Molecular Dissection of the Folding Mechanism of the α Subunit of Tryptophan Synthase: An Amino-Terminal Autonomous Folding Unit Controls Several Rate-Limiting Steps in the Folding of a Single Domain Protein

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Abstract
The α subunit of tryptophan synthase (αTS) from Escherichiacoli is a 268-residue 8-stranded β/α barrel protein. Two autonomous folding 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) Biochemistry18, 4827−4835]. The larger, amino-terminal fragment, αTS(1−188), was overexpressed and independently purified, and its equilibrium and kinetic folding properties were studied by absorbance, fluorescence, and near- and far-UV circular dichroism spectroscopies. The native state of the fragment unfolds cooperatively in an apparent two-state transition with a stability of 3.98 ± 0.19 kcal mol-1 in the absence 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 shows two kinetic phases which arise from the presence of two discrete native state populations. Additionally, the fragment exhibits a significant burst phase in unfolding, indicating that a fraction of the folded state ensemble under native conditions has properties similar to those of the equilibrium intermediate populated at 3 M urea in full-length αTS. Refolding of αTS(1−188) is also complex, exhibiting two detectable kinetic phases and a burst phase that is complete within 5 ms. The two slowest isomerization phases observed in the refolding of the full-length protein are absent in the fragment, suggesting that these phases reflect contributions from the carboxy-terminal segment. The folding mechanism of αTS(1−188) appears to be a simplified version of the mechanism for the full-length protein [Bilsel, O., Zitzewitz, J. A., Bowers, K.E, and Matthews, C. R.(1999) Biochemistry38, 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-pathway intermediates that exist for both full-length αTS and αTS(1−188) may reflect the preponderance of local interactions in the β/α barrel motif.