Molecular dissection of the folding mechanism of the alpha subunit of tryptophan synthase: An amino-terminal autonomous folding unit controls severalrate-limiting steps in the folding of a single domain protein

The alpha subunit of tryptophan synthase (alpha TS) from Escherichia coli i s a 268-residue 8-stranded beta/alpha barrel protein. Two autonomous foldin g units, comprising the first six strands (residues 1-188) and the last two strands (residues 189-268), have been previously identified in this single structural domain protein by tryptic digestion [Higgins, W., Fairwell, T., and Miles, E. W. (1979) Biochemistry 18, 4827-4835]. The larger, amino-ter minal fragment, alpha TS(1-188), was overexpressed and independently purifi ed, and its equilibrium and kinetic folding properties were studied by abso rbance, fluorescence, and near- and far-UV circular dichroism spectroscopie s. The native state of the fragment unfolds cooperatively in an apparent tw o-state transition with a stability of 3.98 +/- 0.19 kcal mol(-1) in the ab sence of denaturant and a corresponding m value of 1.07 +/- 0.05 kcal mol(- 1) M-1. Similar to the full-length protein, the unfolding of the fragment s hows two kinetic phases which arise from the presence of two discrete nativ e state populations. Additionally, the fragment exhibits a significant burs t phase in unfolding, indicating that a fraction of the folded state ensemb le under native conditions has properties similar to those of the equilibri um intermediate populated at 3 M urea in full-length alpha TS. Refolding of alpha TS(1-188) is also complex, exhibiting two detectable kinetic phases and a burst phase that is complete within 5 ms. The two slowest isomerizati on phases observed in the refolding of the full-length protein are absent i n the fragment, suggesting that these phases reflect contributions from the carboxy-terminal segment. The folding mechanism of alpha TS(1-188) appears to be a simplified version of the mechanism for the full-length protein [B ilsel, O., Zitzewitz, J. A., Bowers, K.E, and Matthews, C. R.(1999) Biochem istry 38, 1018-1029]. Four parallel channels in the full-length protein are reduced to a pair of channels that most likely reflect a cis/trans proline isomerization reaction in the amino-terminal fragment. The off- and on-pat hway intermediates that exist for both full-length alpha TS and alpha TS(1- 188) may reflect the preponderance of local interactions in the beta/alpha barrel motif.