Folding mechanism of the alpha-subunit of tryptophan synthase, an alpha/beta barrel protein: global analysis highlights the interconversion of multiple native, intermediate, and unfolded forms through parallel channels.


Abstract

A variety of techniques have been used to investigate the urea-induced kinetic folding mechanism of the alpha-subunit of tryptophan synthase from Escherichia coli. A distinctive property of this 29 kDa alpha/beta barrel protein is the presence of two stable equilibrium intermediates, populated at approximately 3 and 5 M urea. The refolding process displays multiple kinetic phases whose lifetimes span the submillisecond to greater than 100 s time scale; unfolding studies yield two relaxation times on the order of 10-100 s. In an effort to understand the populations and structural properties of both the stable and transient intermediates, stopped-flow, manual-mixing, and equilibrium circular dichroism data were globally fit to various kinetic models. Refolding and unfolding experiments from various initial urea concentrations as well as forward and reverse double-jump experiments were critical for model discrimination. The simplest kinetic model that is consistent with all of the available data involves four slowly interconverting unfolded forms that collapse within 5 ms to a marginally stable intermediate with significant secondary structure. This early intermediate is an off-pathway species that must unfold to populate a set of four on-pathway intermediates that correspond to the 3 M urea equilibrium intermediate. Reequilibrations among these conformers act as rate-limiting steps in folding for a majority of the population. A fraction of the native conformation appears in less than 1 s at 25 degrees C, demonstrating that even large proteins can rapidly traverse a complex energy surface. Study holds ProTherm entries: 16277, 16278, 16279 Extra Details: Unfolding from varying initial urea concentrations. 0.2 mM K2EDTA and 1 mM beta-ME were added in the experiment. Alpha-subunit. tryptophan synthase, alpha/beta barrel, folding mechanism, parallel channels

Submission Details

ID: aeWEsWYS4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:47 p.m.

Version: 1

Publication Details
Bilsel O;Zitzewitz JA;Bowers KE;Matthews CR,Biochemistry (1999) Folding mechanism of the alpha-subunit of tryptophan synthase, an alpha/beta barrel protein: global analysis highlights the interconversion of multiple native, intermediate, and unfolded forms through parallel channels. PMID:9893998
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