Dimerization propensity of the β₁-adrenergic receptor in lipid nanodiscs probed by DEER and single-molecule...

G protein-coupled receptors (GPCRs) comprise a large class of membrane proteins that mediate cellular responses to a wide range of external signals and as such constitute major drug targets. While oligomerization has been shown to play a well-established role in modulating signaling for class C GPCRs (e.g., the glutamate and GABA receptors), the functional relevance of oligomerization for class A receptors, such as the β₁-adrenergic receptor (β₁AR), remains unclear. Here, we have examined the influence of the membrane mimetic environment on the dimerization propensity of β₁AR using a combination of pulsed Q-band double electron-electron resonance spectroscopy and single-molecule fluorescence brightness measurements in an Anti-Brownian Elektrokinetic trap. While β₁AR is predominantly monomeric in docecyl-β-D-maltoside (DDM) micelles, reconstitution of β₁AR in lipid nanodiscs preferentially favors symmetric parallel dimers. Using nanodiscs of different diameters we observed a clear size-dependent increase in the dimer fraction, reaching over 50% of the β₁AR molecules in large (~12.5 nm diameter) nanodiscs. Addition of cholesteryl hemisuccinate, an analog of cholesterol, suppresses β₁AR dimerization in lipid nanodiscs, recapitulating the behavior in DDM micelles. This work provides quantitative evidence that β₁AR possesses an intrinsic, membrane sensitive predisposition for dimerization, and highlights the importance of spatial membrane constraints in the modulation of class A GPCR dimerization.