In-plane homogeneity and lipid dynamics in tethered bilayer lipid membranes (tBLMs)

Abstract

Tethered bilayer lipid membranes (tBLMs) were prepared by the self-assembly of thiolated lipidic anchor molecules on gold, followed by phospholipid precipitation via rapid solvent exchange. They were characterized by their in-plane structure, dynamics and dielectric properties. We find that the in-plane homogeneity and resistivity of the tBLMs depend critically on a well-controlled sample environment during the rapid solvent-exchange procedure. The in-plane dynamics of the systems, assessed by fluorescence correlation spectroscopy (FCS) as the diffusivity of free, labeled phospholipid dissolved in the membrane, depend on the density of the lipidic anchors in the bilayer leaflet proximal to the substrate as well as on details of the molecular structure of the anchor lipid. In DOPC tBLMs in which tethers are laterally dilute (sparsely tethered bilayer lipid membranes, stBLMs), measured diffusivities, D ≈ 4 μm2 s−1, are only slightly greater than those reported in physisorbed bilayers (M. Przybylo, J. Sykora, J. Humpolíckova, A. Benda, A. Zan and M. Hof, Langmuir, 2006, 22, 9096–9099). However, when we distinguish label diffusion in the proximal and in the distal bilayer leaflets, we observe distinct diffusivities, D ≈ 2 μm2 s−1 and 7 μm2 s−1, respectively. The value observed in the distal leaflet is identical to that in free membranes. stBLMs completed with phytanoyl lipids (DPhyPC) show consistently lower label diffusivity than those completed with unsaturated chains (DOPC). As the length of the tether chain increases, a reduction in the apparent diffusivity is observed, which we interpret as an increased propensity of the proximal bilayer leaflet to host free lipid. We also investigated preparation conditions that control whether the tBLMs are laterally homogeneous, as assessed by optical microscopy. In laterally heterogeneous bilayers, the label diffusivity varies only by a factor of ~2 to 4, indicating that the regions in the bilayers with different label solubilities do not correspond to distinct phases, such as a fluid phase coexisting with a gel phase.

Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

“…stBLMs incorporate a single lipidic bilayer membrane of controlled composition that is tethered to a planar electrode-a 0.05-m-thick Au film on a glass or Si substrate-via short ethylene oxide anchors (53), thereby providing a hydrated environment on both sides of the membrane. Thus, the bilayer retains its intrinsic fluidity (54), is virtually defect free (52), and is resilient to external manipulations, such as buffer exchanges, as monitored by electrochemical impedance spectroscopy (EIS) (55). In combination, SPR performed on stBLMs offers unique advantages for the study of viral assembly processes on membranes.…”

Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

“…1) incorporates a single lipidic bilayer membrane of wellcontrolled composition that is tethered to a planar electrode-a 0.05-m-thick Au film on a glass or Si substrate-via short ethyleneoxide anchors (31), thereby providing a hydrated environment on both sides of the membrane. Thereby, the bilayer retains its intrinsic fluidity (32), is virtually defect-free (29), and is resilient to external manipulations such as buffer exchanges, as monitored by electrochemical impedance spectroscopy (33). Its lipidic composition can be precisely controlled (34) and, in particular, functionally important lipids such as PI(4,5)P 2 can be incorporated into the membrane.…”

“…This may be important, since binding studies that use soluble short-chain PI(4,5)P 2 tend to underestimate binding (26). On the other hand, the low defect density of stBLMs suppresses inflated readings in SPR assays that can occur when protein adsorbs to membrane defects (32). Here, we used dioleoylglycerophosphocholine (DOPC), instead of the more physiologically relevant palmitoyl-oleoyl-PC (POPC), as a background component for bilayers because it forms membranes of higher quality more consistently than POPC.…”

Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering

“…2) A model that places proteins randomly on the surface (39) predicts mean nearest neighbor distances of 1.6 or 2.25 protein diameters, respectively, for area coverages of 20% (myrG55) or 10% (G55). Although dynamic protein-protein contacts are indeed likely on the fluid lipid bilayer of an stBLM (29), which may result in cis pairing due to PDZ interactions if protein orientation is not appropriately confined, unspecific interactions are rare as each membrane-bound protein still has ample space for lateral diffusion.…”

“…The aforementioned stBLMs feature a nanometer-thick hydrated layer between the solid surface and the lipid bilayer (19,25) that keeps the bilayer in a physiologically relevant inplane fluid state (29). We used purified preparations of the N-myristoylated and unmyristoylated GRASP domain of GRASP55 (residues 1-208).…”