Abstract
Multilayer lipid membranes perform many important functions in biology, such as electrical isolation (myelination of axons), increased surface area for biocatalytic purposes (thylakoid grana and mitochondrial cristae), and sequential processing (golgi cisternae). Here we develop a simple layer-by-layer methodology to form lipid multilayers via vesicle rupture onto existing supported lipid bilayers (SLBs) using poly l-lysine (PLL) as an electrostatic polymer linker. The assembly process was monitored at the macroscale by quartz crystal microbalance with dissipation (QCM-D) and the nanoscale by atomic force microscopy (AFM) for up to six lipid bilayers. By varying buffer pH and PLL chain length, we show that longer chains (≥300 kDa) at pH 9.0 form thicker polymer supported multilayers, while at low pH and shorter length PLL, we create close packed layers (average lipid bilayers separations of 2.8 and 0.8 nm, respectively). Fluorescence recovery after photobleaching (FRAP) and AFM were used to show that the diffusion of lipid and three different membrane proteins in the multilayered membranes has little dependence on lipid stack number or separation between membranes. These approaches provide a straightforward route to creating the complex membrane structures that are found throughout nature, allowing possible applications in areas such as energy production and biosensing while developing our understanding of the biological processes at play.
Highlights
Multilayered lipid membrane assemblies are utilized throughout nature and are involved in a wide range of energy producing pathways, from the double membranes surrounding mitochondria and Gram-negative bacteria to the stacked thylakoid membranes of photosynthetic plant chloroplasts.[1,2] The benefits of multiple membrane structures lie with the ability to amplify and compartmentalize single membrane functions in series
To follow the binding of poly L-lysine (PLL) and the formation of membranes, quartz crystal microbalance with dissipation (QCM-D) was used, a tool that has proved to be fundamental in the understanding of how supported lipid bilayers (SLBs) are formed.[30,31]
In addition to linking the lipid bilayers, the PLL serves as a thin polymer cushion, providing an attractive and biomimetic environment for transmembrane proteins
Summary
Multilayered lipid membrane assemblies are utilized throughout nature and are involved in a wide range of energy producing pathways, from the double membranes surrounding mitochondria and Gram-negative bacteria to the stacked thylakoid membranes of photosynthetic plant chloroplasts.[1,2] The benefits of multiple membrane structures lie with the ability to amplify and compartmentalize single membrane functions in series. To form the second bilayer, a POPC/POPG (3:1) vesicle solution (tip sonicated as above) in buffer (without CaCl2) was added at 0.5 mg/ mL and left to incubate for 1 h followed by rinsing 20 times with buffer. HFMO3 and CYP2D6 proteins were reconstituted into liposomes using biobeads following a previously described protocol by Reed et al.[27] Briefly, 3:1 POPC/POPG lipid solutions were bathsonicated until clarified in a 0.1 mL solution of a 250 mM MOPS pH 7.2 containing 75 mM MgCl2 and 5% (w/v) cholate detergent. The resuspended vesicle solutions containing either MtrCAB, hFMO3, or CYP2D6 were subsequently extruded using an Avanti Polar Lipids Mini Extruder (Alabaster, AL) through a 200 nm membrane This step was to homogenize vesicle sizes from the resuspended solution, aiding planar bilayer formation. UV−vis was used to measure the characteristic adsorption bands of each protein, allowing the determination of the protein concentration in the extruded proteoliposomes via the appropriate extinction coefficient.[27,28] enzyme assays were used to confirm that hFMO3 and CYP2D6 retained their enzymatic activity after reconstitution.[27,29]
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