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.