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Affinity Capturing and Surface Enrichment of a Membrane Protein Embedded in a Continuous Supported Lipid Bilayer.

ABSTRACT: Investigations of ligand-binding kinetics to membrane proteins are hampered by their poor stability and low expression levels, which often translates into sensitivity-related limitations impaired by low signal-to-noise ratios. Inspired by affinity capturing of water-soluble proteins, which utilizes water as the mobile phase, we demonstrate affinity capturing and local enrichment of membrane proteins by using a fluid lipid bilayer as the mobile phase. Specific membrane-protein capturing and enrichment in a microfluidic channel was accomplished by immobilizing a synthesized trivalent nitrilotriacetic acid (tris-NTA)-biotin conjugate. A polymer-supported lipid bilayer containing His₆-tagged β-secretase (BACE) was subsequently laterally moved over the capture region by using a hydrodynamic flow. Specific enrichment of His₆-BACE in the Ni²⁺-NTA-modified region of the substrate resulted in a stationary three-fold increase in surface coverage, and an accompanied increase in ligand-binding response.

SUBMITTER: Gunnarsson A

PROVIDER: S-EPMC5062009 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Affinity Capturing and Surface Enrichment of a Membrane Protein Embedded in a Continuous Supported Lipid Bilayer.

Gunnarsson Anders A Simonsson Nyström Lisa L Burazerovic Sabina S Gunnarsson Jenny J Snijder Arjan A Geschwindner Stefan S Höök Fredrik F

ChemistryOpen 20160822 5

Investigations of ligand-binding kinetics to membrane proteins are hampered by their poor stability and low expression levels, which often translates into sensitivity-related limitations impaired by low signal-to-noise ratios. Inspired by affinity capturing of water-soluble proteins, which utilizes water as the mobile phase, we demonstrate affinity capturing and local enrichment of membrane proteins by using a fluid lipid bilayer as the mobile phase. Specific membrane-protein capturing and enric ...[more]

PMID: 27777836

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Project description:Biomolecular devices based on photo-responsive proteins have been widely proposed for medical, electrical, and energy storage and production applications. Also, bacteriorhodopsin (bR) has been extensively applied in such prospective devices as a robust photo addressable proton pump. As it is a membrane protein, in principle, it should function most efficiently when reconstituted into a fully fluid lipid bilayer, but in many model membranes, lateral fluidity of the membrane and protein is sacrificed for electrochemical addressability because of the need for an electroactive surface. Here, we reported a biomolecular photoactive device based on light-activated proton pump, bR, reconstituted into highly fluidic microcavity-supported lipid bilayers (MSLBs) on functionalized gold and polydimethylsiloxane cavity array substrates. The integrity of reconstituted bR at the MSLBs along with the lipid bilayer formation was evaluated by fluorescence lifetime correlation spectroscopy, yielding a protein lateral diffusion coefficient that was dependent on the bR concentration and consistent with the Saffman-Delbrück model. The photoelectrical properties of bR-MSLBs were evaluated from the photocurrent signal generated by bR under continuous and transient light illumination. The optimal conditions for a self-sustaining photoelectrical switch were determined in terms of protein concentration, pH, and light switch frequency of activation. Overall, a significant increase in the transient current was observed for lipid bilayers containing approximately 0.3 mol % bR with a measured photo-current of 250 nA/cm2. These results demonstrate that the platforms provide an appropriate lipid environment to support the proton pump, enabling its efficient operation. The bR-reconstituted MSLB model serves both as a platform to study the protein in a highly addressable biomimetic environment and as a demonstration of reconstitution of seven-helix receptors into MSLBs, opening the prospect of reconstitution of related membrane proteins including G-protein-coupled receptors on these versatile biomimetic substrates.

Dataset Information

Affinity Capturing and Surface Enrichment of a Membrane Protein Embedded in a Continuous Supported Lipid Bilayer.

Publications

Affinity Capturing and Surface Enrichment of a Membrane Protein Embedded in a Continuous Supported Lipid Bilayer.

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