Linked Life Data

10644743

Source:http://linkedlifedata.com/resource/pubmed/id/10644743

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
4
pubmed:dateCreated
2000-2-29
pubmed:abstractText
Whereas the mechanism of GroEL/GroES-mediated protein folding has been extensively studied, the role of these chaperonins in oligomeric protein assembly remains poorly understood. In the present study, we investigated the interaction of the chaperonins with an alphabeta heterodimeric intermediate during the alpha(2)beta(2) assembly of human mitochondrial branched-chain alpha-ketoacid dehydrogenase/decarboxylase (BCKD). Incubation of the recombinant His(6)-tagged BCKD in 400 mM KSCN for 45 min at 23 degrees C caused a complete dissociation of the alpha(2)beta(2) heterotetramers into inactive alphabeta heterodimers. Dilution of the denaturant resulted in a rapid recovery of BCKD independent of the chaperonins GroEL/GroES. Prolonged incubation of BCKD in 400 mM KSCN resulted in the generation of nonproductive or "bad" heterodimers, which were unable to undergo spontaneous reactivation but capable of binding to GroEL to form a stable GroEL-alphabeta complex. Incubation of this complex with GroES and Mg-ATP led to the slow reactivation of BCKD with a second-order rate constant k = 480 M(-1) s(-1). Mixing experiments with radiolabeled and unlabeled protein substrates provided direct evidence that GroEL/GroES promote dissociation and subunit exchange between bad heterodimers. This was accompanied by the transformation of bad heterodimers to their "good" or productive counterparts. The good heterodimers were capable of spontaneous dimerization to initially form an inactive heterotetrameric species, followed by conversion to active heterotetramers. However, a large fraction of bad heterodimers were regenerated and rebound to GroEL. The cycle was perpetuated until the reconstitution of active BCKD was complete. Our data support the thesis that chaperonins GroEL/GroES mediate iterative annealing of nonproductive assembly intermediates at the quaternary structure level. This step is essential for an efficient subsequent higher order oligomerization.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
28
pubmed:volume
275
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2786-94
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:year
2000
pubmed:articleTitle
GroEL/GroES promote dissociation/reassociation cycles of a heterodimeric intermediate during alpha(2)beta(2) protein assembly. Iterative annealing at the quaternary structure level.
pubmed:affiliation
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't