Fig. 1.
Negatively charged lipids inhibit β2AR−Gi coupling in detergent. a mB−β2AR emission spectra in DDM + CHS micelles (5:1 DDM:CHS mole ratio) in the absence of epinephrine (APO) and in the presence of epinephrine, ± G protein (Gs or Gi3). Arrows point to the lambda max value, i.e. the wavelength where mB emission intensity is greatest. Inset shows how Gs coupling alters the structure of mB−β2AR, highlighting the change in position of monobromobimane (mB) at C265 of transmembrane 6 (TM6) (Inactive: PDB 5JQH74; Active: PDB 3SN669). b Structures of CHS and phospholipids, with net charge indicated. c Effect of phospholipid on mB−β2AR interaction with G protein (Gi1, Gi2, Gi3, and Gs), read out as an increase in lambda max. Interaction was assessed in the absence of lipid (5:1 DDM:CHS mole ratio) and in the presence of POPG, POPS, POPC, or POPE (5:1:1 DDM:CHS:Lipid mole ratio). Multiplicity adjusted P values were computed by two-way ANOVA followed by Dunnett’s post hoc test between indicated groups. d Selected mB−β2AR emission spectra from panel (c). e Effect of CHS on mB−β2AR interaction with 1 μM Gi3. Interaction was assessed in the presence of CHS (5:1:1 DDM:POPE:CHS mole ratio) or in the absence of CHS (5:1 DDM:POPE mole ratio). Multiplicity adjusted P values were computed by two-way ANOVA followed by Sidak’s post hoc test between indicated groups. a–e mB−β2AR concentration is 300 nM. Data are mean of three independent experiments. f β2AR-induced GTP turnover for Gi3 (± 200 μM epinephrine, EPI) in DDM + POPS (5:1 DDM:POPS mole ratio) and in DDM + POPE (5:1 DDM:POPE mole ratio). Free GTP was assayed after 12 min. Luminescence signals were normalized relative to the condition with Gi3 alone (see Supplementary Fig. 4). Data are mean ± s.e.m. of four independent experiments. Multiplicity adjusted P values were computed by two-way ANOVA followed by Sidak’s post hoc test between indicated groups. Source data are provided in the Source Data File