Functionalized Amphipols: A Versatile Toolbox Suitable for Applications of Membrane Proteins in Synthetic Biology

International audienc

Active plasma membrane P-type H⁺-ATPase reconstituted into nanodiscs is a monomer

Plasma membrane H(+)-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H(+)-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins

Synthesis of a polyhistidine-bearing amphipol and its use for immobilizing membrane proteins

International audienc

Principle of time-resolved Scintillation Proximity Assay (SPA) to quantify stability of high-affinity radio-ligand binding to GPCRs.

<p>A) Receptors are immobilized on the SPA beads. When radioligands bind to immobilized receptors, energy from the radioisotope decay is efficiently transferred to the scintillator contained in the beads, thus generating a detectable light signal. If the buffer, including detergent, causes the loss of receptor affinity to radiolabeled ligand, the signal will start to decrease irreversibly. The decay of high-affinity ligand binding to receptors can be measured when the same assay is counted repeatedly over time. B) Comparison of desalting gravity flow separation columns (blue circles) and SPA (black circles) to measure decrease in binding of [³H]-DPN to KOR over time at room temperature. KOR was solubilized in 1%/0.2% DDM/CHS (w/v) and diluted in the assay to 0.1%/0.02% DDM/CHS (w/v). Specific binding was normalized to initial binding. As control of assay stability, the signal of bead-immobilized [³H]-biotin was measured in parallel (red circles). Error bars are standard error of the mean from four (gravity flow columns) or three (SPA) independent experiments each done in triplicates. To quantify the stability of ligand binding, data was fitted to an exponential decay function (solid and dashed lines).</p

Quantification of high-affinity ligand binding to KOR in detergent micelles or when reconstituted in Nanodiscs.

<p>Quantification of high-affinity ligand binding to KOR in detergent micelles or when reconstituted in Nanodiscs.</p

Pharmacology of KOR in isolated <i>P</i>. <i>pastoris</i> membrane and when reconstituted in Nanodiscs.

<p>A) Saturation binding assay of [³H]-DPN binding to KOR in membrane (black triangles) or KOR reconstituted in Nanodiscs (open triangles). From the specific binding the affinity constant, <i>K</i>_<i>D</i>, of [³H]-DPN binding to KOR was calculated by fitting to a one-site binding model (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150658#pone.0150658.t002" target="_blank">Table 2</a>) B) Competition binding experiments with KOR antagonist naloxone and C) KOR agonist dynorphin A (1–17). Increasing concentrations of ligand competed the binding of 0.8 nM [³H]-DPN binding to KOR in <i>P</i>. <i>pastoris</i> cell membrane (black marks) or reconstituted in Nanodiscs (open marks). The calculated binding constants (<i>K</i>_<i>i</i>) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150658#pone.0150658.t002" target="_blank">Table 2</a>. Data shown are representative experiments of specific binding normalized to initial binding of [³H]-DPN binding to KOR. Data points are shown with standard deviation of triplicate measurements in one assay. All binding experiments were repeated at least three times.</p

Comparison of binding affinities (<i>K</i>_<i>D</i>, <i>K</i>_<i>i</i>) of KOR in <i>P</i>. <i>pastoris</i> membranes or in Nanodiscs.

<p>Comparison of binding affinities (<i>K</i>_<i>D</i>, <i>K</i>_<i>i</i>) of KOR in <i>P</i>. <i>pastoris</i> membranes or in Nanodiscs.</p

Reconstitution of KOR in Nanodiscs.

<p> A) SEC chromatogram (Superdex200) representing UV_{280 nm} traces from resolution of empty Nanodiscs (black dotted line) and of KOR/Nanodisc complexes (black line). KOR/Nanodisc sample eluted as two peaks. The second peak on the chromatogram corresponded to Nanodiscs containing active KOR, confirmed by specific binding of [³H]-DPN (10 nM) to KOR in eluted fractions (red circles). The column was calibrated with standard proteins indicated by arrows. The Stokes diameters of empty Nanodiscs and KOR/Nanodisc complexes were calculated to be 11.9 nm and 14.1 nm respectively. B) SDS-PAGE detection of KOR reconstituted in Nanodiscs. Left: Coomassie brilliant blue staining, KOR (66 kDa) was visible around ≈ 75 kDa while the MSP1E3D1 protein (32.6 kDa) was visible at ≈ 30 kDa. Right: The presence of KOR was confirmed by western blot detection with streptavidin alkaline phosphatase binding to the C-terminal biotin-tag of KOR. Original uncropped and unadjusted gel and blot can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150658#pone.0150658.s002" target="_blank">S2 Fig</a>.</p

Time-resolved stability of KOR in detergent or when reconstituted in Nanodiscs.

<p>Stability of high-affinity ligand binding (10 nM [³H]-DPN ≈ 10 x <i>K</i>_<i>D</i>) to KOR in 0.1%/0.02% DDM/CHS (w/v) micelles (open diamonds) or to KOR reconstituted in Nanodiscs (black diamonds) at 23°C. Specific [³H]-DPN binding was normalized to initial ligand binding at time zero where incubation at RT was initiated. Data are average of at least three experiments each performed in triplicates. Error bars represents standard error of the mean. Data were fitted to a double exponential decay curve to calculate the half-life of high-affinity ligand binding to KOR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150658#pone.0150658.t001" target="_blank">Table 1</a>).</p

Isolated KOR reconstituted in Nanodiscs activated G_i protein.

<p>BODIPY FL GTPγS was used to confirm Gα_i protein activation by KOR after isolation and reconstitution into Nanodiscs. The intrinsic activity of G proteins was measured in the presence of empty Nanodiscs (light grey, Empty ND). Compared to KOR/Nanodisc sample without agonist stimulation (KOR/ND, dark grey), the fluorescence from BODIPY FL GTPγS binding to Gα_i increased by 56% when the KOR/Nanodisc sample was stimulated with 1 μM dynorphin A (1–17) (KOR/ND + Dyn A, dark grey). Error bars represent standard error of the mean between two independent experiments.</p