Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding.

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Abstract
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 .beta.2 subunit of tryptophan synthetase [tryptophan synthase, L-serine hydro-lyase (adding indoleglycerol-phosphate), EC 4.2.1.20] was subjected to controlled proteolysis. The resulting protein is a dimer, the protomer of which contains 2 nonoverlapping polypeptide chains of MW 12,000 and 29,000. Though inactive, the nicked protein is 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 [phosphate]) and substrates, which 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 folding process in the normal .beta.2 subunit.