title: Refolding and recognition of mitochondrial malate dehydrogenase by Escherichia coli chaperonins cpn 60 (groEL) and cpn10 (groES).
abstract: In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). When the enzyme is initially denatured with 3 M guanidinium chloride, chaperonin-assisted refolding is 100% efficient. C.d. spectroscopy reveals that malate dehydrogenase is almost unfolded in 3 M guanidinium chloride, suggesting that a state with little or no residual secondary structure is the optimal 'substrate' for chaperonin-assisted refolding. Malate dehydrogenase denatured to more highly structured states proves to refold less efficiently with chaperonin assistance. The enzyme is shown not to aggregate under the refolding conditions, so that losses in refolding efficiency result from irreversible misfolding. Evidence is advanced to suggest that the chaperonins are unable to rescue irreversibly misfolded malate dehydrogenase. A novel use is made of 100 K Centricon concentrators to study the binding of [14C]acetyl-labelled malate dehydrogenase to groEL by an ultrafiltration binding assay. Analysis of the data by Scatchard plot shows that acetyl-malate dehydrogenase, which has previously been extensively unfolded with guanidinium chloride, binds to groEL at a specific binding site(s). At saturation, one acetyl-malate dehydrogenase homodimer (two polypeptides) is shown to bind to each groEL homooligomer with a binding constant of approx. 10 nM.
authors.affiliation: Department of Chemistry, Imperial College of Science, Technology and Medicine, South Kensington, London, U.K.
authors.forename: J P
authors.initials: JP
authors.lastname: Hutchinson
authors.forename: T S
authors.initials: TS
authors.lastname: el-Thaher
authors.forename: A D
authors.initials: AD
authors.lastname: Miller
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D002021:Buffers; D018834:Chaperonin 60; D002942:Circular Dichroism; D004926:Escherichia coli; D006360:Heat-Shock Proteins / Q000378:metabolism / Q000494:pharmacology*; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D013552:Swine; D014462:Ultrafiltration
chemical_list: D001426:Bacterial Proteins; D002021:Buffers; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D008291:Malate Dehydrogenase