Show simple item record

dc.contributor.authorPappenberger, G
dc.contributor.authorMcCormack, EA
dc.contributor.authorWillison, KR
dc.date.accessioned2018-09-03T13:02:37Z
dc.date.issued2006-07-07
dc.identifier2
dc.identifier.citationJOURNAL OF MOLECULAR BIOLOGY, 2006, 360 pp. 484 - 496
dc.identifier.issn0022-2836
dc.identifier.urihttps://repository.icr.ac.uk/handle/internal/2507
dc.identifier.doi10.1016/j.jmb.2006.05.003
dc.description.abstractThe eukaryotic cytosolic chaperonin CCT is an essential ATP-dependent protein folding machine whose action is required for folding the cytoskeletal proteins actin and tubulin, and a small number of other substrates, including members of the WD40-propellor repeat-containing protein family. An efficient purification protocol for CCT from Saccharomyces cerevisiae has been developed. It uses the calmodulin binding peptide as an affinity tag in an internal loop in the apical domain of the CCT3 subunit, which is predicted to be located on the outside of the double-ring assembly. This purified yeast CCT was used for a novel quantitative actin-folding assay with human beta-actin or yeast ACT1p protein folding intermediates, Ac-I, pre-synthesised in an Escherichia coli translation system. The formation of native actin follows approximately a first-order reaction with a rate constant of about 0.03 min(-1). Yeast CCT catalyses the folding of yeast ACT1p and human beta-actin with nearly identical rate constants and yields. The results from this controlled CCT-actin folding assay are consistent with a model where CCT and Ac-I are in a binding pre-equilibrium with a rate-limiting binding step, followed by a faster ATP-driven processing to native actin. In this pure in vitro system, the human beta-actin mutants, D244S and G150P, show impaired folding behaviour in the manner predicted by our sequence-specific recognition model for CCT-actin interaction. (c) 2006 Elsevier Ltd. All rights reserved.
dc.format.extent484 - 496
dc.languageeng
dc.language.isoeng
dc.publisherACADEMIC PRESS LTD ELSEVIER SCIENCE LTD
dc.titleQuantitative actin folding reactions using yeast CCT purified via an internal tag in the CCT3/gamma subunit
dc.typeJournal Article
rioxxterms.versionofrecord10.1016/j.jmb.2006.05.003
rioxxterms.licenseref.startdate2006-07-07
rioxxterms.typeJournal Article/Review
dc.relation.isPartOfJOURNAL OF MOLECULAR BIOLOGY
pubs.notesaffiliation: Willison, KR (Reprint Author), Inst Canc Res, Chester Beatty Labs, Ctr Cell & Mol Biol, Canc Res UK, 237 Fulham Rd, London SW3 6JB, England. Inst Canc Res, Chester Beatty Labs, Ctr Cell & Mol Biol, Canc Res UK, London SW3 6JB, England. keywords: actin; protein folding; eukaryotic chaperonin; CCT keywords-plus: EUKARYOTIC CYTOSOLIC CHAPERONIN; ANAPHASE-PROMOTING COMPLEX; CYTOPLASMIC CHAPERONIN; BETA-ACTIN; AFFINITY PURIFICATION; PROTEIN-PURIFICATION; INDIVIDUAL SUBUNITS; CRYSTAL-STRUCTURE; BINDING-SITES; II CHAPERONIN research-areas: Biochemistry & Molecular Biology web-of-science-categories: Biochemistry & Molecular Biology author-email: [email protected] number-of-cited-references: 42 times-cited: 41 usage-count-last-180-days: 1 usage-count-since-2013: 6 journal-iso: J. Mol. Biol. doc-delivery-number: 061AW unique-id: ISI:000238844600019 da: 2018-09-03
pubs.notesNot known
pubs.organisational-group/ICR
pubs.organisational-group/ICR/Primary Group
pubs.organisational-group/ICR/Primary Group/ICR Divisions
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Closed research teams
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Closed research teams/Wigley Group
pubs.organisational-group/ICR
pubs.organisational-group/ICR/Primary Group
pubs.organisational-group/ICR/Primary Group/ICR Divisions
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Closed research teams
pubs.organisational-group/ICR/Primary Group/ICR Divisions/Closed research teams/Wigley Group
pubs.volume360
pubs.embargo.termsNot known
icr.researchteamWigley Groupen_US
dc.contributor.icrauthorMccormack, Elizabeth Anneen
dc.contributor.icrauthorCrook, Timothyen


Files in this item

FilesSizeFormatView

There are no files associated with this item.

This item appears in the following collection(s)

Show simple item record