Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding.
Globular proteins often appear to consist of distinct compact "domains," and the assumption is frequently implicitly made that these domains correspond to intermediate structures in the folding process. If this assumption is correct, the polypeptide fragment that builds up a domain should be able to spontaneously fold into its native conformation even when isolated. In an attempt to isolate and study such a fragment, the beta2 subunit of tryptophan synthetase [tryptophan synthase, L-serine hydro-lyase (adding indoleglycerol-phosphate), EC 4.2.1.20] has been subjected to controlled proteolysis. The resulting protein is shown to be a dimer, the protomer of which contains two nonoverlapping polypeptide chains of molecular weights 12,000 and 29,000. Though inactive, the nicked protein is shown to be in a conformation that closely resembles that of the original enzyme, since it still can form an enzyme-bound intermediate of the catalytic reactions. The fluorescence of this intermediate is used to characterize the binding sites for the cofactor (pyridoxal-P) and substrates, which are shown to exist on the nicked protein. The possibility is discussed of using the fragments isolated from the nicked protein to study individual steps of the enzymatic reaction, intracistronic complementation, and the folding process in the normal beta2 subunit.
D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007211:Indoles; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D012694:Serine; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin; D014367:Tryptophan Synthase / Q000302:isolation & purification / Q000378:metabolism
D007211:Indoles; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012694:Serine; D014357:Trypsin; D014367:Tryptophan Synthase