PMID:9893998
| Citation |
Bilsel, O, Zitzewitz, JA, Bowers, KE and Matthews, CR (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. Biochemistry 38:1018-29 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1021/bi982365q |
| Keywords |
Circular Dichroism; Dose-Response Relationship, Drug; Escherichia coli/enzymology; Kinetics; Models, Chemical; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Thermodynamics; Tryptophan Synthase/chemistry; Tryptophan Synthase/metabolism; Urea/chemistry; Urea/metabolism |
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