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Related Concept Videos

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.
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Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...

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Related Experiment Video

Updated: Jun 27, 2026

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions
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Triple Labeling Resolves a GPCR Intermediate State by Using Three-Color Single Molecule FRET.

Léo Bonhomme1, Ecenaz Bilgen2, Caroline Clerté1

  • 1Centre de Biologie Structurale (CBS), University of Montpellier, CNRS, INSERM, Montpellier 34090, France.

Journal of the American Chemical Society
|May 15, 2025
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Summary

This study introduces a novel triple-labeling method for precisely tracking protein movements. This advance allows researchers to observe dynamic protein conformational changes with unprecedented detail, revealing new insights into complex biological processes.

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Area of Science:

  • Structural Biology
  • Biochemistry
  • Molecular Biophysics

Background:

  • Correlating individual conformational changes in dynamic protein complexes is difficult due to reliance on averaged structural data.
  • Single-molecule Förster Resonance Energy Transfer (smFRET) is effective for monitoring conformational dynamics.
  • Simultaneous distance measurements using three probes in smFRET can correlate domain movements but require site-specific labeling.

Purpose of the Study:

  • To develop an orthogonal triple-labeling strategy for site-specific attachment of three probes.
  • To overcome limitations of native amino acid functionalities in protein labeling.
  • To enable high-resolution, multi-distance monitoring of dynamic protein complexes.

Main Methods:

  • Orthogonal triple-labeling strategy combining genetic code expansion and bioorthogonal labeling.
  • Utilized two distinct noncanonical amino acids and an enzymatic self-labeling SNAP tag.
  • Established a 3-color sensor on the human metabotropic glutamate receptor 2 (a GPCR).

Main Results:

  • Successfully demonstrated site-specific attachment of three probes without interfering with native functionalities.
  • Applied the method to the dimeric, multidomain G protein-coupled neuroreceptor, mGluR2.
  • Identified a previously uncharacterized conformational intermediate state of mGluR2 using 3-color smFRET.

Conclusions:

  • The proposed orthogonal triple-labeling strategy is effective for precise multi-probe labeling of complex proteins.
  • This method overcomes significant challenges in site-specific labeling for advanced smFRET studies.
  • The findings provide new insights into the conformational dynamics of GPCRs, exemplified by mGluR2.