2 CONFORMATIONAL STATES OF BETA-LACTAMASE BOUND TO GROEL - A BIOPHYSICAL CHARACTERIZATION

Escherichia coli RTEM beta-lactamase, in which both cysteine residues which form the single disulfide bond have been mutated to alanine resi dues, can form stable reversible complexes with GroEL under two differ ent sets of conditions. Starting with the GdmCl-denatured enzyme, it i s bound to GroEL in a state where no protons are protected against exc hange with (H2O)-H-2, as determined by electrospray ionization mass sp ectrometry (ESI-MS). Ln contrast, when native protein is destabilized at high temperature and added to GroEL, a conformation is bound with 1 8 protected protons after two hours of exchange. While the high-temper ature complex can form both with the wild-type enzyme (with intact dis ulfide bond) and the Cys-Ala double mutant, only the latter protein ca n form a complex starting from GdmCl denatured states. Thus, two diffe rent sets of conformations of the same protein can be bound, depending both on the conditions used to form the complex and on the intrinsic stability of the intermediate recognized by GroEL, and we have charact erized the properties of both complexes. (C) 1998 Academic Press Limit ed.