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Structure of voltage-dependent anion channel-tethered bilayer lipid membranes determined using neutron reflectivity.

ABSTRACT: Neutron reflectivity (NR) has emerged as a powerful technique to study the structure and behavior of membrane proteins at planar lipid interfaces. Integral membrane proteins (IMPs) remain a significant challenge for NR owing to the difficulty of forming complete bilayers with sufficient protein density for scattering techniques. One strategy to achieve high protein density on a solid substrate is the capture of detergent-stabilized, affinity-tagged IMPs on a nitrilotriacetic acid (NTA)-functionalized self-assembled monolayer (SAM), followed by reconstitution into the lipids of interest. Such protein-tethered bilayer lipid membranes (ptBLMs) have the notable advantage of a uniform IMP orientation on the substrate. Here, NR is used to provide a structural characterization of the ptBLM process from formation of the SAM to capture of the detergent-stabilized IMP and lipid reconstitution. The mitochondrial outer-membrane voltage-dependent anion channel (VDAC), which controls the exchange of bioenergetic metabolites between mitochondria and the cytosol, was used as a model ?-barrel IMP. Molecular dynamics simulations were used for comparison with the experimental results and to inform the parameters of the physical models describing the NR data. The detailed structure of the SAM is shown to depend on the density of the NTA chelating groups. The relative content of detergent and protein in surface-immobilized, detergent-stabilized VDAC is measured, while the reconstituted lipid bilayer is shown to be complete to within a few percent, using the known atomic structure of VDAC. Finally, excess lipid above the reconstituted bilayer, which is of consequence for more indirect structural and functional studies, is shown to be present.

SUBMITTER: Hoogerheide DP

PROVIDER: S-EPMC6317592 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Structure of voltage-dependent anion channel-tethered bilayer lipid membranes determined using neutron reflectivity.

Hoogerheide David P DP Noskov Sergei Yu SY Kuszak Adam J AJ Buchanan Susan K SK Rostovtseva Tatiana K TK Nanda Hirsh H

Acta crystallographica. Section D, Structural biology 20181130 Pt 12

Neutron reflectivity (NR) has emerged as a powerful technique to study the structure and behavior of membrane proteins at planar lipid interfaces. Integral membrane proteins (IMPs) remain a significant challenge for NR owing to the difficulty of forming complete bilayers with sufficient protein density for scattering techniques. One strategy to achieve high protein density on a solid substrate is the capture of detergent-stabilized, affinity-tagged IMPs on a nitrilotriacetic acid (NTA)-functiona ...[more]

PMID: 30605136

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Project description:Interfacing nanoelectronic devices with cell membranes can enable multiplexed detection of fundamental biological processes (such as signal transduction, electrophysiology, and import/export control) even down to the single ion channel level, which can lead to a variety of applications in pharmacology and clinical diagnosis. Therefore, it is necessary to understand and control the chemical and electrical interface between the device and the lipid bilayer membrane. Here, we develop a simple bottom-up approach to assemble tethered bilayer lipid membranes (tBLMs) on silicon wafers and glass slides, using a covalent tether attachment chemistry based on silane functionalization, followed by step-by-step stacking of two other functional molecular building blocks (oligo-poly(ethylene glycol) (PEG) and lipid). A standard vesicle fusion process was used to complete the bilayer formation. The monolayer synthetic scheme includes three well-established chemical reactions: self-assembly, epoxy-amine reaction, and EDC/NHS cross-linking reaction. All three reactions are facile and simple and can be easily implemented in many research labs, on the basis of common, commercially available precursors using mild reaction conditions. The oligo-PEG acts as the hydrophilic spacer, a key role in the formation of a homogeneous bilayer membrane. To explore the broad applicability of this approach, we have further demonstrated the formation of tBLMs on three common classes of (nano)electronic biosensor devices: indium-tin oxide-coated glass, silicon nanoribbon devices, and high-density single-walled carbon nanotubes (SWNT) networks on glass. More importantly, we incorporated alemethicin into tBLMs and realized the real-time recording of single ion channel activity with high sensitivity and high temporal resolution using the tBLMs/SWNT network transistor hybrid platform. This approach can provide a covalently bonded lipid coating on the oxide layer of nanoelectronic devices, which will enable a variety of applications in the emerging field of nanoelectronic interfaces to electrophysiology.

Dataset Information

Structure of voltage-dependent anion channel-tethered bilayer lipid membranes determined using neutron reflectivity.

Publications

Structure of voltage-dependent anion channel-tethered bilayer lipid membranes determined using neutron reflectivity.

Similar Datasets

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