KINETIC CHARACTERIZATION OF EARLY IMMUNOREACTIVE INTERMEDIATES DURING THE REFOLDING OF GUANIDINE-UNFOLDED ESCHERICHIA-COLI TRYPTOPHAN SYNTHASE BETA-2 SUBUNITS

This paper deals with stopped-flow studies on the kinetics of the regain of immunoreactivity toward five distinct monoclonal antibodies during the folding of the guanidine-unfolded β2 subunit of Escherichia coli tryptophan synthase and of two complementary proteolytic fragments of β, F1 (N-terminal; Mw = 29000) and F2 (C-terminal; Mw = 12000). It is shown that, while selected as being “specific” for the native protein, these antibodies are all able to recognize early folding intermediates. The two antigenic determinants carried by the F2 domain and the antigenic site carried by the hinge peptide linking F1 and F2 are present so early during the folding process that their kinetics of appearance could not be followed. On the contrary, the rate constants of appearance of two “native-like” epitopes, carried by F1, could be determined during the folding of β chains. The rate constant of appearance of the epitope to antibody 19 was found to be k = 0.065 s−1 at 12 °C. This value is very similar to that we reported previously for the appearance of an early epitope to the same antibody during the folding of acid-denatured β chains. Thus, in spite of the important structural differences between guanidine-unfolded and acid-denatured β chains, the same early folding events seem to be involved in the appearance of this epitope. The rate constant was found to be significantly smaller (k = 0.02 s−1 at 12 °C) for the appearance of the epitope to antibody 9. This shows that the regain of immunoreactivity is not concerted within the F, domain. Finally, it was observed that the epitopes to antibodies 9 and 19 appear significantly more rapidly during the folding of the isolated F1 fragment than during the folding of complete β chains. Thus, in the complete protein, the rest of the polypeptide chain interferes with the folding of the F1 domain even in the early stages leading to the appearance of the immunoreactivity. © 1990, American Chemical Society. All rights reserved.