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Related Concept Videos

Ligand Binding Sites02:40

Ligand Binding Sites

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
Labeling DNA Probes03:31

Labeling DNA Probes

DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
Ligand Binding and Linkage00:49

Ligand Binding and Linkage

Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...

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Related Experiment Video

Updated: May 15, 2026

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells
14:02

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

Published on: April 9, 2018

Mapping a ligand binding site using genetically encoded photoactivatable crosslinkers.

Amy Grunbeck1, Thomas Huber, Thomas P Sakmar

  • 1Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, USA.

Methods in Enzymology
|January 22, 2013
PubMed
Summary

Researchers optimized photocrosslinking technology to identify ligand binding sites on G protein-coupled receptors (GPCRs). This method precisely maps receptor residues interacting with bound ligands, advancing understanding of GPCR signaling mechanisms.

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Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

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Related Experiment Videos

Last Updated: May 15, 2026

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells
14:02

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

Published on: April 9, 2018

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors
16:16

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

Published on: September 13, 2013

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling
10:49

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Published on: September 20, 2016

Area of Science:

  • Biochemistry and Molecular Biology
  • Pharmacology
  • Structural Biology

Background:

  • G protein-coupled receptors (GPCRs) are crucial for diverse biological processes.
  • Understanding how ligands trigger GPCR responses requires identifying ligand-binding sites.
  • Current methods for mapping receptor-ligand interactions have limitations.

Purpose of the Study:

  • To optimize a targeted photocrosslinking technology for precise mapping of GPCR ligand-binding sites.
  • To describe a method for introducing photoactivable crosslinkers into GPCRs.
  • To demonstrate the application of this technology for identifying binding sites of various ligands.

Main Methods:

  • Utilized amber stop codon suppression technology to introduce photoactivable crosslinkers into GPCRs.
  • Optimized targeted photocrosslinking to detect residues near bound ligands.
  • Applied the method to identify binding sites for a fluorescein-tagged peptide and a tritium-labeled small molecule ligand.

Main Results:

  • Successfully introduced photoactivable crosslinkers into GPCRs using amber stop codon suppression.
  • The optimized photocrosslinking technology accurately detected receptor residues within a precise distance of bound ligands.
  • Demonstrated the method's efficacy in identifying binding sites for different types of ligands.

Conclusions:

  • The described photocrosslinking method provides a powerful tool for precisely mapping GPCR ligand-binding sites.
  • This technique enhances the understanding of GPCR-ligand interactions and signaling mechanisms.
  • The optimized method is applicable to various ligands, facilitating broader research in GPCR pharmacology.