UNFOLDING PROPERTIES OF TRYPTOPHAN-CONTAINING ALPHA-SUBUNITS OF THE ESCHERICHIA-COLI TRYPTOPHAN SYNTHASE

The urea-induced unfolding of the Escherichia coli tryptophan synthase alpha-subunit is examined via fluorescence measurements with tryptoph an-containing alpha-subunit mutants, constructed by in vitro mutagenes is, Early unfolding studies with urea and guanidine suggested that the wild type protein unfolded in a two-step process with a stable interm ediate composed of a native alpha-1 folding unit (residues 1-188) and a completely unfolded alpha-2 folding unit (residues 189-268), Recentl y, more detailed spectroscopic and calorimetric data from the Matthews and Yutani groups indicate that such a structure for the intermediate s seems unlikely, Previously, we described the introduction of Trp res idues as unfolding reporter groups separately into each of the folding domains and showed that these proteins are wild type enzymatically an d in their stability to urea, The unfolding behavior of these alpha-su bunits, monitored by fluorescence intensity changes at the discrete em ission lambda(max) for each, in both equilibrium and kinetic experimen ts, suggest that: (a) both folding units commence unfolding simultaneo usly (near 2 M urea); (b) the larger alpha-1 unit unfolds in a multist ep process, initially yielding a partially unfolded intermediate form which subsequently appears to unfold progressively to completion; and (c) the smaller alpha-2 unit unfolds in a single step event, These res ults are also clearly incompatible with the early proposals on the str ucture of the intermediate, It is suggested here that the intermediate is heterogeneous, consisting of a stable, partially unfolded form of alpha-1 attached to either a completely folded or completely unfolded form of alpha-2, These results are consistent with and provide an adde d dimension to the recent description of the proposed structure of the intermediate.