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

Study Summary

Number of data points 6
Proteins Tryptophan synthase alpha chain ; Tryptophan synthase alpha chain
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: m details:Additives urea from varying initial urea concentrations. For intermediate to unfolding. 0. ; Experimental Assay: dG_H2O details:Additives urea from varying initial urea concentrations. For intermediate to unfoldin ; Experimental Assay: m details:Additives urea from varying initial urea concentrations. For native to intermediate. 0.2 m ; Experimental Assay: dG_H2O details:Additives urea from varying initial urea concentrations. For native to intermediate. ; Experimental Assay: m details:Additives K2EDTA (0.2 mM), beta-ME (1 mM), ; Experimental Assay: dG_H2O details:Additives K2EDTA (0.2 mM), beta-ME (1 mM),
Libraries Mutations for sequence MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIIDTLIEAGADALELGIPFSDPLADGPTIQNATLRAFAAGVTPAQCFEMLALIRQKHPTIPIGLLMYANLVFNKGIDEFYAQCEKVGVDSVLVADVPVEESAPFRQAALRHNVAPIFICPPNADDDLLRQIASYGRGYTYLLSRAGVTGAENRAALPLNHLVAKLKEYNAAPPLQGFGISAPDQVKAAIDAGAAGAISGSAIVKIIEQHINEPEKMLAALKVFVQPMKAATRS

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 UniProtKB Entries

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