Nanobodies were cloned into the periplasmic expression vector pET26b, containing an amino terminal signal sequence and a carboxy-terminal 8×histidine tag and transformed into BL21(DE3) Rosetta2 E. coli (Novagen). Cells were induced in Terrific Broth at an OD600 of 0.8 with 1 mM IPTG and incubated with shaking at 22 °C for 24 hours. Periplasmic protein was obtained by osmotic shock and the nanobodies were purified using nickel–nitrilotriacetic acid (Ni-NTA) chromatography9 (link). For crystallography, Nb6B9 was digested overnight with 1:50 (w/w) carboxypeptidase A (Sigma) to remove the His tag, then purified by size exclusion chromatography over a Sephadex S200 size exclusion column.
Surface plasmon resonance experiments were conducted with a Biacore T100 at 25 °C. Protein concentrations were determined by 280 nm absorbance with a Nanodrop2000 spectrometer (Thermo Scientific). Biotinylated BI167107-bound β2AR was immobilized on an SA sensorchip (GE) at an Rmax of approximately 40 response units (RU). Biotinylated tiotropium-bound M2 muscarinic receptor was immobilized with an RU value matching that of the reference surface to control for nonspecific binding. Measurements were made using serial dilutions of Nb80 or Nb6B9 in HBSM (10 mM HEPES pH 7.4, 150 mM NaCl, 0.01% MNG) using single-cycle kinetics. All data were analyzed with the Biacore T100 evaluation software version 2.0 with a 1:1 Langmuir binding model.
Radioligand binding assays were performed using purified β2AR reconstituted into HDL particles comprised of apolipoprotein A1 and a 3:2 (mol:mol) mixture of POPC:POPG lipid22 (link). Binding experiments with G-protein were performed as previously described8 (link). Binding reactions were 500 μL in volume, and contained 50 fmol functional receptor, 0.5 nM 3H dihydroalprenolol (3H-DHA), 100 mM NaCl, 20 mM HEPES pH 7.5, 0.1% bovine serum albumin, and ligands and nanobodies as indicated. Reactions were mixed, then incubated for four hours at room temperature prior to filtration with a Brandel 48-well harvester onto a filter pre-treated with 0.1% polyethylenimine. Radioactivity was measured by liquid scintillation counting. All measurements were performed in triplicate, and are presented as means ± SEM.
Surface plasmon resonance experiments were conducted with a Biacore T100 at 25 °C. Protein concentrations were determined by 280 nm absorbance with a Nanodrop2000 spectrometer (Thermo Scientific). Biotinylated BI167107-bound β2AR was immobilized on an SA sensorchip (GE) at an Rmax of approximately 40 response units (RU). Biotinylated tiotropium-bound M2 muscarinic receptor was immobilized with an RU value matching that of the reference surface to control for nonspecific binding. Measurements were made using serial dilutions of Nb80 or Nb6B9 in HBSM (10 mM HEPES pH 7.4, 150 mM NaCl, 0.01% MNG) using single-cycle kinetics. All data were analyzed with the Biacore T100 evaluation software version 2.0 with a 1:1 Langmuir binding model.
Radioligand binding assays were performed using purified β2AR reconstituted into HDL particles comprised of apolipoprotein A1 and a 3:2 (mol:mol) mixture of POPC:POPG lipid22 (link). Binding experiments with G-protein were performed as previously described8 (link). Binding reactions were 500 μL in volume, and contained 50 fmol functional receptor, 0.5 nM 3H dihydroalprenolol (3H-DHA), 100 mM NaCl, 20 mM HEPES pH 7.5, 0.1% bovine serum albumin, and ligands and nanobodies as indicated. Reactions were mixed, then incubated for four hours at room temperature prior to filtration with a Brandel 48-well harvester onto a filter pre-treated with 0.1% polyethylenimine. Radioactivity was measured by liquid scintillation counting. All measurements were performed in triplicate, and are presented as means ± SEM.
