Ned from the National Center for Biotechnology Information (NCBI) protein database. The corresponding DNA sequences were codon optimized for E. coli expression by DNA2.0 and cloned into a pJexpress414 vector. Information about all the sequences is shown in Table S1. RTS 500 ProteoMaster Kits were purchased from Roche Molecular Diagnostics. The fluorescent-labeled antagonists for ADRB2 and DRD1 were purchased from CellAura Technologies.Lipid PreparationSmall unilamellar vesicles of DMPC were prepared by probe sonicating a 25 mg/mL aqueous Finafloxacin web solution of DMPC on ice until optical clarity was achieved, typically for 15 minutes. Two minutes of centrifugation at 13,700 RCF was used to remove any metal contamination from the sonication probe tip. DMPC small unilamellar vesicles were added to the cell-free reaction at a final concentration of 2 mg/mL.Expression, Purification and Solubility Tests 
of GPCR-NLP ComplexesA myriad of commercial cell-free kits, to include mammalian, yeast, 3PO web insect, E. coli and others, are available for providing cell-free expressed proteins. [31,39] For this study preparative 1 mL reactions were carried out using the Roche RTS 500 ProteoMaster Kit to optimize for protein yield combined with functional ligand binding. Lyophilized reaction components (Lysate, Reaction Mix, Amino Acid Mix, and Methionine) were dissolved in reconstitution buffer and combined as specified by the manufacturer. DMPC was used either as non-labeled or as fluorescently labeled by mixing 99.5 DMPC and 0.5 Texas RedH DHPE (Molar concentration). To co-express GPCRs and D49A1, different ratios of plasmids were added to the lysate mixture along with added DMPC vesicles for screening. The reactions were incubated at 30uC overnight. SDS-PAGE was used to confirm the protein yields [30] and determine the optimized plasmid ratios between the D49A1 plasmid and GPCR expression vector. The optimized ratios 100:1 for NK1R: D49A1, 20:1 for ADRB2: D49A1 and 20:1 for DRD1: D49A1 were used to produce GPCR-NLP complexes for characterization. To characterize the solubilities of GPCRs produced by cell-free coexpression method, the relative fluorescence units (RFU) were measured for the supernatant solution after spinning the vials for 10 minutes at an rpm of 13K and then taking the spectrum using a Nanodrop ND-3300. [13]. Immobilized metal affinity chromatography was used to isolate the protein complex of interest (D49A1 or D49A1 associated with GPCR of interest) using the D49A1 encoded His tag. None of the GPCR proteins contained a His tag. The soluble fraction (,1 mL) was mixed with Ni-NTA Superflow resin (1 ml of 50 slurry, Qiagen) according to the manufacturer’s protocol using native purification conditions with the following modifications: 12column volumes of 10 mM imidazole in PBS buffer was used toGPCRs Supported in Nanolipoprotein Discswash the column. A total of 6 mL 1326631 of elution buffer (400 mM imidazole in 
PBS) were used to elute the bound protein in 1-mL aliquots. All of the elution fractions were combined, concentrated and buffer exchanged into TBS using a 100 kDa molecular weight sieve filter (Vivascience) to achieve a final volume of ,200 mL. This material was used for all subsequent characterization assays. The purification quality was confirmed by SDS PAGE. [30].Dot Blot Assays of Fluorescence Labeled Ligands Binding with GPCR-NLP ComplexesA total of 100 mL of 100 nM fluorescence labeled ligands for NK1R, ADRB2 and DRD1 were mixed with 100 mL ,0.2 nM.Ned from the National Center for Biotechnology Information (NCBI) protein database. The corresponding DNA sequences were codon optimized for E. coli expression by DNA2.0 and cloned into a pJexpress414 vector. Information about all the sequences is shown in Table S1. RTS 500 ProteoMaster Kits were purchased from Roche Molecular Diagnostics. The fluorescent-labeled antagonists for ADRB2 and DRD1 were purchased from CellAura Technologies.Lipid PreparationSmall unilamellar vesicles of DMPC were prepared by probe sonicating a 25 mg/mL aqueous solution of DMPC on ice until optical clarity was achieved, typically for 15 minutes. Two minutes of centrifugation at 13,700 RCF was used to remove any metal contamination from the sonication probe tip. DMPC small unilamellar vesicles were added to the cell-free reaction at a final concentration of 2 mg/mL.Expression, Purification and Solubility Tests of GPCR-NLP ComplexesA myriad of commercial cell-free kits, to include mammalian, yeast, insect, E. coli and others, are available for providing cell-free expressed proteins. [31,39] For this study preparative 1 mL reactions were carried out using the Roche RTS 500 ProteoMaster Kit to optimize for protein yield combined with functional ligand binding. Lyophilized reaction components (Lysate, Reaction Mix, Amino Acid Mix, and Methionine) were dissolved in reconstitution buffer and combined as specified by the manufacturer. DMPC was used either as non-labeled or as fluorescently labeled by mixing 99.5 DMPC and 0.5 Texas RedH DHPE (Molar concentration). To co-express GPCRs and D49A1, different ratios of plasmids were added to the lysate mixture along with added DMPC vesicles for screening. The reactions were incubated at 30uC overnight. SDS-PAGE was used to confirm the protein yields [30] and determine the optimized plasmid ratios between the D49A1 plasmid and GPCR expression vector. The optimized ratios 100:1 for NK1R: D49A1, 20:1 for ADRB2: D49A1 and 20:1 for DRD1: D49A1 were used to produce GPCR-NLP complexes for characterization. To characterize the solubilities of GPCRs produced by cell-free coexpression method, the relative fluorescence units (RFU) were measured for the supernatant solution after spinning the vials for 10 minutes at an rpm of 13K and then taking the spectrum using a Nanodrop ND-3300. [13]. Immobilized metal affinity chromatography was used to isolate the protein complex of interest (D49A1 or D49A1 associated with GPCR of interest) using the D49A1 encoded His tag. None of the GPCR proteins contained a His tag. The soluble fraction (,1 mL) was mixed with Ni-NTA Superflow resin (1 ml of 50 slurry, Qiagen) according to the manufacturer’s protocol using native purification conditions with the following modifications: 12column volumes of 10 mM imidazole in PBS buffer was used toGPCRs Supported in Nanolipoprotein Discswash the column. A total of 6 mL 1326631 of elution buffer (400 mM imidazole in PBS) were used to elute the bound protein in 1-mL aliquots. All of the elution fractions were combined, concentrated and buffer exchanged into TBS using a 100 kDa molecular weight sieve filter (Vivascience) to achieve a final volume of ,200 mL. This material was used for all subsequent characterization assays. The purification quality was confirmed by SDS PAGE. [30].Dot Blot Assays of Fluorescence Labeled Ligands Binding with GPCR-NLP ComplexesA total of 100 mL of 100 nM fluorescence labeled ligands for NK1R, ADRB2 and DRD1 were mixed with 100 mL ,0.2 nM.
