Substoichiometric amounts of the molecular chaperones GroEL and GroES prevent thermal denaturation and aggregation of mammalian mitochondrial malate dehydrogenase in vitro.
The molecular chaperones GroEL and GroES were produced at very high levels in Escherichia coli, purified, and shown to protect pig mitochondrial malate dehydrogenase (MDH) against thermal inactivation in vitro. The apparent rate of MDH inactivation at 37 degrees C was reduced by a factor of at least 5 in a process which required only GroEL, GroES, and ATP. GroEL alone did not protect MDH against thermal inactivation but kept the denatured protein soluble and thereby prevented its aggregation. Reactivation of this soluble and inactive form of MDH could be achieved by addition of GroES even after 120 days of storage at -20 degrees C. Protection could be extended for more than 24 hr at 37 degrees C and was observed at molar ratios of chaperones to MDH as low as 1:4, suggesting that GroEL and GroES perform multiple turnovers in the absence of auxiliary chaperones. The availability of these chaperones in large quantities combined with the apparent promiscuity of GroEL binding shows great potential for stabilization of many proteins for which thermostable variants are not available. We speculate that GroEL and GroES perform similar protective roles in vivo and thereby increase the half-life of proteins which otherwise might aggregate under physiological conditions.
Hartman
D J
DJ
Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia.
Surin
B P
BP
Dixon
N E
NE
Hoogenraad
N J
NJ
Høj
P B
PB
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D018835:Chaperonin 10; D018834:Chaperonin 60; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D006358:Hot Temperature; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*; D008954:Models, Biological; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D015183:Restriction Mapping; D013552:Swine
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D008291:Malate Dehydrogenase