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

Sequence Assay Result Units