Urea-induced unfolding of the alpha subunit of tryptophan synthase: evidence for a multistate process.

The urea-induced unfolding of the alpha subunit of tryptophan synthase from E. coli was monitored by optical spectroscopy and by urea-gradient gel electrophoresis. Three independent lines of evidence support the conclusion that one or more stable intermediates are present in this process: (i) Satisfactory fits of the equilibrium unfolding transitions obtained from difference spectroscopy at 286 nm and circular dichroism spectroscopy at 222 nm require a model which involves a stable intermediate in addition to the native and unfolded forms. (ii) Kinetic studies of the change in the extinction coefficient at 286 nm show that while the unfolding is well described by a single exponential change the refolding kinetics are complex. The nature of the dependence of the refolding kinetics on the initial concentration of urea supports the conclusion that at least one stable intermediate exists. (iii) The patterns obtained from urea-gradient gel electrophoresis experiments on the alpha subunit show that at least one and possibly two stable intermediates are involved; the intermediates have markedly different degrees of compactness. A kinetic model for the folding of the alpha subunit, consistent with all of these results, can be formulated.