Guanidine hydrochloride induced unfolding of the alpha subunit of tryptophan synthase and of the two alpha proteolytic fragments: evidence for stepwise unfolding of the two alpha domains.
The relationship between the domain structure of the alpha subunit of Escherichia coli tryptophan synthase and the mechanism of unfolding of the alpha subunit is investigated. Previous studies of the unfolding of the alpha subunit by increasing concentrations of guanidine hydrochloride or urea detected a partially unfolded form of the alpha subunit at intermediate concentrations of either denaturant. The possibility that this partially unfolded form of the alpha subunit results from the preferential unfolding of one of the two domains of the alpha subunit is now investigated. This study utilizes two proteolytic fragments of the alpha subunit, alpha-1 and alpha-2, which have been shown to refold independently and to correspond to two domains of the alpha subunit. The effects of guanidine hydrochloride concentration on the separate alpha-1 and alpha-2 fragments, on the intact alpha subunit, and on the derivative nicked by trypsin (alpha') are compared by measuring ellipticity at 222 nm and by measuring the susceptibility of tyrosyl residues to chemical modification. The results show that guanidine hydrochloride induced unfolding of the alpha subunit results from the stepwise unfolding of the two domains: the alpha-2 fragment and the corresponding domain in the intact alpha subunit are unfolded by low concentrations of guanidine hydrochloride; the alpha-1 fragment and the corresponding domain in the intact alpha subunit are unfolded by higher concentrations of guanidine hydrochloride.
D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology; D006146:Guanidines / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D014367:Tryptophan Synthase / Q000378:metabolism*
D006146:Guanidines; D007093:Imidazoles; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014367:Tryptophan Synthase; C012160:N-acetylimidazole