Ja. Zitzewitz et al., Identifying the structural boundaries of independent folding domains in the alpha subunit of tryptophan synthase, a beta/alpha barrel protein, PROTEIN SCI, 8(6), 1999, pp. 1200-1209
Two equilibrium intermediates have previously been observed in the urea den
aturation of the alpha subunit of tryptophan synthase (alpha TS) from Esche
richia coli, an eight-stranded beta/alpha barrel protein. In the current st
udy, a series of aminoterminal fragments were characterized to probe the el
ementary folding units that may be in part responsible for this complex beh
avior. Stop-codon mutagenesis was used to produce eight fragments ranging i
n size from 105-214 residues and containing incremental elements of seconda
ry structure. Equilibrium studies by circular dichroism indicate that all o
f these fragments are capable of adopting secondary structure. All except f
or the shortest fragment fold cooperatively. The addition of the fourth, si
xth, and eighth beta-strands leads to distinct increases in structure, coop
erativity, and/or stability, suggesting that folding involves the modular a
ssembly of beta alpha beta supersecondary structural elements. One-dimensio
nal NMR titrations at high concentrations of urea, probing the environment
around His92, were also performed to test for the presence of residual stru
cture in the fragments. All fragments that contained the first four beta al
pha units of structure exhibited a cooperative unfolding transition at high
concentrations of urea with significant but reduced stability relative to
the full-length protein. These results suggest that the residual structure
in alpha TS requires the participation of hydrophobic residues in multiple
beta-strands that span the entire sequence.