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

A variety of techniques have been used to investigate the urea-induced kine tic folding mechanism of the a-subunit of tryptophan synthase from Escheric hia coli. A distinctive property of this 29 kDa alpha/beta barrel protein i s the presence of two stable equilibrium intermediates, populated at approx imately 3 and 5 M urea, The refolding process displays multiple kinetic pha ses whose lifetimes span the submillisecond to greater than 100 s time scal e; unfolding studies yield two relaxation times on the order of 10-100 s. I n an effort to understand the populations and structural properties of both the stable and transient intermediates, stopped-flow, manual-mixing, and e quilibrium circular dichroism data were globally fit to various kinetic mod els. Refolding and unfolding experiments from various initial urea concentr ations as well as forward and reverse double-jump experiments were critical for model discrimination. The simplest kinetic model that is consistent wi th all of the available data involves four slowly interconverting unfolded forms that collapse within 5 ms to a marginally stable intermediate with si gnificant secondary structure. This early intermediate is an off-pathway sp ecies that must unfold to populate a set of four on-pathway intermediates t hat correspond to the 3 M urea equilibrium intermediate. Reequilibrations a mong these conformers act as rate-limiting steps in folding for a majority of the population. A fraction of the native conformation appears in less th an 1 s at 25 degrees C, demonstrating that even large proteins can rapidly traverse a complex energy surface.