Fig 1.
Scheme of a peptide hormone (yellow) bound family B GPCR (blue) in lipid bilayer (grey).
Fig 2.
Scheme of a receptor (blue) incorporated in a nanodisc.
A nanodisc is a lipid bilayer (grey) surrounded by two membrane scaffold proteins (green).
Fig 3.
The purification method of GLP1R using nanodiscs.
GLP1R tagged with the 1D4 epitope is expressed in a mammalian expression system; membrane fractions are isolated and then solubilized using detergent; solubilized membrane fractions are incubated with phospholipid and membrane scaffold proteins (MSPs); Bio-Beads are added to remove detergents and to initiate nanodisc assembly; GLP1R incorporated in nanodiscs are bound to antibody-conjugated resin and unbound nanodiscs or non-GLP1R components are washed off; 1D4 peptide was used to elute purified GLP1R-ND.
Fig 4.
Fluorescently labeled peptides for ligand binding assays.
(A) Sequences of FAM-labeled GLP-1-(7–37) and Ex-4; (B) Chemical structure of the fluorescent dye 5(6)-carboxyfluorescein (FAM); (C) Chemical structure of BODIPY-FL-GTPγS.
Fig 5.
Expression and purification of GLP1R.
(A) Western blot showing the transfection and expression of GLP1R in HEK293S cells. The two major SDS-resistant bands at ~50 kDa and ~100 kDa belong to the monomer and dimer states of GLP1R; (B) UV-visible spectrum of purified GLP1R in nanodiscs.
Fig 6.
(A) SDS-PAGE gel of purified GLP1R-ND, with two major bands at ~28 kDa and ~50 kDa representing MSP and GLP1R respectively; (B) Transmission electron microscopy (TEM) images of GLP1R-ND. The average size is ~18 nm; (C) Mass-based size distribution of isolated GLP1R-ND and wash-off unbound nanodiscs collected during chromatographic purification measured by dynamic light scattering (DLS).
Fig 7.
Ligand binding activity of GLP1R-ND.
Titration curves of FAM labeled (A) GLP-1(7–37) and (B) Ex-4 with GLP1R incorporated in nanodiscs (GLP1R-ND) and with nanodiscs without the receptor (empty-ND). The FAM labeled peptide concentration was kept constant at 55 nM. The fluorescence was monitored at excitation/emission 497 nm/518 nm with slit widths 5 nm/5 nm. Each data point is an average of three anisotropy values measured using three distinct preparations of purified receptor with standard deviation shown as errors.
Fig 8.
(A) The fluorescence intensity monitored at excitation/emission 500 nm/512 nm with slit widths 2.5 nm/5 nm for the reaction mixture of BODIPY-FL-GTPγS and Gs: addition of ligand GLP-1 alone shows no Gs activation (black), addition of GLP1R-ND alone shows an increase in fluorescence intensity, indicating the basal activity (brown), and addition of GLP1R-ND together with the GLP-1-(7–37) ligand (red) and addition of GLP1R-ND together with the Ex-4 ligand (blue) show an increase in intensity above basal level; (B) The activation of Gs by GLP1R-ND upon binding to GLP-1 and Ex-4 after subtraction of the basal activity.