Molecular-scale structural and functional characterization of sparsely tethered bilayer lipid membranes (original) (raw)
Related papers
Formation of tethered bilayer lipid membranes probed by various surface sensitive techniques
Biointerphases, 2009
Tethered bilayer lipid membranes are promising biomimetic architectures. Their formation has been investigated using four different surface sensitive techniques, including optical, acoustic, and electrical methods. The lipid bilayers are built in a two-step procedure; the proximal layer is formed by self-assembly and is then completed to a bilayer by fusion with small vesicles. The different technical approaches revealed specific aspects of the layer formation processes, namely, first a fast adsorption process followed by a longer rearrangement period. Similar phenomena have been observed for the vesicle fusion process. The results allow for a more controlled assembly protocol for the preparation of highly insulating lipid membranes.
A New Lipid Anchor for Sparsely Tethered Bilayer Lipid Membranes
Langmuir, 2009
Mixed self-assembled monolayers (SAMs) of β-mercaptoethanol and the new synthetic lipid 1,2-di-O-palmityl-3-[ω-mercapto-nona(ethylene oxide) glycerol], FC16, were investigated for their ability to form sparsely-tethered bilayer lipid membranes (stBLMs) completed with various phospholipids. We investigated the structural and functional properties of FC16-based stBLMs and compared these to stBLMs prepared using a previously characterized synthetic lipid, 1,2-di-O-myristyl-3-[ω-mercaptohexa(ethylene oxide) glycerol] (WC14). FC16-based stBLMs show increased resistivity to ion transfer and an increase in the submembrane space of ≈ 0.5 nm. Importantly, FC16-based stBLMs formed well-defined, complete bilayers with charged phospholipids such as POPG. In these, POPG, incorporates into the outer monolayer leaflet in the same ratio as in the immersion solution, but is excluded from the inner leaflet. In all cases we investigated thus far, the area densities of the lipids within the bilayers were on average close to those in free bilayer membranes. For charged phospholipids, FC16 appears to provide a distinct advantage over WC14 for the formation of well-defined stBLMs.
Structural Analysis of Tethered Bilayer Lipid Membranes
Langmuir, 2010
Solid supported membrane systems have been established as biomimetic architectures, which allow for the systematic investigation of various membrane-related processes. Especially tethered bilayer lipid membranes have been a successful concept. They consist of a lipid bilayer that is covalently anchored to a solid substrate through a spacer group. The submembrane part, which is defined by the spacer group, is important especially for the biological activity of incorporated membrane proteins. Anchor lipids with different spacer and anchor groups have been synthesized, and the resulting membrane structures have been investigated by neutron reflectivity. The different molecular architectures had a significant effect on both the amount of water incorporated in the spacer region and the electrical properties of the bilayer. A detailed understanding of the structure-function relationship allows for an optimized design of the molecular architecture with respect to possible applications, for example an optimized protein incorporation.
The Journal of Physical Chemistry B, 2007
The formation of tethered lipid bilayer membranes (tBLMs) from unilamelar vesicles of egg yolk phosphatidylcholine (EggPC) on mixed self-assembled monolayers (SAMs) from varying ratios of 6-mercaptohexanol and EO(3)Cholesteryl on gold has been monitored by simultaneous attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS). The influence of the lipid orientation (and hence the anisotropy) of lipids on a gold film on the dichroic ratio was studied by simulations of spectra with a matrix method for anisotropic layers. It is shown that for certain tilt angles of the dielectric tensor of the adsorbed anisotropic layer dispersive and negative absorption bands are possible. The experimental data indicate that the structure of the assemblies obtained varies with varying SAM composition. On SAMs with a high content of EO(3)Cholesteryl, tBLMs with reduced fluidity are formed. For SAMs with a high content of 6-mercaptohexanol, the results are consistent with the adsorption of flattened vesicles, and spherical vesicles have been found in a small range of surface compositions. The kinetics of the adsorption process is consistent with the assumption of spherical vesicles as long-living intermediates for surfaces of a high 6-mercaptohexanol content. No long-living spherical vesicles have been detected for surfaces with a large fraction of EO(3)Cholesteryl tethers. The observed differences between the surfaces suggest that for the formation of tBLMs (unlike supported BLMs) no critical surface coverage of vesicles is needed prior to lipid bilayer formation.
Structure and Properties of Tethered Bilayer Lipid Membranes with Unsaturated Anchor Molecules
Langmuir, 2013
The self-assembled monolayers (SAMs) of new lipidic anchor molecule HC18 [Z-20-(Z-octadec-9-enyloxy)-3, 6,9,12,15,18,22-heptaoxatetracont-31-ene-1-thiol] and mixed HC18/β-mercaptoethanol (βME) SAMs were studied by spectroscopic ellipsometry, contact angle measurements, reflection−absorption infrared spectroscopy, and electrochemical impedance spectroscopy (EIS) and were evaluated in tethered bilayer lipid membranes (tBLMs). Our data indicate that HC18, containing a double bond in the alkyl segments, forms highly disordered SAMs up to anchor/βME molar fraction ratios of 80/20 and result in tBLMs that exhibit higher lipid diffusion coefficients relative to those of previous anchor compounds with saturated alkyl chains, as determined by fluorescence correlation spectroscopy. EIS data shows the HC18 tBLMs, completed by rapid solvent exchange or vesicle fusion, form more easily than with saturated lipidic anchors, exhibit excellent electrical insulating properties indicating low defect densities, and readily incorporate the pore-forming toxin αhemolysin. Neutron reflectivity measurements on HC18 tBLMs confirm the formation of complete tBLMs, even at low tether compositions and high ionic lipid compositions. Our data indicate that HC18 results in tBLMs with improved physical properties for the incorporation of integral membrane proteins (IMPs) and that 80% HC18 tBLMs appear to be optimal for practical applications such as biosensors where high electrical insulation and IMP/peptide reconstitution are imperative.
Stable insulating tethered bilayer lipid membranes
2008
Tethered bilayer lipid membranes have been shown to be an excellent model system for biological membranes. Coupling of a membrane to a solid supports creates a stable system that is accessible for various surface analytical tools. Good electrical sealing properties also enable the use of the membranes in practical sensing applications. The authors have shown that tethered membranes have extended lifetimes up to several months. Air-stability of the bilayer can be achieved by coating the membrane with a hydrogel. The structure of a monolayer and its stability under applied dc potentials have been investigated by neutron scattering.
Nanostructural determination of a lipid bilayer tethered to a gold substrate
The European physical journal. E, Soft matter, 2016
Tethered lipid bilayer membranes (tBLM) are planar membranes composed of free lipids and molecules tethered to a solid planar substrate providing a useful model of biological membranes for a wide range of biophysical studies and biotechnological applications. The properties of the tBLM depend on the free lipids and on the chemistry of the tethering molecules. We present a nanoscale characterization of a tBLM composed of deuterated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (d-DMPC) free lipids, benzyl disulfide undecaethylene glycol phytanol (DLP) tethering molecules, and benzyl disulfiide tetraethylene glycol polar spacer molecules (PSM) used to control the areal density of tethering molecules through coadsorption. The use of selected isotopic substitution provides a way to distinguish the conformation and location of the tethered lipids from the free lipids and to elucidate how the two components influence the structure of the tBLM. These findings provide useful information to op...
Electrochimica Acta, 2019
Two 4-thio pseudo-glycolipids, a disulfide and a thiol, were synthesized as candidates to build tethered bilayer lipid membranes (tBLMs) on gold (111) surface. Monolayers of the disulfide and thiol were built on gold surfaces using the self-assembly and Langmuir-Blodgett (LB) transfer methods. Monolayers were prepared in several solvents and at various temperatures; their quality was assessed by differential capacitance. Best monolayers were achieved when the disulfide was self-assembled in ethanol. The quality of such monolayers was further improved by allowing the assembly to proceed under stirring of the solution at increased temperatures. The charge number per adsorbed molecule and surface concentration of disulfide on gold (111) surface were determined by chronocoulometry. A DPhPC/disulfide tBLM was then built by vesicle fusion of 1,2-diphytanyl-sn-glycero-3-phosphocholine (DPhPC) on top of the disulfide monolayer. The minimum capacitance of the gold electrode with tBLM (1.3 F cm-2 2) was close to the value of a real cell membrane and the AFM force spectroscopy measurements showed that the DPhPC/disulfide tBLM had a thickness of 6.2 ± 0.6 nm consistent with the thickness expected for a bilayer. Finally, the potential of the tBLM to study transmembrane proteins was assessed by investigating the reconstitution of gramicidin A into the membrane by polarization modulation infrared absorption spectroscopy (PM-IRRAS). These results demonstrate that the disulfide is a good candidate to construct tBLMs on gold surface and study transmembrane proteins.
Solid supported lipid membranes: New concepts for the biomimetic functionalization of solid surfaces
Biointerphases, 2008
Surface-layer (S-layer) supported lipid membranes on solid substrates are interfacial architectures mimicking the supramolecular principle of cell envelopes which have been optimized for billions of years of evolution in most extreme habitats. The authors implement this biological construction principle in a variety of layered supramolecular architectures consisting of a stabilizing protein monolayer and a functional phospholipid bilayer for the design and development of new types of solid-supported biomimetic membranes with a considerably extended stability and lifetimecompared to existing platforms-as required for novel types of bioanalytical sensors. First, Langmuir monolayers of lipids at the water/air interface are used as test beds for the characterization of different types of molecules which all interact with the lipid layers in various ways and, hence, are relevant for the control of the structure, stability, and function of supported membranes. As an example, the interaction of S-layer proteins from the bulk phase with a monolayer of a phospholipid synthetically conjugated with a secondary cell wall polymer (SCWP) was studied as a function of the packing density of the lipids in the monolayer. Furthermore, SCWPs were used as a new molecular construction element. The exploitation of a specific lectintype bond between the N-terminal part of selected S-layer proteins and a variety of glycans allowed for the buildup of supramolecular assemblies and thus functional membranes with a further increased stability. Next, S-layer proteins were self-assembled and characterized by the surface-sensitive techniques, surface plasmon resonance spectroscopy and quartz crystal microbalance with dissipation monitoring. The substrates were either planar gold or silicon dioxide sensor surfaces. The assembly of S-layer proteins from solution to solid substrates could nicely be followed in-situ and in real time. As a next step toward S-layer supported bilayer membranes, the authors characterized various architectures based on lipid molecules that were modified by a flexible spacer separating the amphiphiles from the anchor group that allows for a covalent coupling of the lipid to a solid support, e.g., using thiols for Au substrates. Impedance spectroscopy confirmed the excellent charge barrier properties of these constructs with a high electrical resistance. Structural details of various types of these tethered bimolecular lipid membranes were studied by using neutron reflectometry. Finally, first attempts are reported to develop a code based on a SPICE network analysis program which is suitable for the quantitative
Tethered Lipid Bilayers on Electrolessly Deposited Gold for Bioelectronic Applications
Biomacromolecules, 2006
This paper presents the formation of a novel biomimetic interface consisting of an electrolessly deposited gold film overlaid with a tethered bilayer lipid membrane (tBLM). Self-assembly of colloidal gold particles was used to create an electrolessly deposited gold film on a glass slide. The properties of the film were characterized using field-effect scanning electron microscopy, energy dispersive spectroscopy, and atomic force microscopy. Bilayer lipid membranes were then tethered to the gold film by first depositing an inner molecular leaflet using a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate], 1,2-di-O-phytanyl-snglycero-3-phosphoethanolamine (DPGP), and cystamine in ethanol onto a freshly prepared electrolessly deposited gold surface. The outer leaflet was then formed by the fusion of liposomes made from DPGP or 1,2-dioleoylsn-glycero-3-phosphocholine on the inner leaflet. To provide functionality, two membrane biomolecules were also incorporated into the tBLMs: the ionophore valinomycin and a segment of neuropathy target esterase containing the esterase domain. Electrochemical impedance spectroscopy, UV/visible spectroscopy, and fluorescence recovery after pattern photobleaching were used to characterize the resulting biomimetic interfaces and confirm the biomolecule activity of the membrane. Microcontact printing was used to form arrays of electrolessly deposited gold patterns on glass slides. Subsequent deposition of lipids yielded arrays of tBLMs. This approach can be extended to form functional biomimetic interfaces on a wide range of inexpensive materials, including plastics. Potential applications include high-throughput screening of drugs and chemicals that interact with cell membranes and for probing, and possibly controlling, interactions between living cells and synthetic membranes. In addition, the gold electrode provides the possibility of electrochemical applications, including biocatalysis, bio-fuel cells, and biosensors.