Multilayered Lipid Membrane Stacks for Biocatalysis Using Membrane Enzymes

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.

Model Lipid Membranes Assembled from Natural Plant Thylakoids into 2D Microarray Patterns as a Platform to Assess the Organization and Photophysics of Light‐Harvesting Proteins

“…Random (Brownian) motion in 2D is expected to lead to an overall migration of membrane proteins from a high concentration in the thylakoids to a lower concentration in the hybrid membranes, [ 27 ] tending toward a lower energy state of dynamic equilibrium. This interpretation is in agreement with other studies which observed lipid and cofactor diffusion between multilayers of stacked model membranes [ 28 ] and bears similarities to the dynamic protein rearrangements which occur in natural thyalkoids. [ 7 , 10 ]…”

Model Lipid Membranes Assembled from Natural Plant Thylakoids into 2D Microarray Patterns as a Platform to Assess the Organization and Photophysics of Light‐Harvesting Proteins

“…Random (Brownian) motion in 2D is expected to lead to an overall migration of membrane proteins from a high concentration in the thylakoids to a lower concentration in the hybrid membranes, [ 27 ] tending toward a lower energy state of dynamic equilibrium. This interpretation is in agreement with other studies which observed lipid and cofactor diffusion between multilayers of stacked model membranes [ 28 ] and bears similarities to the dynamic protein rearrangements which occur in natural thyalkoids. [ 7 , 10 ]…”

Understanding the photophysics and structural organization of photosynthetic proteins using model lipid membranes assembled from natural plant thylakoids

“…It is unlikely that proteins could transfer vertically between stacked lipid multilayers (because this would expose hydrophobic portions of the protein to the polar solvent which would be thermodynamically unfavourable), so it seems probable that proteins and thylakoid lipids migrate laterally between lipid bridges that connect the ruptured thylakoids to patches of putative hybrid membrane. This interpretation is in agreement with other studies which observed lipid and cofactor diffusion between multilayers of stacked model membranes 63 and bears similarities to the dynamic protein rearrangements which occur in natural thyalkoids. 12,24 After hybrid membrane formation and stabilization (washing to remove any loosely associated material), photobleaching experiments suggested that the majority of proteins and lipids were able to diffuse laterally within the membrane (Supplementary Figure S7), suggesting that the lateral motion of proteins/lipids was not significantly impeded by interactions with the substrate.…”

Electrochemical Biosensors Based on Membrane-Bound Enzymes in Biomimetic Configurations

“…The cyclic voltammograms (CVs) performed showed a linear increase in the MQ7 current and in its peak area with the number of lipid bilayers deposited on the electrode, indicating an increase in the number of quinones that interact with the electrode. The same behavior was observed for UQ10 [ 68 ]. In the CVs, electrocatalytic H 2 oxidation coincided with the ubiquinol oxidation peak, confirming that the electronic transfer between MBH and the electrode was mediated by the quinone pool ( Figure 4 b), with a considerable stability of the immobilized membrane enzyme ( Figure 4 c).…”

Model Lipid Membranes Assembled from Natural Plant Thylakoids into 2D Microarray Patterns as a Platform to Assess the Organization and Photophysics of Light‐Harvesting Proteins

“…Random (Brownian) motion in 2D is expected to lead to an overall migration of membrane proteins from a high concentration in the thylakoids to a lower concentration in the hybrid membranes, [ 27 ] tending toward a lower energy state of dynamic equilibrium. This interpretation is in agreement with other studies which observed lipid and cofactor diffusion between multilayers of stacked model membranes [ 28 ] and bears similarities to the dynamic protein rearrangements which occur in natural thyalkoids. [ 7 , 10 ]…”

Understanding the photophysics and structural organization of photosynthetic proteins using model lipid membranes assembled from natural plant thylakoids

“…It is unlikely that proteins could transfer vertically between stacked lipid multilayers (because this would expose hydrophobic portions of the protein to the polar solvent which would be thermodynamically unfavourable), so it seems probable that proteins and thylakoid lipids migrate laterally between lipid bridges that connect the ruptured thylakoids to patches of putative hybrid membrane. This interpretation is in agreement with other studies which observed lipid and cofactor diffusion between multilayers of stacked model membranes 63 and bears similarities to the dynamic protein rearrangements which occur in natural thyalkoids. 12,24 After hybrid membrane formation and stabilization (washing to remove any loosely associated material), photobleaching experiments suggested that the majority of proteins and lipids were able to diffuse laterally within the membrane (Supplementary Figure S7), suggesting that the lateral motion of proteins/lipids was not significantly impeded by interactions with the substrate.…”

Electrochemical Biosensors Based on Membrane-Bound Enzymes in Biomimetic Configurations

“…The cyclic voltammograms (CVs) performed showed a linear increase in the MQ7 current and in its peak area with the number of lipid bilayers deposited on the electrode, indicating an increase in the number of quinones that interact with the electrode. The same behavior was observed for UQ10 [ 68 ]. In the CVs, electrocatalytic H 2 oxidation coincided with the ubiquinol oxidation peak, confirming that the electronic transfer between MBH and the electrode was mediated by the quinone pool ( Figure 4 b), with a considerable stability of the immobilized membrane enzyme ( Figure 4 c).…”

Model Lipid Membranes Assembled from Natural Plant Thylakoids into 2D Microarray Patterns as a Platform to Assess the Organization and Photophysics of Light‐Harvesting Proteins

“…Random (Brownian) motion in 2D is expected to lead to an overall migration of membrane proteins from a high concentration in the thylakoids to a lower concentration in the hybrid membranes, [ 27 ] tending toward a lower energy state of dynamic equilibrium. This interpretation is in agreement with other studies which observed lipid and cofactor diffusion between multilayers of stacked model membranes [ 28 ] and bears similarities to the dynamic protein rearrangements which occur in natural thyalkoids. [ 7 , 10 ]…”

Understanding the photophysics and structural organization of photosynthetic proteins using model lipid membranes assembled from natural plant thylakoids

“…It is unlikely that proteins could transfer vertically between stacked lipid multilayers (because this would expose hydrophobic portions of the protein to the polar solvent which would be thermodynamically unfavourable), so it seems probable that proteins and thylakoid lipids migrate laterally between lipid bridges that connect the ruptured thylakoids to patches of putative hybrid membrane. This interpretation is in agreement with other studies which observed lipid and cofactor diffusion between multilayers of stacked model membranes 63 and bears similarities to the dynamic protein rearrangements which occur in natural thyalkoids. 12,24 After hybrid membrane formation and stabilization (washing to remove any loosely associated material), photobleaching experiments suggested that the majority of proteins and lipids were able to diffuse laterally within the membrane (Supplementary Figure S7), suggesting that the lateral motion of proteins/lipids was not significantly impeded by interactions with the substrate.…”

Electrochemical Biosensors Based on Membrane-Bound Enzymes in Biomimetic Configurations

“…The cyclic voltammograms (CVs) performed showed a linear increase in the MQ7 current and in its peak area with the number of lipid bilayers deposited on the electrode, indicating an increase in the number of quinones that interact with the electrode. The same behavior was observed for UQ10 [ 68 ]. In the CVs, electrocatalytic H 2 oxidation coincided with the ubiquinol oxidation peak, confirming that the electronic transfer between MBH and the electrode was mediated by the quinone pool ( Figure 4 b), with a considerable stability of the immobilized membrane enzyme ( Figure 4 c).…”