Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese.
In vitro refolding of the monomeric mitochondrial enzyme, rhodanese (thiosulfate sulfurtransferase; EC 2.8.1.1) is facilitated by molecular chaperonins. The four components: two proteins from Escherichia coli, chaperonin 60 (groEL) and chaperonin 10 (groES), MgATP, and K+, are necessary for the in vitro folding of rhodanese. These were previously shown to be necessary for the in vitro folding of ribulose-1,5-bisphosphate carboxylase at temperatures in excess of 25 degrees C (Viitanen, P. V., Lubben, T. H., Reed, J., Goloubinoff, P., O'Keefe, D. P., and Lorimer, G. H. (1990) Biochemistry 29, 5665-5671). The labile folding intermediate, rhodanese-I, which rapidly aggregates at 37 degrees C in the absence of the chaperonins, can be stabilized by forming a binary complex with chaperonin 60. The discharge of the binary chaperonin 60-rhodanese-I complex, results in the formation of active rhodanese, and requires the presence of chaperonin 10. Optimal refolding is associated with a K(+)-dependent hydrolysis of ATP. At lower protein concentrations and 25 degrees C, where aggregation is reduced, a fraction of the rhodanese refolds to an active form in the absence of the chaperonins. This spontaneous refolding can be arrested by chaperonin 60. There is some refolding (approximately equal to 20%) when ATP is replaced by nonhydrolyzable analogs, but there is no refolding in the presence of ADP or AMP. ATP analogs may interfere with the interaction of rhodanese-I with the chaperonins. Nondenaturing detergents facilitate rhodanese refolding by interacting with exposed hydrophobic surfaces of folding intermediates and thereby prevent aggregation (Tandon, S., and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618). The chaperonin proteins appear to play a similar role in as much as they can replace the detergents. Consistent with this view, chaperonin 60, but not chaperonin 10, binds 2-3 molecules of the hydrophobic fluorescent reporter, 1,1'-bi(4-anilino)naphthalene-S,5'-disulfonic acid, indicating the presence of hydrophobic surfaces on chaperonin 60. The number of bound probe molecules is reduced to 1-2 molecules when chaperonin 10 and MgATP are added. The results support a model in which chaperonins facilitate folding, at least in part, by interacting with partly folded intermediates, thus preventing the interactions of hydrophobic surfaces that lead to aggregation.
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
J A
JA
Mendoza
E
E
Rogers
G H
GH
Lorimer
P M
PM
Horowitz
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001426:Bacterial Proteins / Q000494:pharmacology*; D018835:Chaperonin 10; D018834:Chaperonin 60; D018833:Chaperonins; D006360:Heat-Shock Proteins / Q000494:pharmacology; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D011506:Proteins / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D011506:Proteins; D000255:Adenosine Triphosphate; D014508:Urea; D013884:Thiosulfate Sulfurtransferase; D018833:Chaperonins