Figure 1.
MT1 expression yields obtained with ten combinations of cDNA constructs and host systems.
Expression levels of the human MT1 receptor were assessed using a [3H]-melatonin ligand binding assay. N: number of independent experiments. Schematic representations of the evaluated expression vectors use the following abbreviations: MT1, human MT1 receptor; His, 10-histidine tag; attB1 and attB2, recombination sites of the Gateway system; Tev, tobacco etch virus protease cleavage site; Trx protein, thioredoxin protein; α-F, sequence signal of the Saccharomyces cerevisiae α-Factor; Flag, flag-epitope tag; Biotin, biotinylation domain from Propionibacterium shermanii; Gαi1, αi1 subunit of G protein; ss, signal sequence from influenza hemagglutinin gene; YFP, yellow fluorescent protein; and 2-Strep, double Strep tag.
Figure 2.
Detergent screening for MT1 extraction from Pichia pastoris membranes.
P. Pastoris membranes were solubilized in the presence of a panel of detergent concentrations indicated on the figures. Solubilized proteins were then partially purified on Ni-NTA Spin columns, and finally analyzed using a [3H]-melatonin binding assay (A) and analytical size exclusion chromatography (SEC) (B and C). A, MB: P. pastoris membranes expressing MT1 receptor. B and C, Protein absorbance profiles measured at 280 nm; white triangles: MT1 oligomers; black triangles: MT1 monomers.
Figure 3.
Characterization of MT1 samples purified in the presence of CHAPS or Fos14.
P. pastoris membranes were solubilized with 1% CHAPS (A) or 0.25% Fos14 (B), and purified in the presence of the indicated concentration of detergents using a two-step purification approach consisting of anti-flag affinity chromatography followed by size exclusion chromatography. 1 and 5: Size exclusion chromatography profile. Red arrows indicate SEC elution fractions F17, corresponding to MT1 oligomers, and F22, corresponding to MT1 monomers. 2 and 6: SDS-PAGE of SEC elution fractions F17 and F22 colored with Coomassie Blue (left) and revealed by anti-Flag immunodetection (right). 3 and 7: Saturation ligand binding experiments with [3H]-melatonin on SEC elution fraction F17 for CHAPS and F17 or F22 for Fos14. 4 and 8: Negative staining electron microscopy on SEC elution fractions F17 for CHAPS and F22 for Fos14.
Figure 4.
Characterization of samples purified in a mixture of CHAPS and Fos14 detergents.
P. pastoris membranes were solubilized and purified using a two-step purification approach (anti-flag affinity chromatography followed by SEC). A, Samples were solubilized in a mixture of 0.25% Fos14/0.1% CHAPS and purified in presence of 0.1% Fos14/0.1% CHAPS. 1: SDS-PAGE of SEC elution fractions F17 and F22 colored with Coomassie Blue (left) and revealed by anti-Flag immunodetection (right). 2: Saturation ligand binding experiments with [3H]-melatonin on SEC elution fraction F22. 3: Negative staining electron microscopy on SEC elution fraction F22. B, Samples were solubilized in a mixture of 0.25% Fos14/1% CHAPS, purified in the presence of 0.1% Fos14/0.5% CHAPS, and analyzed by negative staining electron microscopy.
Figure 5.
Correlation plots between binding affinities of MT1 receptors in CHO membranes, in P. pastoris membranes, and purified.
A, pKi correlation of MT1 in P. pastoris membranes vs. MT1 in CHO membranes. B, pKi correlation of MT1 purified in Fos14/CHAPS vs. MT1 in CHO membranes. C, pKi correlation of MT1 in P. pastoris membranes vs. MT1 purified in Fos14/CHAPS. Processed data are presented in Table 1. Pearson's correlation analyses revealed r coefficients of 0.908 (p<0.0001, n = 4), 0.840 (p<0.0001, n = 4), and 0.840 (p<0.0001, n = 4) for A, B, and C, respectively.
Table 1.
Comparison of binding affinities (Ki) of MT1 receptors in CHO and Pichia pastoris (P.p.) membranes, and purified in a 0.1% Fos14/0.1% CHAPS mixture.
Figure 6.
Gi coupling of MT1 reconstituted in lipid nanodiscs.
Kinetics of GTPγS binding to Gi in the Gαiβ1γ2 trimer triggered by MT1R-containing nanodiscs in the absence of ligand (open circles) or in the presence of 10 µM of the melatonin agonist (closed circles). The kinetics measured in the presence of empty nanodiscs is given as open squares. These data were treated as described in the Materials and Methods section to extract the basal exchange rates.