A Tethered Bilayer Assembled on Top of Immobilized Calmodulin to Mimic Cellular Compartmentalization

International audienceBACKGROUND: Biomimetic membrane models tethered on solid supports are important tools for membrane protein biochemistry and biotechnology. The supported membrane systems described up to now are composed of a lipid bilayer tethered or not to a surface separating two compartments: a "trans" side, one to a few nanometer thick, located between the supporting surface and the membrane; and a "cis" side, above the synthetic membrane, exposed to the bulk medium. We describe here a novel biomimetic design composed of a tethered bilayer membrane that is assembled over a surface derivatized with a specific intracellular protein marker. This multilayered biomimetic assembly exhibits the fundamental characteristics of an authentic biological membrane in creating a continuous yet fluid phospholipidic barrier between two distinct compartments: a "cis" side corresponding to the extracellular milieu and a "trans" side marked by a key cytosolic signaling protein, calmodulin. METHODOLOGY/PRINCIPAL FINDINGS: We established and validated the experimental conditions to construct a multilayered structure consisting in a planar tethered bilayer assembled over a surface derivatized with calmodulin. We demonstrated the following: (i) the grafted calmodulin molecules (in trans side) were fully functional in binding and activating a calmodulin-dependent enzyme, the adenylate cyclase from Bordetella pertussis; and (ii) the assembled bilayer formed a continuous, protein-impermeable boundary that fully separated the underlying calmodulin (trans side) from the above medium (cis side). CONCLUSIONS: The simplicity and robustness of the tethered bilayer structure described here should facilitate the elaboration of biomimetic membrane models incorporating membrane embedded proteins and key cytoplasmic constituents. Such biomimetic structures will also be an attractive tool to study translocation across biological membranes of proteins or other macromolecules

AC binding to the CaM/tethered lipid bilayer structures.

<p>The different surface constructions (in a, amine-coated surface alone; in b, CaM-coated surface at a density of 45±5 ng/cm², in c and d, CaM-coated surface (at 45±5 ng/cm²) overlaid with a continuous tethered bilayer membrane, <i>t</i>BLM) were incubated with AC (48 nM or 240 nM) in HBS buffer for 30 min, and extensively washed first with HBS buffer, then with 0.1% Triton X-100 and finally with HBS buffer again. In d, the <i>t</i>BLM was disrupted by washing with 0.1% Triton X-100 before incubation with AC. AC binding to different surface constructions was monitored by measuring its enzymatic activity (expressed as initial rate of cAMP formation) with a colorimetric assay.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019101#pone.0019101-Laine1" target="_blank">[19]</a> The results shown are the mean from 6 independent experiments.</p

AC activation by the immobilized CaM.

<p>(A) Initial rates of AC activity (v_i in nmoles of cAMP/min) as a function of AC concentration (nM) in the solution deposited (or injected) on the indicated surfaces: cysteamine coated surface (red curve) and cysteamine-coated surface bearing 45±5 ng/cm² of immobilized CaM (blue curve). The data and standard deviations were from at least six measurements with independent surfaces. (B) AC binding to the immobilized CaM was not impaired by the membrane solubilization procedure. Initial rates of AC activity (v_i in nmoles of cAMP/min) as a function of AC concentration in the solution (nM) deposited (or injected) onto the calmodulin coated amino-surfaces (45±5 ng CaM/cm²), were quantified either after washing of the surface with 5 mL of HBS buffer (white bars) or after washing of the surface with 2 mL of 0.1% Triton X-100 in HBS buffer followed by 5 mL of HBS buffer (blue bars). The enzymatic reaction medium (600 µL) was deposited onto the surface after removal of the HBS buffer and kinetics of Pi production were determined as mentioned above. The average and the standard deviations are from three measurements on independent surfaces.</p

SPR quantification of AC binding to the different type of surfaces.

<p>AC binding to the different type of surfaces: cysteamine monolayer (red curve); cysteamine monolayer partially derivatized with CaM at a surface coverage of 45±5 ng/cm² (blue curve); and tethered bilayer assembled over the CaM coated surface (at 45±5 ng/cm²; green curve). The amounts of AC bound to the different surfaces (in ng/cm²) as a function of the AC concentrations injected onto the surfaces, were determined by SPR spectroscopy. The average and standard deviation values were calculated from three independent measurements.</p

CaM immobilization on cysteamine gold surfaces.

<p>Amount of immobilized CaM per surface (ng/cm²) as a function of the concentration of the CaM solution (µg/mL) injected over the amine-coated gold surface. The protein surface coverage was calculated from SPR measurements; the averages and standard deviations were calculated from 8 independent measurements.</p

Tethered membrane fluidity.

<p>The lipid diffusion coefficients and the mobile fractions of the biomimetic constructions are given as a function of the initial CaM coverage. They are calculated from six independent measurements.</p

Evaluation of the lipid assembly fluidity.

<p>(A) Fluorescence photobleaching images as a function of time for lipid assemblies formed onto cysteamine-coated surface with different CaM coverages (45, 65 and 110 ng/cm²). (B) Fluorescence recovery curves after photobleaching of tethered lipid assemblies built on the surface with different CaM surface coverage: 45, 65 and 110 ng/cm².</p

Scheme of the tethered bilayer formed over CaM molecules immobilized on top of an amine-coated surface.

<p>Scheme of the tethered bilayer formed over CaM molecules immobilized on top of an amine-coated surface.</p