Abstract
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.
Highlights
Molecular machines, capable of reversible molecular motion or vectorial charge transport in response to optical or electrochemical stimuli, are widely proposed as constituents of Boolean logic gates for high density data storage and processing or for signal processing in imaging and sensing
A new approach to a switchable photoelectric device is described based on photoactivated light-driven proton transfer across bR reconstituted into a microcavity-supported lipid bilayers (MSLBs)
A reliable method for reconstituting this seven-pass protein into the MSLB is described exploiting the LB monolayer over pore assembly followed by proteoliposome fusion
Summary
Capable of reversible molecular motion or vectorial charge transport in response to optical or electrochemical stimuli, are widely proposed as constituents of Boolean logic gates for high density data storage and processing or for signal processing in imaging and sensing. We report on a method of reconstitution of bR at an MSLB using a hybrid two-step method involving fusion of proteoliposomes containing bR to pre-deposited lipid monolayers spanned over aqueous-filled microcavity arrays This method could reliably be used to reconstitute different densities of bR to the MSLB permitting investigation of the biophysical properties of formed artificial membranes and the photoelectrical activity of bR as a function of the concentration. BR-MSLBs were spanned across buffer-filled microcavity arrays using a combination of Langmuir−Blodgett (LB) and vesicle fusion methods, as described previously.[32,33] Briefly, approximately 50 μL of DOPC (1 mg/mL in chloroform) was deposited onto the air−water interface of LB trough (NIMA 102D) and the solvent allowed to evaporate for 15 min. All measurements were conducted in 0.1 M KCl as the supporting electrolyte
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