Size exclusion chromatography

For analytical scale size exclusion chromatography (SEC), approx. 10 – 30 µg of protein in 50 µl SEC buffer were loaded on a analytical scale SEC column (Superdex 200, 3.2/30 increase or Superdex 200, 3.2/30, GE Life Sciences) pre-equilibrated with 2-3 CV of SEC-buffer at a flow rate of 50 – 75 µl/min.

Sample elution was performed at the same flow rate and protein content during elution was traced by absorbance at 280 nm. For preparative SEC, 1 – 9 mg in 500 µl SEC-buffer were loaded on a semi-preparative SEC column (Superdex 200, 10/300, GE Life Sciences) pre-equilibrated with SEC-buffer at a flow rate of 500 µl/min. Sample elution was performed at a flow rate of 500 µl/min and protein content during elution was traced by absorbance at 280 nm. All samples were centrifuged for 10 min at 18,000 x g to ensure removal of potential precipitates. SEC analysis was performed on a semi-automated FPLC system (Äkta purifier, GE Life Sciences) at 16°C.

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3.6.4 Protein quantification

Quantification of total protein concentration by Bradford assay

Protein concentration in the E. coli extract was determined by Bradford assay. Therefore, 10 µl of the sample were incubated with 990 µl of Bradford solution for 3 min at room temperature and absorbance at 595 nm was measured and blank subtracted and compared to a reference curve previously determined with BSA standard solutions.

Quantification of protein and peptide concentration by UV absorbance

The concentration of purified proteins and Endothelin-1 peptide was routinely determined by measuring the light absorbance of the sample at 280 nm and applying the Lambert-Beer law. Extinction coefficients and molecular masses of the respective proteins were calculated from the amino acid sequence, assuming complete oxidation of all cysteine residues (Tab. 3.4).

Tab. 3.4 Molar extinction coefficients for protein and peptide quantification.

Construct Molar extinction coefficient

280 nm (M^-1 cm^-1)

Molecular mass (g mol^-1)

Human - 1AR-sfGFP-His10 84,060 63,314

Human ts- - 1AR-sfGFP-His10 86,580 63,260

Human ts-fl- 1AR-sfGFP-His10 92,330 80,428

Turkey - 1AR-sfGFP-His6 90,590 62,987

Turkey ts- - 1AR-sfGFP-His6 95,310 70,342

Turkey ts- - 1AR-His10 71,555 36,442

Cyclic ET-1 7,240 2,491

ETA-sfGFP-His6 85,145 78,396

ETB-sfGFP-His6 90,520 72,887

Ts-ETB C-sfGFP-His6 84,645 70,313

MSP1E3D1 28,420 31,962

roGFP1-iE 23,380 30,632

DsbA 28,420 24,461

DsbC 17,420 26,978

Quantification of GFP-concentration by fluorescence

Receptor concentration in protein mixtures was routinely determined by the fluorescence intensity of the c-terminal sfGFP-tag. 6 µl of the sample were diluted in a total of 300 µl GFP-buffer in a black 96-well plate and incubated at room temperature for 1 h. Fluorescence emission intensity at 510 nm was measured during excitation at 495 nm on a fluorescence reader (Genius Pro, Tecan) and compared with reference curve previously determined with purified GFP.

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3.6.5 Radioligand filter binding assay

Determination of ligand binding sites

1 - 1000 nM of non-purified receptor (according to its GFP fluorescence) were incubated with 50 - 200 nM ³H-dihydroalprenolol (³H-alprenolol) (Biotrend) or 0.5 nM ¹²⁵I-Tyr13-Endothelin-1 (¹²⁵I-ET-1) for 30 - 60 min at room temperature in binding buffer. GF/B glass fibre filters (Merck-Millipore) were prepared by incubation with 150 µl of 0.3% (w/v) polyethyleneimine for 30 min and subsequent 4-times washing with 150 µl of pre-wash buffer. Incubated samples were applied on the prepared glass fibre filters and soaked through to bind receptor-ligand complexes on the filter membrane. To remove unbound ligand, filters were washed 8 times with ice-cold wash buffer. Filters were collected and retained radioactivity was counted on a liquid scintillation counter (Hidex, Finland) after addition of liquid scintillation cocktail. Unspecific binding was determined by incubation of the receptor with a large excess of cold ligand (4 µM Endothelin-1 or 40 µM alprenolol) for 30-60 min in binding buffer prior to radioligand filter binding assay. Filter assay was performed in a 96-well format on a vacuum manifold (MultiScreen HTS, Merck-Millipore).

To ensure complete determination of ligand binding sites, the receptor concentration was chosen in way that the concentration of ligand binding competent receptor was lower than the concentration of radiolabelled ligand to prevent ligand saturation. If the fraction of ligand binding competent receptor could not be estimated prior to assay performance, suitable conditions had to be determined by titration of the receptor in the radioligand filter-binding assay.

The receptor-bound ligand was determined from the retained radioactivity in the glass fibre filters using the specific activity of the radiolabelled ligand as given from the specifications from the supplier.

Ligand binding sites in the initial sample were then calculated from the sample dilution factor applied in the assay. For analysis of statistical significance, a student t-test (t-test, unpaired, two-tailed) was applied using GraphPad Prism 5.

Determination of ligand affinities

For KD determinations, low concentrations of the non-purified receptors (usually 5 nM for ETB and 50 nM for 1AR) were incubated with increasing concentrations of ³H-alprenolol or ¹²⁵I-ET-1 for 30 - 60 min at room temperature. Unspecific binding was again determined by pre-incubation of the receptor with a large excess of unlabelled ligand and filter-binding assay was performed as described above. A one-site binding model (one-site – total and non-specific binding) was fitted on the obtained data set using GraphPad Prism 5 (GraphPad Software)

For Ki determinations, 100 nM of the non-purified receptor was first incubated with a mixture of 10 nM of ³H-alprenolol and 50 nM of non-labelled alprenolol for 1 h in RA binding buffer at room temperature.

Afterwards, samples were mixed in a one to one ratio with binding buffer with various concentrations of the competitors and incubated for another 60 min at room temperature before ligand separation on a glass fibre filter as described above. A one-site binding model (one site – fit Ki) was fitted on the obtained data set using GraphPad Prism 5. For analysis of statistical significance, a student t-test (t-test, unpaired, two-tailed) was applied using GraphPad Prism 5.

52 Thermostability assays

IMAC purified receptors were incubated for 30 - 45 min at various temperatures using a gradient on a PCR cycler. Samples were centrifuged at 18,000 x g for 10 min at 4°C to remove potential precipitates and remaining ligand binding activity was determined by radioligand filter binding assay as described above. For TM calculation, a dose response model (log (inhibitor) vs. normalized response – variable slope) was fitted on the obtained data set using GraphPad Prism 5.