Expression and characterization of the chaperonin molecular machine from the hyperthermophilic archaeon <i>Pyrococcus furiosus</i>

Chen, Huayou; Chu, Zema; Ma, Yanhe; Zhang, Yi; Yang, Shengli

doi:10.1002/jobm.200610215

Cited by 16 publications

(7 citation statements)

References 25 publications

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“…Malate dehydrogenase (MDH) was purchased from Toyobo. DNA manipulation, site-directed mutagenesis, and DNA sequencing The pET21-PfCPN was constructed as described by Chen et al (2007). Site-directed mutagenesis was achieved by standard methods.…”

Section: Methodsmentioning

confidence: 99%

“…The supernatant identified by SDS-PAGE was incubated at 80°C for 30 min. Denatured proteins were then removed by centrifugation as described previously (Chen et al 2007). After SDS-PAGE identification, the clear supernatant was loaded onto Toyopearl GigaCap Q-650M equilibrated with buffer A (25 mM HEPES pH 7.5, 50 mM, 1 mM PMSF).…”

Section: Methodsmentioning

confidence: 99%

“…G345D exhibited substantially reduced ATP and ADP hydrolysis activity compared with the wild-type PfCPN. Both ATP and ADP hydrolysis activities of wild-type and mutated PfCPN were analyzed at 80°C, which gives the highest ATPase activity (Chen et al 2007). Mg 2?…”

Section: Atp/adp Hydrolysis Activities Of Wild-type and Mutated Pfcpnmentioning

confidence: 99%

“…We have already constructed pET21-PfCPN (Chen et al 2007) and mutated PfCPN, pET21-PfCPN (G345D), was prepared by site-directed mutagenesis.…”

Section: Preparation Of Wild-type and Mutated Pfcpnmentioning

confidence: 99%

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Gly-345 plays an essential role in Pyrococcus furiosus chaperonin function

et al. 2011

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Atp/adp Hydrolysis Activities Of Wild-type and Mutated Pfcpnmentioning

confidence: 99%

“…We have already constructed pET21-PfCPN (Chen et al 2007) and mutated PfCPN, pET21-PfCPN (G345D), was prepared by site-directed mutagenesis.…”

Section: Preparation Of Wild-type and Mutated Pfcpnmentioning

confidence: 99%

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Gly-345 plays an essential role in Pyrococcus furiosus chaperonin function

et al. 2011

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“…PfCPN was expressed and purified as described by Chen et al (2007). Micrococcus lysodeikticus was a gift from the Institute of Microbiology, Chinese Academy of Sciences.…”

Section: Samples and Reagentsmentioning

confidence: 99%

Characterization of ATPase activity of class II chaperonin from the hyperthermophilic archaeon Pyrococcus furiosus

et al. 2009

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To understand how molecular damage under harsh environmental conditions can be controlled, we investigated the properties of ATPase activity of the chaperonin molecular machinery from the hyperthermophilic archaeon Pyrococcus furiosus (PfCPN). PfCPN ATPase activity depended on K(+) and Mg(2+) and its optimal pH was 7.5. PfCPN had almost no ADPase activity. ADP strongly competitively inhibited PfCPN ATPase activity. Inhibition of PfCPN ATPase decreased its chaperonin activity in protecting lysozyme from heat-induced inactivation.

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Thermosome: A Group II Chaperonin of Archaea

Yamamoto

Yohda

2016

Encyclopedia of Life Sciences

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Chaperonin, heat shock protein 60, plays an important role in the proteostasis of cytosol. Chaperonins are divided into two groups: group I and group II. Group II chaperonins exist in eukaryotes and archaea, and these chaperonins are named CCT/TRiC and Thermosome, respectively. Group II chaperonins have almost the same structure as group I chaperonins. The main difference is the existence of a helical protrusion, which constitutes a built‐in lid of the cavity. Group II chaperonin captures an unfolded protein in the cavity in the open conformation and changes to the closed conformation in an ATP‐dependent manner, which triggers folding of the captured protein. As Thermosome is relatively stable and simple compared with CCT, the conformational change mechanism and also the interaction with co‐chaperone, Prefoldin, have been studied in detail using Thermosome. The conformational change procedure of Thermosome will give insights on protein folding mechanism by group II chaperonins. Key Concepts Group II chaperonin is an essential cytosolic molecular chaperone in eukaryotes and archaea. The archaeal group II chaperonin is named Thermosome. Group II chaperonin has a built‐in lid for the central cavity. As ATP induces a conformational change from the open to the closed conformation, twisting of the ring occurs. Group II chaperonin cooperates with a co‐chaperone, Prefoldin, which captures an unfolded protein and transfers it to the central cavity of the group II chaperonin.

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Expression and characterization of the chaperonin molecular machine from the hyperthermophilic archaeon Pyrococcus furiosus

Cited by 16 publications

References 25 publications

Gly-345 plays an essential role in Pyrococcus furiosus chaperonin function

Gly-345 plays an essential role in Pyrococcus furiosus chaperonin function

Characterization of ATPase activity of class II chaperonin from the hyperthermophilic archaeon Pyrococcus furiosus

Thermosome: A Group II Chaperonin of Archaea

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