Raw DEER data for the beta1-adrenergic receptor in lipid nanodiscs

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 b₁-adrenergic receptor (b₁AR), remains unclear. Here we have examined the influence of the membrane mimetic environment on the dimerization propensity of b₁AR using a combination of pulsed Q-band double electron-electron resonance (DEER) EPR spectroscopy and single-molecule fluorescence brightness measurements in an Anti-Brownian ELektrokinetic (ABEL) trap. While b₁AR is predominantly monomeric in docecyl-b-D-maltoside (DDM) micelles, reconstitution of b₁AR 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 b₁AR molecules in large (~12.5 nm diameter) nanodiscs. Addition of cholesteryl hemissucinate, an analog of cholesterol, suppresses b₁AR dimerization in lipid nanodiscs, recapitulating the behavior in DDM micelles. This work provides quantitative evidence that b₁AR possesses an intrinsic, membrane sensitive predisposition for dimerization, and highlights the importance of spatial membrane constraints in the modulation of class A GPCR dimerization.