BINDING, ENCAPSULATION AND EJECTION - SUBSTRATE DYNAMICS DURING A CHAPERONIN-ASSISTED FOLDING REACTION

Mitochondrial malate dehydrogenase (mMDH) folds more rapidly in the pr esence of GroEL, GroES and Am than it does unassisted. The increase in folding rate as a function of the concentration of GroEL-ES reaches a maximum at a stoichiometry which is approximately equimolar (mMDH sub units:GroEL oligomer) and with an apparent dissociation constant K' fo r the GroE acceptor state of at least 1 x 10(-8) M. However, even at c haperonin concentrations which are 4000 x K', i.e. at negligible conce ntrations of free mMDH, the observed folding rate of the substrate rem ains at its optimum, showing not only that folding occurs in the chape ronin-mMDH complex but also that this rate is uninhibited by any inter actions with sites on GroEL. Despite the ability of mMDH to fold on th e chaperonin, trapping experiments show that its dwell time on the com plex is only 20 seconds. This correlates with both the rate of Am turn over and the du ell time of GroES on the complex and is only similar t o 5% of the time taken for the substrate to commit to the folded state . The results imply that ATP drives the chaperonin complex through a c ycle of three functional states: (1) an acceptor complex in which the unfolded substrate is bound tightly; (2) an encapsulation state in whi ch it is sequestered but direct protein-protein contact is lost so tha t folding can proceed unhindered; and (3) an ejector state which force s dissociation of the substrate whether folded or not. (C) 1997 Academ ic Press Limited.