Two equilibrium intermediates have previously been
observed in the urea denaturation of the α subunit
of tryptophan synthase (αTS) from Escherichia coli,
an eight-stranded β/α barrel protein. In the current
study, a series of amino-terminal fragments were characterized
to probe the elementary folding units that may be in part
responsible for this complex behavior. Stop-codon mutagenesis
was used to produce eight fragments ranging in size from
105–214 residues and containing incremental elements
of secondary structure. Equilibrium studies by circular
dichroism indicate that all of these fragments are capable
of adopting secondary structure. All except for the shortest
fragment fold cooperatively. The addition of the fourth,
sixth, and eighth β-strands leads to distinct increases
in structure, cooperativity, and/or stability, suggesting
that folding involves the modular assembly of βαβ
supersecondary structural elements. One-dimensional NMR
titrations at high concentrations of urea, probing the
environment around His92, were also performed to test for
the presence of residual structure in the fragments. All
fragments that contained the first four βα 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 αTS requires
the participation of hydrophobic residues in multiple β-strands
that span the entire sequence.