title: 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.
authors.affiliation: Department of Chemistry, Center for Biomolecular Structure and Function, The Pennsylvania State University, University Park 16802, USA.
authors.forename: O
authors.initials: O
authors.lastname: Bilsel
authors.forename: J A
authors.initials: JA
authors.lastname: Zitzewitz
authors.forename: K E
authors.initials: KE
authors.lastname: Bowers
authors.forename: C R
authors.initials: CR
authors.lastname: Matthews
mesh_terms: D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008956:Models, Chemical; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000378:metabolism*; D014508:Urea / Q000737:chemistry / Q000378:metabolism
chemical_list: D014508:Urea; D014367:Tryptophan Synthase