Specific Structure Appears at the N terminus in the Sub-millisecond Folding Intermediate of the Alpha Subunit of Tryptophan Synthase, a TIM Barrel Protein

Competing views of the products of sub-millisecond folding reactions observed in many globular proteins have been ascribed either to the formation of discrete, partially folded states or to the random collapse of the unfolded chain under native-favoring conditions. To test the validity of these alternative interpretations for the stopped-flow burst-phase reaction in the (βα)8, TIM barrel motif, a series of alanine replacements were made at five different leucine or isoleucine residues in the alpha subunit of tryptophan synthase (αTS) from Escherichia coli. This protein has been proposed to fold, in the sub-millisecond time range, to an off-pathway intermediate with significant stability and ∼50% of the far-UV circular dichroism (CD) signal of the native conformation. Individual alanine replacements at any of three isoleucine or leucine residues in either α1, β2 or β3 completely eliminate the off-pathway species. These variants, within 5 ms, access an intermediate whose properties closely resemble those of an on-pathway equilibrium intermediate that is highly populated at moderate urea concentrations in wild-type αTS. By contrast, alanine replacements for leucine residues in either β4 or β6 destabilize but preserve the off-pathway, burst-phase species. When considered with complementary thermodynamic and kinetic data, this mutational analysis demonstrates that the sub-millisecond appearance of CD signal for αTS reflects the acquisition of secondary structure in a distinct thermodynamic state, not the random collapse of an unfolded chain. The contrasting results for replacements in the contiguous α1/β2/β3 domain and the C-terminal β4 and β6 strands imply a heterogeneous structure for the burst-phase species. The α1/β2/β3 domain appears to be tightly packed, and the C terminus appears to behave as a molten-globule-like structure whose folding is tightly coupled to that of the α1/β2/β3 domain.