partially unfolded intermediate form which subsequently appears to unfold progressively to completion; and (c) the smaller alpha-2 unit unfolds in a single step event. These results are also clearly incompatible with the early proposals on the structure of the intermediate. It is suggested here that the intermediate is heterogeneous, consisting of a stable, partially unfolded form of alpha-1 attached to either a completely folded or completely unfolded form of alpha-2. These results are consistent with and provide an added dimension to the recent description of the proposed structure of the intermediate. Study holds ProTherm entries: 5202, 5203, 5204 Extra Details: additive : Na2EDTA(0.2 mM),N -> I tryptophan; two-step process; stable intermediate; alpha-subunit

Submission Details

ID: ZodcrJPh

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:29 p.m.

Version: 1

Publication Details
Choi SG;Hardman JK,J. Biol. Chem. (1995) Unfolding properties of tryptophan-containing alpha-subunits of the Escherichia coli tryptophan synthase. PMID:7499309
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