Kinetics of appearance of an early immunoreactive species during the refolding of acid-denatured Escherichia coli tryptophan synthase .beta.2 subunit

Abstract

A reversible acid-denaturation process of the .beta.2 subunit of Escherichia coli tryptophan synthase has been set up. The acid-denatured state has been physically characterized: though not in a random-coiled conformation, it is extensively denatured. The renaturation of this denatured state of .beta.2 has been observed in a stopped-flow system, in the presence of a monoclonal antibody directed against native .beta.2. It is shown that the association occurs very early in the folding of .beta.2. The association rate constants of the antibody with the immunoreactive folding intermediate and with native .beta.2 are the same (3 .times. 105 M-1 .cntdot. s-1). But at high antibody concentrations the formation of the antigen/antibody complex is rate limited by a rapid (5.4 .times. 10-2 s-1) isomerization of refolding .beta. chains. This isomerization appears to reflect the formation of at least part of the epitope recognized by the antibody during the folding of .beta.2. Further conformational adjustments occurring later in the folding pathway would then allow the ultimate structuring of the epitope.