Characterization of an early intermediate in the folding of the alpha subunit of tryptophan synthase by hydrogen exchange measurement.
The development of the hydrogen bonding network in the early stages of the folding of the alpha subunit of tryptophan synthase was monitored with a hydrogen exchange technique. The orders of magnitude difference between the rapid conversions of the unfolded forms to two stable intermediates (milliseconds) and the subsequent slow conversions of the intermediates to the native form (greater than 100 s) was used to selectively label with tritium the hydrogen bonds that form in the first 30 s of folding at 0 degree C. Rapid removal of the tritiated solvent by gel filtration ensured that hydrogen bonds formed in subsequent folding reactions would be unlabeled. Limited proteolysis and separation of peptides by high-pressure liquid chromatography permitted the determination of the amount of label retained in individual peptides by scintillation counting. Peptides 1-70 and 71-188, which when covalently linked comprise the stable amino domain in the native conformation, retain 91% and 93%, respectively, of the label retained when the protein is allowed to completely refold in tritiated solvent. Peptide 189-268, the marginally stable carboxyl domain, only retains 43% of the label. The striking difference in retention of label confirms the independent folding of these two domains and shows that the kinetic intermediates that appear in the folding of alpha subunit correspond to structural domains in the native conformation. The near-equality of the labeling of the two peptides comprising the amino domain shows that this domain folds as a single entity and that subdomain folding is unlikely.(ABSTRACT TRUNCATED AT 250 WORDS)
D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology; D006860:Hydrogen Bonding; D007700:Kinetics; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D014357:Trypsin; D014367:Tryptophan Synthase / Q000378:metabolism*
D000596:Amino Acids; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014357:Trypsin; D014367:Tryptophan Synthase