Construct design and expression
Constructs of all chimeras were synthesized by GenScript and then cloned into a modified pFastBac1 vector (Invitrogen) containing an HA signal sequence and a FLAG tag at the N-terminus, and a 10x His tag at the C-terminus. For A2AAR constructs, fusion proteins in the ICL3 were initially inserted between residues L208 and R222. The C-terminus was truncated to residue A317 to minimize flexibility. For β2AR, fusion proteins in the ICL3 were inserted between residues L230 and K263. The C-terminus was truncated to residue K348. The N-terminus was left intact in both receptors. Recombinant baculoviruses were generated with the Bac-to-Bac system (Invitrogen) and used to infect Sf9 cells. 50 μL of P0 baculovirus was used to infect 5 mL of Sf9 cells at a density of 2-3 × 106 cells/mL. Cells were grown at 27 °C for 48 h prior to being harvested.
For small scale analysis of chimeric receptors, 5 mL of frozen Sf9 cells were thawed in buffer containing 20 mM HEPES pH 7.5, 10 mM NaCl, 10 mM MgCl2, 20 mM KCl, and Protease Inhibitor cocktail tablets (1 tablet per 50 mL buffer, Roche). Dounce homogenization and centrifugation at 14,000 × g was carried out to disrupt intact cells and to remove unwanted soluble and membrane associated proteins, and repeated twice with the addition of 1 M NaCl in the buffer. Remaining membranes containing receptors were resuspended in buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mg/mL iodoacetamide, and an appropriate ligand (1 mM timolol for β2AR constructs and 4 mM theophylline for A2AAR constructs). Membranes were allowed to rock for 30 min at 4 °C, followed by solubilization with 0.5% (w/v) n-Dodecyl-β-D-Maltoside (DDM, Affymetrix) and 0.1% (w/v) cholesteryl hemisuccinate (CHS, Sigma) for 2-4 h at 4 °C. Solubilized membranes were spun down at 14,000 × g for 45 min, and the resulting supernatant was incubated with 50-100 μL TALON IMAC resin (Clontech) overnight in the presence of 20 mM imidazole. TALON resin was spun down at 500 × g, resuspended in buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 25 mM imidazole, and applied to a gravity column (Poly-Prep, Bio-Rad). Protein was washed with an additional 20 column volumes (CV) of the same resuspension buffer, and then eluted with buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 0.05% (w/v) DDM, 0.01% (w/v) CHS, and 250 mM imidazole. Fractions containing protein as determined by colorimetric Bradford assay were pooled together. For A2AAR constructs, 800 mM NaCl was used in place of 150 mM NaCl. All purification buffers were pre-chilled on ice and purifications carried out at 4 °C.
For purifications intended for crystallization trials, the above procedure was used with the following modifications: 1) 1 L of frozen Sf9 cells was used, and TALON resin bed volume was increased to 0.5 mL, typically providing approximately 1-2 mg of purified receptor, 2) for β2AR constructs, 1 mM timolol was present in all buffers during and after solubilization, up until reconstitution in LCP. 20 μL PNGaseF (New England Biolabs) was added after the initial 20 CV wash and allowed to incubate overnight before elution to deglycosylate the receptor. 3) for A2AAR constructs, 4 mM theophylline was present during solubilization, and exchanged for 100 μM ZM241385 on TALON resin and maintained in all subsequent steps up until reconstitution in LCP.
For purifications intended for FRAP studies, the following modifications were made: Packed TALON resin was resuspended in 5 mL wash buffer, followed by the addition of 5-10 μL of 5 mg/mL Cy3-mono NHS ester (GE Healthcare) dissolved in dimethylformamide. Protein was allowed to incubate with the dye at 4 °C for 2-3 h in darkness. Free dye was then removed by flowing additional wash buffer through resin until the pink color could no longer be visually observed in the flowthrough, followed by elution as usual. All steps after dye labeling were carried out in darkness to prevent photobleaching.
SDS-PAGE and immunoblotting
Protein samples were mixed with LDS sample buffer (Novex), separated on 10% Bis-Tris gels (Novex) at 130 volts, and stained with Coomassie SimplyBlue SafeStain (Novex). For Western blots, proteins were transferred from gels to nitrocellulose membranes using the iBlot Dry Blotting System and probed with monoclonal anti-FLAG M2 alkaline phosphatase antibody (Sigma) for at least 1 h. Membranes were then washed in PBS, and antibodies were visualized with SIGMAFAST BCIP/NBT (Sigma).
Analytical size exclusion chromatography
Purified receptor constructs were applied to a Sepax Nanofilm SEC-250 column (4.6 × 250 mm) using an Agilent model 1200 HPLC system at a flowrate of 0.5 mL/min and signal detection set to 350 nm. Prior to sample injection the column was equilibrated in 50 mM HEPES pH 7.5, 500 mM NaCl, 2% (v/v) glycerol, 0.05% (w/v) DDM (Affymetrix) and 0.01% (w/v) CHS (Sigma). Sample reservoirs and column were maintained at 4 °C throughout analysis.
N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) dye (Invitrogen) was dissolved in DMSO (Sigma) at 4 mg/mL as a stock solution for future use. The stock solution was kept at -80°C and diluted 1:40 in dye dilution solution (10 mM buffer, 500 mM NaCl, 10% (v/v) glycerol, 0.025% (w/v) DDM and 0.005% (w/v) CHS) before use. The thermal denaturation assay was performed with total volume of 200 μL sample in a quartz fluorometer cuvette (Starna Cells, Inc., Atascadero, CA). Receptor (4 μg) was diluted in the appropriate buffer solution to a final volume of 200 μL. 5 μL of the diluted dye solution was then added to the protein solution and incubated for 30 min at 4 °C in darkness. The mixed solution was transferred to a cuvette and the data were collected by a Cary Eclipse spectrofluorometer (Varian, USA) with a temperature ramping rate of 2 °C/min. The excitation wavelength was 387 nm and the emission wavelength was 463 nm. All assays were performed over a temperature range starting from 20 °C to 90 °C. The stability data were processed with GraphPad Prism (GraphPad Software, San Diego, CA, USA).
In meso crystallization methods for membrane proteins have been described in detail (Caffrey and Cherezov, 2009
). Protein solution was mixed with a molten lipid mixture of 10:1 (w/w) monoolein:cholesterol, at a ratio of 40% (w/w) protein with 60% (w/w) lipid in a custom syringe mixer. A syringe containing reconstituted LCP was loaded onto an automated crystallization robot (NT8-LCP, Formulatrix) and 35-50 nL of LCP dispensed onto 96-well glass sandwich plates (Marienfeld), and then overlaid with 800 nL of precipitant solution. Drops were manually sealed with a coverslip and incubated and imaged at 20 °C in an automatic incubator/imager, RockImager 1000 (Formulatrix). Precipitants were all from homemade screens made and aliquoted into 96-well plates. Crystals of both β2
AR-BRIL and A2A
AR -BRIL grew within 7 d.
Cy3-labeled protein for LCP-FRAP analysis was reconstituted into LCP and dispensed onto glass sandwich plates as described in the crystallization section. The setup for automated high-throughput LCP-FRAP analysis has been described (Xu et al., 2011a
). Briefly, LCP sandwich plates were loaded onto a custom built LCP-FRAP station consisting of a Zeiss AxioImager A1 fluorescent microscope, a Micropoint dye cell laser (Photonic Instruments), a cooled CCD FireWire camera CoolSnap HQ2 (Photometrics), and an automated XYZ microscope stage MS-2000 (Applied Scientific Instrumentation). Each LCP drop was then bleached by firing 15-20 laser pulses at a 25 Hz pulse rate. Fluorescence images were taken immediately before and after bleaching. After a recovery period of 30 minutes, images were taken again to determine fluorescence recovery, and analyzed by ImagePro Advanced Microscopy Suite (Media Cybernetics).
X-ray data collection
Crystallographic data were collected on the 23ID-B/D beamline (GM/CA CAT) of the Advanced Photon Source at the Argonne National Laboratory using a 10 μm collimated minibeam at a wavelength of 1.0330 Å and a MarMosaic 300 detector. To reduce radiation damage, crystals were translated to a fresh position, if possible, or replaced after collecting 5-10 frames at 3 s exposure and 1° oscillation with unattenuated beam. For details on the A2A
AR-BRIL structure determination, please refer to Liu et al. (2012)