ABSTRACT
Compared to the group I chaperonins, such as Escherichia coli GroEL, which facilitate protein folding, many aspects of the functional mechanism of archaeal group II chaperonins are unclear. Sequence homology between the chaperonin from Pyrococcus furiosus (PfCPN) and other group II chaperonins, together with the homo-oligomeric nature of PfCPN, suggest that PfCPN may serve as a model to clarify the role of the homologous position Gly-345 in the chaperonin-mediated protein folding. Here, we show that the purified chaperonin mutant in which the conserved residue Gly-345 is replaced by Asp (G345D) displays only about 25% ATP/ADP hydrolysis activities of the wild-type in the presence of Co(2+) and has a reduced capacity to promote folding of denatured malate dehydrogenase in vitro. This may be a reflection that Gly-345 plays an essential role in conformational change and protein refolding by archaeal group II chaperonins.
Subject(s)
Archaeal Proteins/metabolism , Glycine/chemistry , Group II Chaperonins/metabolism , Pyrococcus furiosus/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Archaeal Proteins/chemistry , Archaeal Proteins/genetics , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Group II Chaperonins/chemistry , Group II Chaperonins/genetics , Microscopy, Electron, Transmission , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Protein Folding , Pyrococcus furiosus/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The chaperonin molecular machine from hyperthermophilic archaeon Pyrococcus furiosus was studied in this paper. The Pyrococcus furiosus chaperonin gene (PfCPN) was amplified by PCR from the Pyrococcus furiosus genomic DNA, and expressed in Escherichia coli BL21-Codonplus(DE)(3)-RIL. The recombinant PfCPN was purified to homogeneity by using ion-exchange and size-exclusion chromatography. It was found that the ATPase activity of the PfCPN was highest at 88 degrees C, and there existed a nested cooperativity of the ATPase activity of the PfCPN. This result suggested that nested allosteric behavior may be common to chaperonin molecular machines from archaea. The half-life (t(1/2)) of the ATPase activity of the PfCPN at 100 degrees C was about 60 min. The PfCPN displayed chaperone activity in preventing lysozyme from thermal inactivation. This chaperone activity was in an ATP-dependent manner.
