Abstract
Multilayered or stacked lipid membranes are a common principle in biology and have various functional advantages compared to single‐lipid membranes, such as their ability to spatially organize processes, compartmentalize molecules, and greatly increase surface area and hence membrane protein concentration. Here, a supramolecular assembly of a multilayered lipid membrane system is reported in which poly‐l‐lysine electrostatically links negatively charged lipid membranes. When suitable membrane enzymes are incorporated, either an ubiquinol oxidase (cytochrome bo 3 from Escherichia coli) or an oxygen tolerant hydrogenase (the membrane‐bound hydrogenase from Ralstonia eutropha), cyclic voltammetry (CV) reveals a linear increase in biocatalytic activity with each additional membrane layer. Electron transfer between the enzymes and the electrode is mediated by the quinone pool that is present in the lipid phase. Using atomic force microscopy, CV, and fluorescence microscopy it is deduced that quinones are able to diffuse between the stacked lipid membrane layers via defect sites where the lipid membranes are interconnected. This assembly is akin to that of interconnected thylakoid membranes or the folded lamella of mitochondria and has significant potential for mimicry in biotechnology applications such as energy production or biosensing.
Highlights
Supported lipid bilayers (SLBs) have been widely advocated for biotechnological applications and frequently used as modelDouble or multilayered membranes, along with their asso- cell membranes in fundamental studies
Initial structural characterization of the membrane stacks was performed on glass and mica using fluorescence microscopy and atomic force microscopy (AFM), respectively
After addition of PLL, discrete regions with twofold fluorescent intensity are evident (Figure 2A,B). These patches are seen by AFM (Figure 2D), and the height and fluorescence intensity are consistent with the formation of two lipid bilayer membranes on top of each other
Summary
Supported lipid bilayers (SLBs) have been widely advocated for biotechnological applications and frequently used as model. The potential of SLBs to mimic complex multilayer membrane assemblies has been reported,[8] yet the development of consistent methodologies has been limited, and only few model systems are described that investigate protein behavior in multiple mem-. We created multilayers of membrane enzymes in a native-like lipid environment using the LBL assembly of bacterial membrane extracts at gold electrodes. With each deposition of a membrane layer, we see the catalytic activity increasing as the total amount of enzyme on the surface increases. This biomimetic system demonstrates how the stacking of membranes can proportionally increase the concentration of active membrane proteins at surfaces
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