title: Purification and characterization of chaperonin 60 and chaperonin 10 from the anaerobic thermophile Thermoanaerobacter brockii.
abstract: Chaperonin 60 and chaperonin 10 (GroEL and GroES homologues, respectively) have been isolated from extracts of the anaerobic thermophile Thermoanaerobacter brockii. A simple and rapid purification for chaperonin 60 made use of hydrophobic and anion-exchange chromatographies, and could be readily scaled up; approximately 2 mg pure chaperonin 60 was obtained/g cells. In contrast with all other prokaryotic chaperonin 60 proteins that have been studied, which are tetradecamers, including those from Thermus sp., the T. brockii protein is a heptamer, and as isolated was not in association with chaperonin 10. The preparation is readily crystallized using 2-propanol or poly(ethylene glycol) with MgCl2. The N-terminal amino acid sequence of this preparation is similar to other thermophilic chaperonin 60 proteins. Chaperonin 10 was purified from the flow-through of the first hydrophobic column (which bound chaperonin 60) using a more hydrophobic adsorbent to remove contaminating proteins, followed by anion-exchange chromatography. Chaperonin 10 was obtained with a yield of approximately 10% that of chaperonin 60. The subunit molecular mass of chaperonin 10 determined by electrospray mass spectrometry is 10254 +/- 0.4 Da, which is very similar to the molecular mass of Escherichia coli GroES. Similarly, the subunit size of chaperonin 60 determined by mass spectrometry is very similar to that of GroEL, at 57949 +/- 10 Da. T. brockii chaperonin 60 has an ATPase activity that is suppressed by chaperonin 10, and the two proteins together are active in protein-folding assays. Mitochondrial malate dehydrogenase was successfully refolded at 37 degrees C after denaturation in guanidine hydrochloride, using T. brockii chaperonin 60 and chaperonin 10, or chaperonin 60 and E. coli GroES. The denatured enzyme was protected from aggregation by association with chaperonin 60. Guanidine-hydrochloride-denatured preparations of isocitrate dehydrogenase and secondary alcohol dehydrogenase isolated from T. brockii were also refolded at 60-65 degrees C. In each case, refolding required chaperonin 60, chaperonin 10 and ATP, giving up to 80% regeneration of control activity.
authors.affiliation: Centre for Protein and Enzyme Technology, La Trobe University, Bundoora, Australia.
authors.forename: K N
authors.initials: KN
authors.lastname: Truscott
authors.forename: P B
authors.initials: PB
authors.lastname: Høj
authors.forename: R K
authors.initials: RK
authors.lastname: Scopes
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001421:Bacteria, Anaerobic / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D018835:Chaperonin 10; D018834:Chaperonin 60; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D016988:Gram-Positive Asporogenous Rods, Irregular / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008929:Mitochondria, Heart / Q000201:enzymology; D008969:Molecular Sequence Data; D017510:Protein Folding; D051381:Rats; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D000426:Alcohol Dehydrogenase; D008291:Malate Dehydrogenase; D007521:Isocitrate Dehydrogenase; D000251:Adenosine Triphosphatases