ArticleJournal of the American Chemical Society2025
Triple Labeling Resolves a GPCR Intermediate State by Using Three-Color Single Molecule FRET.
Article in Journal of the American Chemical Society, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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Who cites it
5 citing papers in PubMed.
- Site-Specific Protein Bioconjugation Through Cellular Incorporation of Noncanonical Amino Acids.Angewandte Chemie (International ed. in English) · 2026Review
- ATP-driven conformational dynamics reveal hidden intermediates in a heterodimeric ABC transporter.eLife · 2026Article
- Three-color single-molecule fluorescence resonance energy transfer to study macromolecular dynamics.Current opinion in structural biology · 2026Review
- Single-Molecule Forster Resonance Energy Transfer With a Minimalistic 3D-Printed Setup and Dyes in the Blue-Green Spectral Region.Chemphyschem : a European journal of chemical physics and physical chemistry · 2026Article
- Three-color single-molecule maximum likelihood analysis of folding and binding of diffusing molecules.Biophysical journal · 2025Article
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Authors and funding
8 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The correlation of individual conformational changes in dynamic protein complexes remains challenging as most structural methods rely on averaged information over a large number of molecules. Single molecule FRET is a powerful tool for monitoring such conformational changes. When performed using three distinct probes, it enables the correlation of domain movements by providing up to three simultaneous distance measurements with high temporal resolution. Nevertheless, a major challenge lies in the site-specific attachment of three probes to unique positions within the target protein. Here, we propose an orthogonal triple-labeling strategy that is not compromised by native, reactive amino acid functionalities. It combines genetic code expansion and bioorthogonal labeling of two different noncanonical amino acids with an enzymatic self-labeling SNAP tag. We demonstrate its application by establishment of a 3-color sensor on the human metabotropic glutamate receptor 2, a dimeric, multidomain G protein-coupled neuroreceptor, and describe a previously unknown conformational intermediate state using 3-color single molecule FRET.
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Registered trials
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