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
Ja. Zitzewitz et Cr. Matthews, 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, BIOCHEM, 38(31), 1999, pp. 10205-10214
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.