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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
1
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pubmed:dateCreated |
1997-2-5
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pubmed:abstractText |
Monoclonal antibody (mAb) 4B11 binds to a conformational epitope in the lactose permease that is exposed on the cytoplasmic face of the membrane with a KD of 2.8 x 10(-7) M. By studying binding of 4B11 to permease mutants containing six contiguous His residues in each cytoplasmic loop, inserted factor Xa protease sites, or a C-terminal deletion, the cytoplasmic loops between helices VIII and IX (loop VIII/IX) and between helices X and XI (loop X/XI) are shown to comprise the epitope. Subsequently, Cys-scanning mutagenesis in conjunction with thiol modification was carried out in order to identify specific residues involved in 4B11 recognition. Glu342 and Arg344 in loop X/XI are primary determinants for 4B11 binding, while Ile283 in loop VIII/IX and Phe334 and Lys335 in loop X/XI are secondary determinants. Consistently, binding of avidin to biotinylated single-Cys replacements in loop VIII/IX or loop X/XI blocks 4B11 binding, but avidin binding to biotinylated Cys residues in other cytoplasmic loops or insertion of cytochrome b562 into cytoplasmic loop VI/VII has no significant effect. The studies demonstrate that the last two cytoplasmic loops in lactose permease comprise a discontinuous epitope for monoclonal antibody 4B11 and thereby provide independent evidence for the conclusion that helices VIII-XI are in close proximity.
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Antibodies, Monoclonal,
http://linkedlifedata.com/resource/pubmed/chemical/Avidin,
http://linkedlifedata.com/resource/pubmed/chemical/Biotin,
http://linkedlifedata.com/resource/pubmed/chemical/Carrier Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Epitopes,
http://linkedlifedata.com/resource/pubmed/chemical/Escherichia coli Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/LacY protein, E coli,
http://linkedlifedata.com/resource/pubmed/chemical/Membrane Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Membrane Transport Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Monosaccharide Transport Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Symporters,
http://linkedlifedata.com/resource/pubmed/chemical/lactose permease
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pubmed:status |
MEDLINE
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pubmed:month |
Jan
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pubmed:issn |
0006-2960
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:day |
7
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pubmed:volume |
36
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
274-80
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pubmed:dateRevised |
2004-12-3
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pubmed:meshHeading |
pubmed-meshheading:8993344-Antibodies, Monoclonal,
pubmed-meshheading:8993344-Avidin,
pubmed-meshheading:8993344-Biotin,
pubmed-meshheading:8993344-Blotting, Western,
pubmed-meshheading:8993344-Carrier Proteins,
pubmed-meshheading:8993344-Cell Membrane,
pubmed-meshheading:8993344-Epitopes,
pubmed-meshheading:8993344-Escherichia coli,
pubmed-meshheading:8993344-Escherichia coli Proteins,
pubmed-meshheading:8993344-Gene Expression Regulation, Bacterial,
pubmed-meshheading:8993344-Kinetics,
pubmed-meshheading:8993344-Membrane Proteins,
pubmed-meshheading:8993344-Membrane Transport Proteins,
pubmed-meshheading:8993344-Monosaccharide Transport Proteins,
pubmed-meshheading:8993344-Mutagenesis, Site-Directed,
pubmed-meshheading:8993344-Mutation,
pubmed-meshheading:8993344-Protein Binding,
pubmed-meshheading:8993344-Protein Conformation,
pubmed-meshheading:8993344-Protein Structure, Secondary,
pubmed-meshheading:8993344-Spheroplasts,
pubmed-meshheading:8993344-Symporters
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pubmed:year |
1997
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pubmed:articleTitle |
The last two cytoplasmic loops in the lactose permease of Escherichia coli comprise a discontinuous epitope for a monoclonal antibody.
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pubmed:affiliation |
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90095-1662, USA.
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pubmed:publicationType |
Journal Article
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