FRET-based measurement of GPCR conformational changes

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• G protein-coupled receptors (GPCRs) undergo agonist-dependent interactions with kinases and arrestins.
• Conformational changes in transmembrane domains upon ligand binding are transmitted to the GPCR C-terminus.
• Förster resonance energy transfer (FRET) is a technique to measure distances between protein domains using fluorescent labels.

Purpose of the Study:

• To develop and apply a novel FRET technique for probing conformational changes in GPCRs.
• To investigate ligand-induced distance changes between the beta(2) adrenoceptor (beta(2)-AR) transmembrane helix 6 and its C-terminus.

Main Methods:

• Developed a FRET strategy using site-specific donor (LumioGreen) and acceptor (Alexa Fluor 568) fluorophores.
• Introduced orthogonal labeling chemistries via mutagenesis (FlAsH site) and cysteine modification (C265).
• Analyzed FRET data to determine distances within the beta(2)-AR.

Main Results:

• Established a FRET technique for precise labeling of GPCRs.
• Measured an average distance of 62 Å between TM6 and the C-terminus of beta(2)-AR.
• Observed ligand-specific conformational changes, indicating an extended and unstructured C-terminus.

Conclusions:

• The beta(2)-AR C-terminus is largely extended and unstructured.
• Ligand binding induces conformational changes relevant to arrestin interactions.
• The developed FRET technique is broadly applicable to GPCRs and other membrane proteins for conformational studies.