Radioligand Assay

Bioavailable 25-hydroxyvitamin D was defined as circulating 25-hydroxyvitamin D not bound to vitamin D–binding protein, which is analogous to the definition of bioavailable testosterone.^{28 (link)} Concentrations of bioavailable 25-hydroxyvitamin D were calculated in 1025 homozygotes, for whom we could use a single genotype-specific binding affinity constant on the basis of the presence of a single vitamin D–binding protein variant (see the Methods section in the Supplementary Appendix).^{22 (link)} Calculated concentrations of bioavailable 25-hydroxyvitamin D were validated by direct measurement in a subgroup of homozygous participants with the use of a competitive radioligand-binding assay (see the Methods section and Fig. S2 and S3 in the Supplementary Appendix). Measured and calculated 25-hydroxyvitamin D concentrations were correlated (Pearson's r = 0.81 in 32 Gc1F homozygotes and 0.90 in 13 Gc1S homozygotes; P<0.001 for both relationships) (Fig. S4 in the Supplementary Appendix).

The binding affinity of each FRα mutant was determined by saturation radioligand-binding assay. 40 nM of each mutant in 100 μl binding buffer (25 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Triton X-100) was immobilized in the wells of a protein G-coated 96-well plate (Thermo Scientific) for 40 min. Endogenous ligand was stripped with 100 μl stripping buffer (25 mM acetate acid, pH 3.5, 150 mM NaCl, 0.1% Triton X-100) for 1 min as described previously^{28 (link)}. After neutralizing and washing with 200 μl binding buffer, proteins were incubated for 40 min with 100 μl binding buffer supplemented with the indicated concentrations of [³H]-folic acid (Moravek Biochemicals). FRα-bound [³H]-folic acid was determined by scintillation counting following removal of unbound ligand by two 100-μl washes with binding buffer. K_d was determined by nonlinear regression using GraphPad Prism.

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) chromatography^{9 (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 R_max 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 lipid^{22 (link)}. Binding experiments with G-protein were performed as previously described^{8 (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.