Identifying the structural boundaries of independent folding domains in the alpha subunit of tryptophan synthase, a beta/alpha barrel protein

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.