Triple Labeling Resolves a GPCR Intermediate State by Using Three-Color Single Molecule FRET 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.

Our new study is out !
We introduce a triple labeling strategy (2 non canonical Amino Acids + 2 click chemistries and 1 SNAP-tag) to triple label a G-protein coupled receptor.
Open access in JACS
pubs.acs.org/doi/10.1021/...

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Check our new preprint ! Triple orthogonal labeling of a GPCR with 2 unnatural aminoacids + 1 SNAP-Tag for 3 color #smFRET. We resolve a new intermediate in the activation of metabotropic glutamate receptor 2.
#GPCR #clickchemistry #singlemolecules
www.biorxiv.org/content/10.1...

Structural basis of orientated asymmetry in a mGlu heterodimer - Nature Communications Metabotropic glutamate receptors exist as both homo- and heterodimers. Here, the authors report the mGlu2-4 receptor structures in the inactive, intermediate and active states, and reveal the reason f...

Our new study is out ! We combined #cryoEM and #smFRET to reveal the structural basis of the activation of an heterodimeric #GPCR, the metabotropic glutamate receptor mGluR2-4.
Fantastic collaboration with the teams of Jean-Philippe Pin, Yan Zhang & Jianfeng Liu.
www.nature.com/articles/s41...