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Photo-Cross-Linkers Incorporated into G-Protein-Coupled Receptors in Mammalian Cells: A Ligand Comparison

Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037 (USA) http://wang.salk.edu/
Angewandte Chemie International Edition (Impact Factor: 11.26). 08/2011; 50(35):8077-81. DOI: 10.1002/anie.201102646
Source: PubMed

ABSTRACT Capturing the right ligand at the right spot: A well-balanced system for non-natural amino acid mutagenesis allows the ligand binding sites of a class II G-protein coupled receptor to be mapped and distinct binding domains to be identified for different ligands in the native environment of mammalian cells.

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Available from: Irene Coin, Aug 28, 2015
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    • "On the other hand, some residues that lie in proximity of the docked ligand did not crosslink, which comprise potential ''false negatives'' (Figure S4D). Many of these residues point away from the ligand, whereas others may have conformations favoring intrareceptor crosslinking or solvent quenching (Coin et al., 2011). Although any crosslinking technique can miss existing proximities, false negatives do not impact the quality of the modeling restraints, which are based solely on reliable positive crosslinking signals. "
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    • "The GFP_Y182 TAG reporter gene was encoded on the same plasmid with the orthogonal tRNA Leu CUA (Figure 6A). Three copies of this tRNA expression cassette driven by the H1 promoter were included to increase the UAG suppression efficiency, as we previously demonstrated in mammalian cells (Coin et al., 2011). A red fluorescent protein, mCherry, was coexpressed with the orthogonal LeuRS through the internal ribosome entry site (IRES) on the other plasmid to indicate successful gene delivery in vivo. "
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    • "The GFP_Y182 TAG reporter gene was encoded on the same plasmid with the orthogonal tRNA Leu CUA (Figure 6A). Three copies of this tRNA expression cassette driven by the H1 promoter were included to increase the UAG suppression efficiency, as we previously demonstrated in mammalian cells (Coin et al., 2011). A red fluorescent protein, mCherry, was coexpressed with the orthogonal LeuRS through the internal ribosome entry site (IRES) on the other plasmid to indicate successful gene delivery in vivo. "
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