Linked Life Data

20813910

Source:http://linkedlifedata.com/resource/pubmed/id/20813910

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
10
pubmed:dateCreated
2010-10-4
pubmed:abstractText
Many essential cellular processes depend upon the self-assembly of stable multiprotein entities. The architectures of the vast majority of these protein machines remain unknown because these structures are difficult to obtain by biophysical techniques alone. However, recent progress in defining the architecture of protein complexes has resulted from integrating information from all available biochemical and biophysical sources to generate computational models. Chemical cross-linking is a technique that holds exceptional promise toward achieving this goal by providing distance constraints that reflect the topography of protein complexes. Combined with the available structural data, these constraints can yield three-dimensional models of higher order molecular machines. However, thus far the utility of cross-linking has been thwarted by insufficient yields of cross-linked products and tandem mass spectrometry methods that are unable to unambiguously establish the identity of the covalently labeled peptides and their sites of modification. We report the cross-linking of amino moieties by 1,3-diformyl-5-ethynylbenzene (DEB) with analysis by high resolution electron transfer dissociation. This new reagent coupled with this new energy deposition technique addresses these obstacles by generating cross-linked peptides containing two additional sites of protonation relative to conventional cross-linking reagents. In addition to excellent coverage of sequence ions by electron transfer dissociation, DEB cross-linking produces gas-phase precursor ions in the 4+, 5+, or 6+ charge states that are readily segregated from unmodified and dead-end modified peptides using charge-dependent precursor selection of only quadruply and higher charge state ions. Furthermore, electron transfer induces dissociation of the DEB-peptide bonds to yield diagnostic ion signals that reveal the "molecular ions" of the unmodified peptides. We demonstrate the power of this strategy by cross-linking analysis of the 21-protein, ADP-bound GroEL-GroES chaperonin complex. Twenty-five unique sites of cross-linking were determined.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
1535-9484
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
9
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2306-17
pubmed:dateRevised
2011-10-3
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
Topographic studies of the GroEL-GroES chaperonin complex by chemical cross-linking using diformyl ethynylbenzene: the power of high resolution electron transfer dissociation for determination of both peptide sequences and their attachment sites.
pubmed:affiliation
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, USA.
pubmed:publicationType
Journal Article, Research Support, N.I.H., Extramural