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          Institute: MPI für Hirnforschung     Collection: Neurochemistry (Heinrich Betz)     Display Documents

ID: 10568.0, MPI für Hirnforschung / Neurochemistry (Heinrich Betz)
Affinity labeling of cysteine-mutants evidences contact residues in modeled receptor binding sites
Authors:Perret, P.; Laube, B.; Schemm, R.; Betz, H.; Goeldner, M.; Foucaud, B.
Date of Publication (YYYY-MM-DD):2002
Title of Journal:Journal of Receptor and Signal Transduction Research
Journal Abbrev.:J. Recept. Signal Transduct. Res.
Issue / Number:1-4
Start Page:345
End Page:356
Review Status:Peer-review
Audience:Not Specified
Abstract / Description:To investigate the topology of binding sites in two ionotropic receptors, we have initiated a strategy combining affinity labeling with cysteine-scanning mutagenesis. For the GABA(A) receptor we have used reactive derivatives of non-competitive blockers (NCBs) to explore interacting positions in its channel. The polypeptide positions of the M-2 segment of the alpha(1) subunit which we mutated into cysteine were selected for their established accessibility, as determined by the substituted-cysteine accessibility method (SCAM). Using the Xenopus oocyte expression system, we show that receptors containing mutations V257C and S272C are inactivated by several reactive NCBs. These position-selective inactivations lead to an analysis of NCB binding in the channel. For the NMDA receptor glycine-binding site, the prototype antagonist L- 701,324 was derivatized at different positions with different reactive groups. The receptor positions to mutate into cysteine were selected after a 3-D homology model. The observed receptor inactivations are mutant-and probe-selective, leading to an unambiguous chemical docking of the antagonist pharmacophore and supporting the model. The site-specificity of the inactivating reactions is assessed by protection experiments and by mutant to wild-type (WT) comparisons. The scope and limitations of the method are briefly discussed.
Free Keywords:engineered chemical-coupling; cysteine-scanning; binding-site topology
Comment of the Author/Creator:Date: 2002, FEB-NOV
External Publication Status:published
Document Type:Article
Communicated by:N. N.
Affiliations:MPI für Hirnforschung/Neurochemistry (Abt.: Betz)
External Affiliations:ULP, CNRS UMR 7514, Fac Pharm, Chim Bioorgan Lab, F-67401 Illkirch Graffenstaden, France; Texas A&M Univ, Dept Biol, College Stn, TX 77843 USA
Identifiers:ISI:000179631000024 [ID No:1]
ISSN:1079-9893 [ID No:2]
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