Using Nanodiscs to Create Water‐Soluble Transmembrane Chemoreceptors Inserted in Lipid Bilayers

Selective allosteric coupling in core chemotaxis signaling complexes

“…Tsr-6H, Tar-6H (11), and membrane scaffold protein MSP1D1E3(-) (13) were purified as described, as were CheA, CheW, and CheY (26). Tar-C and Tsr-C (27) were purified from membrane vesicles isolated from cells producing the respective chemoreceptors at high levels (12) and for which the receptor content was determined by 7% polyacrylamide gel electrophoresis, staining with Coomassie Brilliant Blue, and comparison with purified Tar-6H and Tsr-6H standards. Such receptor-containing membranes were solubilized with n-octyl-β-D-glucoside, mixed with equal-molar N-biotinylaminoethyl methanethiosulfonate (Toronto Research Chemicals Inc.), and incubated on ice in the dark for 1 h. After centrifugation for 17 min, 543,000 × g, 4°C in a TLA 100.4 rotor, the supernatant was loaded on a column of SoftLink Soft Release Avidin Resin (Promega V2012), the column washed with 50 mM Tris·HCl (pH 7.5), 100 mM NaCl, 10% (wt/vol) glycerol, and 25 mM cholate, and Tar-C-Biotin or Tsr-C-Biotin eluted with that buffer containing 5 mM dBiotin.…”

“…Nanodiscs containing Tar-6H or Tsr-6H at approximately three receptor dimers per disc were prepared essentially as previously described (12). Discs containing both Tar and Tsr, either Tar-6H and Tsr-C or Tar-C and Tsr-6H, were prepared similarly except that both purified receptors were provided and Nanodiscs were purified by two sequential columns (Fig.…”

“…Using Nanodiscs, individual core complexes can be reconstituted in vitro as water-soluble, active units (Fig. 1A) and their specific features characterized (7,8,11,12). Nanodiscs are small, ∼100-nm-diameter, plugs of lipid bilayer with the hydrophobic region shielded from the aqueous environment by a protective belt of membrane scaffold protein (13), thereby rendering that small plug of bilayer water soluble.…”

Influence of Membrane Lipid Composition on a Transmembrane Bacterial Chemoreceptor

“…Materials-Membrane scaffold protein MSP1D1(Ϫ) (20) and chemoreceptor Tar with a 6-histidine carboxyl-terminal extension (Tar-6H) plus a QEQE arrangement at the four methyl-accepting sites (21) were purified as described (22). A polar extract of total E. coli lipids, individual natural lipids purified from such an extract, and individual synthetic lipids were purchased in chloroform solutions from Avanti Polar Lipids (Alabaster, AL).…”

Bacterial chemoreceptors: high-performance signaling in networked arrays

“…Individual, membrane-inserted dimers can be isolated from interaction with other receptors by placing them in Nanodiscs (Figure 2), which are definedsize, water-soluble plugs of lipid bilayer surrounded by a protein annulus [38,39]. These isolated dimers are efficiently methylated and the rate of methylation is augmented by attractant binding, demonstrating that individual dimers not only bind ligand, but are capable of transmitting ligand-induced conformational changes to the methylation region of the receptor [40].…”