Tethered bilayer lipid membranes self-assembled on mercury electrodes (original) (raw)
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A Peptide-Tethered Lipid Bilayer on Mercury as a Biomimetic System
A novel spacer consisting of a hexapeptide molecule with a high tendency to form a 310-helical structure, which terminates with a sulfydryl group for anchoring to a metal, was tailored for use as a tethered hydrophilic spacer to be interposed between a metal support and a lipid bilayer. The thiol peptide has two triethylenoxy side chains that impart it a satisfactory hydrophilicity and are intended to keep the anchored thiol peptide chains sufficiently apart so as to accommodate water molecules and inorganic ions and to create a suitable environment for the incorporation of integral proteins. This thiol peptide was anchored to a hanging mercury drop electrode. The formation of a phospholipid bilayer on top of the self-assembled thiol peptide was carried out by a novel procedure which exploits the spontaneous tendency of a lipid film to form a bilayer when interposed between two hydrophilic phases. The resulting mercury-supported thiol peptide/lipid bilayer system was characterized by ac voltammetry with phase resolution, chronocoulometry, and impedance spectroscopy. The suitability of this tethered film as a biomembrane model was tested by incorporating ubiquinone-10 and valinomycin.
Incorporation of channel-forming peptides in a Hg-supported lipid bilayer
The channel-forming peptides gramicidin and alamethicin were incorporated in a mercury-supported lipid bilayer composed of a tethered thiolipid monolayer with a self-assembled dioleoylphosphatidylcholine monolayer on top of it. The thiolipid consists of a hexapeptide chain with a high tendency to form a 3 10 -helical structure, which terminates at the N-terminus end with a sulfydryl group for anchoring to the metal while the C-terminus end is covalently linked to the polar head of dimyristolylphosphatidylethanolamine. The hexapeptide moiety has two triethyleneoxy side chains that impart a satisfactory hydrophilicity and are intended to keep the anchored thiolpeptide chains sufficiently apart, so as to accommodate water molecules and inorganic ions and to create a suitable environment for the incorporation of integral proteins. Changes in the conductance of this biomimetic membrane following the incorporation of gramicidin and alamethicin were detected by impedance spectroscopy. The surface dipole potential of the hexapeptide chain and the transmembrane potential of the lipid bilayer were estimated by using a simple electrostatic model of the mercury|solution interphase.
Langmuir, 2005
Tethered bilayer lipid membranes (tBLMs) are described based on the self-assembly of a monolayer on template stripped gold of an archea analogue thiolipid, 2,3-di-o-phytanyl-sn-glycerol-1-tetraethylene glycol-D,L-R-lipoic acid ester lipid (DPTL), and a newly designed dilution molecule, tetraethylene glycol-D,L-Rlipoic acid ester (TEGL). The tBLM is completed by fusion of liposomes made from a mixture of diphytanoylphosphatidyl choline (DPhyPC), cholesterol, and 1,2-diphytanoyl-sn-glycero-3-phosphate (DPhyPG) in a molar ratio of 6:3:1. Melittin and gramicidin are incorporated into these tBLMs as shown by surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) studies. Ionic conductivity at 0 V vs Ag|AgCl, 3 M KCl, measured by EIS measurements are comparable to the results obtained by other research groups. Admittance plots as a function of potential are discussed on a qualitative basis in terms of the kinetics of ion transport through the channels.
A biomimetic membrane was obtained by anchoring a hydrophilic monolayer consisting of a triethyleneoxythiol (EO3) or hexaethyleneoxythiol "spacer" to a hanging mercury drop electrode and by interposing a lipid film previously spread on the surface of an aqueous electrolyte between the spacer-coated electrode and the aqueous solution. The impedance spectrum of this biomimetic membrane and its response to the incorporation of the ion carrier valinomycin and of the physiological quinone ubiquinone-10 indicate that it has a good fluidity and consists of a stable lipid bilayer on top of the hydrophilic spacer acting as an ionic reservoir. Upon incorporation of the channel-forming polypeptide melittin, this model system exhibits a conductance entirely analogous to that reported on black lipid membranes, with the appreciable advantage of a higher stability and resistance to disruption. The surface dipole potential of the EO3 monolayer alone was obtained from the extrathermodynamic "free charge density" q M on EO3-coated mercury, as experienced by the diffuse layer ions. The partial charge transfer from the sulfydryl group of EO3 to mercury was estimated from q M and from the thermodynamic "total charge density" σ M on EO3-coated mercury. *
Mercury-supported hydrophilic spacers with a lipid bilayer on top are used as biomembrane models by incorporating the ion carrier valinomycin and the ion channel melittin in the bilayer. The same films supported by gold are used to incorporate valinomycin. The extrathermodynamic absolute potential difference across the mercury/water interphase is estimated from the difference in charge density between a bare mercury electrode and a mercury electrode coated with a self-assembled dioleoylphosphatidylcholine (DOPC) monolayer. This estimate is confirmed by the potential dependence of the stationary light-on current of purple membrane fragments adsorbed at the DOPC-coated mercury electrode. The surface dipole potential of a mercury-supported self-assembled monolayer of a hydrophilic spacer is estimated from diffuse-layer effects and from the previously determined value of the absolute potential difference across the mercury/ water interphase. The zero potential difference across a DOPC bilayer freely suspended on top of the above hydrophilic spacer is estimated from the potential dependence of the conductance of the mercury-supported spacer/(lipid bilayer) film, upon incorporation of valinomycin and melittin. The latter measurements, besides confirming the previous estimates, are in agreement with those carried out on black lipid membranes, thus showing the validity of this mercury-supported biomembrane model. Finally, the absolute potential difference across the gold/water interphase is estimated from a comparison between the potential dependence of the conductance of mercury-and gold-supported spacer/(lipid bilayer) films incorporating valinomycin.
Electrochemical Investigation of Melittin Reconstituted into a Mercury-Supported Lipid Bilayer
The channel-forming peptide melittin was incorporated into a biomimetic membrane consisting of a mercury electrode coated with a thiolipid monolayer, with a lipid monolayer self-assembled on top of it. The thiolipid consisted of a hydrophilic tetraethyleneoxy chain terminated at one end with a disulfide group, for anchoring to the mercury surface, and covalently linked at the other end to two diphytanyl chains, which formed a lipid bilayer with the overhanging lipid monolayer. The conductance of the lipid bilayer in contact with aqueous 0.1 M KCl was measured by electrochemical impedance spectroscopy over a frequency range from 1 × 10 -2 to 1 × 10 5 Hz and a potential range of 0.7 V for different compositions of the outer lipid monolayer. The conductance increases abruptly above the background level at sufficiently negative applied potentials, attaining a maximum value that increases with the composition of the outer monolayer in the order PC/chol (60:40) < PC < PC/SM/chol (59:15:26) < PS, with PC ) phosphatidylcholine, chol ) cholesterol, SM ) sphingomyelin, and PS ) phosphatidylserine. The higher the maximum conductance, the less negative the applied potential at which it is attained. This behavior is also discussed using a model of the electrified interphase.
Biophysical Journal, 2002
The potential independent limiting flux of hydrated Tl ϩ ions through gramicidin (GR) channels incorporated in phospholipid monolayers self assembled on a hanging mercury-drop electrode is shown to be controlled both by diffusion and by a dehydration step. Conversely, the potential independent limiting flux of dehydrated Tl ϩ ions stemming from Tl amalgam eletrooxidation is exclusively controlled by diffusion of thallium atoms within the amalgam. Modulating the charge on the polar heads of dioleoylphosphatidylserine (DOPS) by changing pH affects the limiting flux of hydrated Tl ϩ ions to a notable extent, primarily by electrostatic interactions. The dipole potential of DOPS and dioleoylphosphatidylcholine (DOPC), positive toward the hydrocarbon tails, does not hinder the translocation step of Tl ϩ ions to such an extent as to make it rate limiting. Consequently, incorporation in the lipid monolayer of phloretin, which decreases such a positive dipole potential, does not affect the kinetics of Tl ϩ flux through GR channels. In contrast, the increase in the positive dipole potential produced by the incorporation of ketocholestanol causes the translocation step to contribute to the rate of the overall process. A model providing a quantitative interpretation of the kinetics of diffusion, dehydration-hydration, translocation, and charge transfer of the Tl ϩ /Tl 0 (Hg) couple through GC channels incorporated in mercury-supported phospholipid monolayers is provided. A cut-off disk model yielding the profile of the local electrostatic potential created by an array of oriented dipoles located in the lipid monolayer along the axis of a cylindrical ion channel is developed.
Journal of Electroanalytical Chemistry, 1998
The preparation of self-assembled alkanethiol monolayers and alkanethiol phospholipid bilayers on a home-made hanging mercury drop electrode is described. Two different experimental procedures are used to prepare alkanethiol monolayers on mercury. Once a stable thiol monolayer is obtained on the mercury electrode, formation of the bilayer is simple and highly reproducible. The bilayers consist of a thiol monolayer anchored to mercury via the sulfur atom, with a second phospholipid monolayer on top of it. Monolayers and bilayers are characterized electrochemically by measuring capacitance and resistance. Both thiol-coated and thiol lipid-coated mercury electrodes present a compact and well-ordered structure, absence of defects and high reproducibility. The permeability of these films to compounds with different degrees of lipophilicity is examined. The results point to a high impermeability of self-assembled alkanethiol monolayers and mixed alkanethiol phospholipid bilayers supported by mercury to lipophilic compounds.
Electrochimica Acta, 2018
The interactions of cationic lipopeptide C 15 H 31 CO-Trp-Lys-D-Leu-Lys with model lipid bilayers supported on gold surface were investigated using electrochemical methods combined with atomic force microscopy imaging and quartz crystal microbalance measurements. It has been found that the mode of the lipopeptide action strongly depends on the net charge of the lipid membrane. In case of the negatively charged bilayers composed of L-a-phosphatidylethanolamines and L-a-phosphatidylglycerols extracted from E. coli bacteria, C 15 H 31 CO-Trp-Lys-D-Leu-Lys molecules initially aggregate on the top of the membrane as a result of the electrostatic attraction between cationic peptide moiety and anionic polar heads of phosphatidylglycerols. Further fusion of the aggregates leads to the swelling and partial disruption of the membrane, while its permeability is substantially increased. Interestingly, the changes in the molecular organization of negatively charged membrane were much more pronounced in the upper leaflet of the bilayer. The effect of C 15 H 31 CO-Trp-Lys-D-Leu-Lys on zwitterionic bilayer composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and cholesterol was definitely different. In this case, the disturbance of the membrane structure was rather subtle. It has been observed that a small fraction of lipopeptide molecules rapidly inserts into the bilayer and perturbation of the assembly is homogenous throughout the film. This was followed by a noticeable decrease in membrane permeability and stiffening of the lipid bilayer. Such sealing effect indicates that the insertion of lipopeptide did not result either in pore formation or membrane rupture.