Thermostable chaperonin from Clostridium thermocellum.
Homologues of the chaperonins Cpn60 and Cpn10 have been purified from the Gram-positive cellulolytic thermophile Clostridium thermocellum. The Cpn60 protein was purified by ATP-affinity chromatography and the Cpn10 protein was purified by gel-filtration, ion-exchange and hydrophobic interaction chromatographies. The identities of the proteins were confirmed by N-terminal sequence analysis and antigenic cross-reactivity. The Cpn60 homologue is a weak, thermostable ATPase (t1/2 at 70 decrees C more than 90 min) with optimum activity (Kcat 0.07 S-1) between 60 degrees C and 70 degrees C. The ATPase activity of the authentic Cpn60 was inhibited by Escherichia coli GroES. The catalytic properties of a recombinant C. thermocellum Cpn60 purified from a GST-Cpn60 fusion protein expressed in E. coli [Ciruela (1995) Ph.D. Thesis, University of Kent] were identical with those of the authentic C. thermocellum Cpn60. Gel-filtration studies show that at room temperature the Cpn60 migrates mainly as a heptamer. Electron microscopy confirms the presence of complexes showing 7-fold rotational symmetry and also reveals a small number of particles that seem to be tetradecamers with a similar structure to E. coli GroEL complexes.
Cross
S J
SJ
Research School of Biosciences, University of Kent at Canterbury, U.K.
Ciruela
A
A
Poomputsa
K
K
Romaniec
M P
MP
Freedman
R B
RB
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D018835:Chaperonin 10 / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D018834:Chaperonin 60 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D002846:Chromatography, Affinity; D003013:Clostridium / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D018835:Chaperonin 10; D018834:Chaperonin 60; D000251:Adenosine Triphosphatases