Linked Life Data

16679316

Source:http://linkedlifedata.com/resource/pubmed/id/16679316

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
29
pubmed:dateCreated
2006-7-17
pubmed:abstractText
The reaction cycles of cytochrome P450s (P450) require input of two electrons. Electrostatic interactions are considered important driving forces in the association of P450s with their redox partners, which in turn facilitates the transfer of the two electrons. In this study, the cross-linking reagent, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), was used to covalently link cytochrome P450 2E1 (CYP2E1) with cytochrome b(5) (b(5)) through the formation of specific amide bonds between complementary charged residue pairs. Cross-linked peptides in the resulting protein complex were distinguished from non-cross-linked peptides using an (18)O-labeling method on the basis that cross-linked peptides incorporate twice as many (18)O atoms as non-cross-linked peptides during proteolysis conducted in (18)O-water. Subsequent tandem mass spectrometric (MS/MS) analysis of the selected cross-linked peptide candidates led to the identification of two intermolecular cross-links, Lys(428)(CYP2E1)-Asp(53)(b(5)) and Lys(434)(CYP2E1)-Glu(56)(b(5)), which provides the first direct experimental evidence for the interacting orientations of a microsomal P450 and its redox partner. The biological importance of the two ion pairs for the CYP2E1-b(5) interaction, and the stimulatory effect of b(5), was confirmed by site-directed mutagenesis. Based on the characterized cross-links, a CYP2E1-b(5) complex model was constructed, leading to improved insights into the protein interaction. The described method is potentially useful for mapping the interactions of various P450 isoforms and their redox partners, because the method is relatively rapid and sensitive, and is capable of suggesting not only protein interacting regions, but also interacting orientations.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
21
pubmed:volume
281
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
20404-17
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:16679316-Acetaminophen, pubmed-meshheading:16679316-Amino Acid Sequence, pubmed-meshheading:16679316-Base Sequence, pubmed-meshheading:16679316-Binding Sites, pubmed-meshheading:16679316-Cytochrome P-450 CYP2E1, pubmed-meshheading:16679316-Cytochromes b5, pubmed-meshheading:16679316-DNA Primers, pubmed-meshheading:16679316-Escherichia coli, pubmed-meshheading:16679316-Humans, pubmed-meshheading:16679316-Kinetics, pubmed-meshheading:16679316-Mass Spectrometry, pubmed-meshheading:16679316-Models, Molecular, pubmed-meshheading:16679316-Molecular Sequence Data, pubmed-meshheading:16679316-Mutagenesis, Site-Directed, pubmed-meshheading:16679316-Oxidation-Reduction, pubmed-meshheading:16679316-Peptide Fragments, pubmed-meshheading:16679316-Protein Conformation, pubmed-meshheading:16679316-Recombinant Proteins, pubmed-meshheading:16679316-Trypsin
pubmed:year
2006
pubmed:articleTitle
Identification of the interactions between cytochrome P450 2E1 and cytochrome b5 by mass spectrometry and site-directed mutagenesis.
pubmed:affiliation
Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, USA.
pubmed:publicationType
Journal Article, Research Support, N.I.H., Extramural