Linked Life Data

11457863

Source:http://linkedlifedata.com/resource/pubmed/id/11457863

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
39
pubmed:dateCreated
2001-9-24
pubmed:abstractText
Experiments were designed to evaluate the proximity of transmembrane helices two (TM2) and eleven (TM11) in the tertiary structure of OxlT, the oxalate:formate exchange transporter of Oxalobacter formigenes. A tandem duplication of the Factor Xa protease cleavage site (IEGRIEGR) was inserted into the central cytoplasmic loop of an OxlT cysteine-less derivative in which an endogenous cleavage site had been eliminated by mutagenesis (R248Q). Using this host, double cysteine derivatives were constructed so as to pair one of seventeen positions in TM2 with one of four positions in TM11. Following treatment of membrane vesicles with Cu(II)(1,10-phenanthroline)(3), molecular iodine, or N,N'-o-phenylenedimaleimide, samples were exposed to Factor Xa, and disulfide bond formation was assessed after SDS-polyacrylamide gel electrophoresis by staining with antibody directed against the OxlT C terminus. In the absence of disulfide bond formation, exposure to Factor Xa revealed the expected C-terminal 22-kDa fragment, a result unaffected by the presence of reductant. By contrast, after disulfide formation, OxlT mobility remained at 35 kDa, and appearance of the 22-kDa fragment required addition of 200 mm dithiothreitol prior to electrophoresis. The four TM11 positions chosen for cysteine substitution lie on a helical face known to interact with substrate. Similarly, TM2 positions supporting disulfide trapping were also confined to a single helical face. We conclude that TM2 and TM11 are in close juxtaposition to one another in the tertiary structure of OxlT.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
28
pubmed:volume
276
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
36681-6
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:11457863-Bacterial Proteins, pubmed-meshheading:11457863-Binding Sites, pubmed-meshheading:11457863-Carrier Proteins, pubmed-meshheading:11457863-Cross-Linking Reagents, pubmed-meshheading:11457863-Cysteine, pubmed-meshheading:11457863-Disulfides, pubmed-meshheading:11457863-Electrophoresis, Polyacrylamide Gel, pubmed-meshheading:11457863-Factor Xa, pubmed-meshheading:11457863-Kinetics, pubmed-meshheading:11457863-Membrane Proteins, pubmed-meshheading:11457863-Membrane Transport Proteins, pubmed-meshheading:11457863-Models, Molecular, pubmed-meshheading:11457863-Mutagenesis, Site-Directed, pubmed-meshheading:11457863-Mutation, pubmed-meshheading:11457863-Oxalobacter formigenes, pubmed-meshheading:11457863-Plasmids, pubmed-meshheading:11457863-Protein Binding, pubmed-meshheading:11457863-Protein Conformation, pubmed-meshheading:11457863-Protein Structure, Secondary, pubmed-meshheading:11457863-Protein Structure, Tertiary, pubmed-meshheading:11457863-Time Factors
pubmed:year
2001
pubmed:articleTitle
Helix proximity in OxlT, the oalate:formate antiporter of oxalobacter formigenes. Cross-linking between TM2 and TM11.
pubmed:affiliation
Department of Physiology, Johns Hopkins Medical School, Baltimore, Maryland 21205, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, Non-P.H.S.