Concentrating Membrane Proteins Using Asymmetric Traps and AC Electric Fields (original) (raw)
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Artificial membrane systems allow researchers to study the structure and function of membrane proteins in a matrix that approximates their natural environment and to integrate these proteins in ex vivo devices such as electronic biosensors, thin-film protein arrays, or biofuel cells. Given that most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In this work, we explored the role of the bilayer surface charge in determining transmembrane protein orientation and functionality during formation of proteoliposomes. We reconstituted a model vectorial ion pump, proteorhodopsin, in liposomes of opposite charges and varying charge densities and determined the resultant protein orientation. Antibody-binding assay and proteolysis of proteoliposomes showed physical evidence of preferential orientation, and functional assays verified the vectorial nature of ion transport in this system. Our results indicate that the manipulation of lipid composition can indeed control orientation of an asymmetrically charged membrane protein, proteorhodopsin, in liposomes.
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Membrane proteins are a class of nanoscopic entities that control the matter, energy, and information transport across cellular boundaries. Electrostatic interactions are shown to direct the rapid co-assembly of proteorhodopsin (PR) and lipids into long-range crystalline arrays. The roles of inherent charge variations on lipid membranes and PR variants with different compositions are examined by tuning recombinant PR variants with different extramembrane domain sizes and charged amino acid substitutions, lipid membrane compositions, and lipid-to-PR stoichiometric ratios. Rational control of this predominantly electrostatic assembly for PR crystallization is demonstrated, and the same principles should be applicable to the assembly and crystallization of other integral membrane proteins.
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Planar asymmetric lipid bilayers composed of phosphatidylethanolamine and phosphatidylglycerol lipids are transferred onto a gold electrode surface. Lipids containing two saturated, one monounsaturated and two monounsaturated hydrocarbon chains compose the model membranes. Results of electrochemically controlled polarization modulation infrared reflection absorption spectroscopy and quartz crystal microbalance with energy dissipation studies reveal two different types of electric potentialdependent structural rearrangements in the bilayers. They are correlated with the geometry of the lipid molecule. Packing parameter correlates the cross-section area of the hydrophobic and hydrophilic parts of amphiphilic molecules. In bilayers composed of lipids with the packing parameter <1, the hydrocarbon chains are tilted with respect to the bilayer plane and the polar head groups are well hydrated. At a threshold potential an abrupt flow of water through the bilayer is connected with membrane dehydration and upward orientation of the chains. In bilayers composed of lipids with packing parameter !1, electric potentials have negligible effect on the membrane structure. A simple rule correlating the packing parameter with molecular scale changes occurring at electrified membranes has a large diagnostic implication for biomimetic studies and our understanding of molecular processes occurring in biological cell membranes.
Biophysical Journal, 2006
The heterologous expression and purification of membrane proteins represent major limitations for their functional and structural analysis. Here we describe a new method of incorporation of transmembrane proteins in planar lipid bilayer starting from 1 pmol of solubilized proteins. The principle relies on the direct incorporation of solubilized proteins into a preformed planar lipid bilayer destabilized by dodecyl-b-maltoside or dodecyl-b-thiomaltoside, two detergents widely used in membrane biochemistry. Successful incorporations are reported at 20°C and at 4°C with three bacterial photosynthetic multisubunit membrane proteins. Height measurements by atomic force microscopy (AFM) of the extramembraneous domains protruding from the bilayer demonstrate that proteins are unidirectionally incorporated within the lipid bilayer through their more hydrophobic domains. Proteins are incorporated at high density into the bilayer and on incubation diffuse and segregate into protein close-packing areas. The high protein density allows high-resolution AFM topographs to be recorded and protein subunits organization delineated. This approach provides an alternative experimental platform to the classical methods of twodimensional crystallization of membrane proteins for the structural analysis by AFM. Furthermore, the versatility and simplicity of the method are important intrinsic properties for the conception of biosensors and nanobiomaterials involving membrane proteins.
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A new concept of solid-supported tethered bilayer lipid membrane (tBLM) for the functional incorporation of membrane proteins is introduced. The incorporated protein itself acts as the tethering molecule resulting in a versatile system in which the protein determines the characteristics of the submembraneous space. This architecture is achieved through a metal chelating surface, to which histidine-tagged (His-tagged) membrane proteins are able to bind in a reversible manner. The tethered bilayer lipid membrane is generated by substitution of protein-bound detergent molecules with lipids using in-situ dialysis or adsorption. The system is characterized by surface plasmon resonance, quartz crystal microbalance, and electrochemical impedance spectroscopy. His-tagged cytochrome c oxidase (CcO) is used as a model protein in this study. However, the new system should be applicable to all recombinant membrane proteins bearing a terminal His-tag. In particular, combination of surface immobilization and membrane reconstitution opens new prospects for the investigation of functional membrane proteins by various surface-sensitive techniques under a defined electric field.
Journal of visualized experiments : JoVE, 2018
Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents. Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol. Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to ...
Electrically Controlling and Optically Observing the Membrane Potential of Supported Lipid Bilayers
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Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors. These versatile model membranes can be made from various components, ranging from simple synthetic phospholipids to complex mixtures of constituents, mimicking the cell membrane with its relevant physiochemical and molecular phenomena. In addition, the high stability of supported lipids bilayers allows for their study via a wide array of experimental probes. In this work, we describe a platform for supported lipid bilayers that is accessible both electrically and optically. We show that the polarization of the supported membrane can be electrically controlled and optically probed using voltage-sensitive dyes. Membrane polarization dynamics is understood through electrochemical impedance spectroscopy and the analysis of the equivalent electrical circuit. We also describe the effect of the conducting electrode layer on ...
Membrane perturbation by an external electric field: a mechanism to permit molecular uptake
European Biophysics Journal, 2007
Electropermeabilisation is a well established physical method, based on the application of electric pulses, which induces the transient permeabilisation of the cell membrane. External molecules, otherwise nonpermeant, can enter the cell. Electropermeabilisation is now in use for the delivery of a large variety of molecules, as drugs and nucleic acids. Therefore, the method has great potential in the fields of cancer treatment and gene therapy. However many open questions about the underlying physical mechanisms involved remain to be answered or fully elucidated. In particular, the induced changes by the effects of the applied field on the membrane structure are still far from being fully understood. The present review focuses on questions related to the current theories, i.e. the basic physical processes responsible for the electropermeabilisation of lipid membranes. It also addresses recent findings using molecular dynamics simulations as well as experimental studies of the effect of the field on membrane components.