Binding, encapsulation and ejection: substrate dynamics during a chaperonin-assisted folding reaction.
Mitochondrial malate dehydrogenase (mMDH) folds more rapidly in the presence of GroEL, GroES and ATP 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 subunits:GroEL oligomer) and with an apparent dissociation constant K' for the GroE acceptor state of at least 1 x 10(-8) M. However, even at chaperonin concentrations which are 4000 x K', i.e. at negligible concentrations of free mMDH, the observed folding rate of the substrate remains at its optimum, showing not only that folding occurs in the chaperonin-mMDH complex but also that this rate is uninhibited by any interactions with sites on GroEL. Despite the ability of mMDH to fold on the chaperonin, trapping experiments show that its dwell time on the complex is only 20 seconds. This correlates with both the rate of ATP turnover and the dwell time of GroES on the complex and is only approximately 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 cycle of three functional states: (1) an acceptor complex in which the unfolded substrate is bound tightly; (2) an encapsulation state in which it is sequestered but direct protein-protein contact is lost so that folding can proceed unhindered; and (3) an ejector state which forces dissociation of the substrate whether folded or not.
Ranson
N A
NA
Molecular Recognition Centre, School of Medical Sciences, University of Bristol, UK.
Burston
S G
SG
Clarke
A R
AR
D000255:Adenosine Triphosphate / Q000378:metabolism; D018835:Chaperonin 10 / Q000378:metabolism; D018834:Chaperonin 60 / Q000378:metabolism; D018833:Chaperonins / Q000378:metabolism*; D005033:Ethylmaleimide / Q000378:metabolism; D005456:Fluorescent Dyes; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D008956:Models, Chemical; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding
D018835:Chaperonin 10; D018834:Chaperonin 60; D005456:Fluorescent Dyes; D006146:Guanidines; D000255:Adenosine Triphosphate; D008291:Malate Dehydrogenase; D018833:Chaperonins; D019791:Guanidine; D005033:Ethylmaleimide