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Mapping a Ligand Binding Site Using Genetically Encoded Photoactivatable Crosslinkers

G protein-coupled receptor (GPCR) signaling complexes are important for mediating many different biological processes. Uncovering the mechanism for how a ligand triggers a GPCR to elicit a specific response is an active area of research. One step toward understanding this mechanism is through identi... Full description

Journal Title: Methods in Enzymology 2013, Vol.520, p.307-322
Main Author: Grunbeck, Amy
Other Authors: Huber, Thomas , Sakmar, Thomas P
Format: Electronic Article Electronic Article
Language: English
Subjects:
Quelle: Gale eBooks
Publisher: United States: Elsevier Science & Technology
ID: ISSN: 0076-6879
Link: https://www.ncbi.nlm.nih.gov/pubmed/23332706
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recordid: cdi_gale_vrl_7002400023
title: Mapping a Ligand Binding Site Using Genetically Encoded Photoactivatable Crosslinkers
format: Article
creator:
  • Grunbeck, Amy
  • Huber, Thomas
  • Sakmar, Thomas P
subjects:
  • Animals
  • Binding site
  • Binding Sites
  • Cellular signal transduction
  • Cross-Linking Reagents - chemistry
  • G proteins
  • GPCR
  • Humans
  • Ligand
  • Membrane proteins
  • Models, Biological
  • Photocrosslink
  • Polymer crosslinking
  • Protein Binding
  • Receptors, G-Protein-Coupled - chemistry
  • Receptors, G-Protein-Coupled - genetics
  • Receptors, G-Protein-Coupled - metabolism
  • Signal transduction
  • Signal Transduction - genetics
  • Signal Transduction - physiology
ispartof: Methods in Enzymology, 2013, Vol.520, p.307-322
description: G protein-coupled receptor (GPCR) signaling complexes are important for mediating many different biological processes. Uncovering the mechanism for how a ligand triggers a GPCR to elicit a specific response is an active area of research. One step toward understanding this mechanism is through identifying a ligand's binding site on a GPCR. We have optimized a targeted photocrosslinking technology to detect the residues in a receptor that are within a precise distance from a bound ligand in the receptor–ligand complex. Here, we describe the method for introducing photoactivable crosslinkers into a GPCR using the amber stop codon suppression technology. In addition, we review the steps to identify the binding site of a fluorescein-tagged peptide ligand and a tritium-labeled small molecule ligand.
language: eng
source: Gale eBooks
identifier: ISSN: 0076-6879
fulltext: fulltext
issn:
  • 0076-6879
  • 1557-7988
url: Link


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descriptionG protein-coupled receptor (GPCR) signaling complexes are important for mediating many different biological processes. Uncovering the mechanism for how a ligand triggers a GPCR to elicit a specific response is an active area of research. One step toward understanding this mechanism is through identifying a ligand's binding site on a GPCR. We have optimized a targeted photocrosslinking technology to detect the residues in a receptor that are within a precise distance from a bound ligand in the receptor–ligand complex. Here, we describe the method for introducing photoactivable crosslinkers into a GPCR using the amber stop codon suppression technology. In addition, we review the steps to identify the binding site of a fluorescein-tagged peptide ligand and a tritium-labeled small molecule ligand.
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subjectAnimals ; Binding site ; Binding Sites ; Cellular signal transduction ; Cross-Linking Reagents - chemistry ; G proteins ; GPCR ; Humans ; Ligand ; Membrane proteins ; Models, Biological ; Photocrosslink ; Polymer crosslinking ; Protein Binding ; Receptors, G-Protein-Coupled - chemistry ; Receptors, G-Protein-Coupled - genetics ; Receptors, G-Protein-Coupled - metabolism ; Signal transduction ; Signal Transduction - genetics ; Signal Transduction - physiology
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abstractG protein-coupled receptor (GPCR) signaling complexes are important for mediating many different biological processes. Uncovering the mechanism for how a ligand triggers a GPCR to elicit a specific response is an active area of research. One step toward understanding this mechanism is through identifying a ligand's binding site on a GPCR. We have optimized a targeted photocrosslinking technology to detect the residues in a receptor that are within a precise distance from a bound ligand in the receptor–ligand complex. Here, we describe the method for introducing photoactivable crosslinkers into a GPCR using the amber stop codon suppression technology. In addition, we review the steps to identify the binding site of a fluorescein-tagged peptide ligand and a tritium-labeled small molecule ligand.
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