Abstract
BackgroundBiomimetic 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 FindingsWe 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).ConclusionsThe 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.
Highlights
We present an improved model of biomimetic tBLM mimicking the three-dimensional architecture of a genuine biological membrane in that it defines a physical boundary between two distinct compartments, i.e. cis and trans sides (Figure 1)
We established and validated the experimental conditions to assemble such a multilayered structure that preserves the functional activity of CaM and ensures the formation of a continuous yet fluid lipid bilayer acting as a proteinimpermeable barrier between two distinct compartments
CaM was covalently coupled to the amino-grafted surface using EDC activation in the presence of calcium (2 mM) to stabilize its active conformation.[12]
Summary
We present an improved model of biomimetic tBLM mimicking the three-dimensional architecture of a genuine biological membrane in that it defines a physical boundary between two distinct compartments, i.e. cis (or external) and trans (internal) sides (Figure 1) This was achieved by assembling a continuous tethered bilayer over a surface derivatized with the protein calmodulin (CaM) to serve as a specific cytoplasmic marker. 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
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