Sensing lipid bilayer formation and expansion with a microfabricated cantilever array

We show that cantilever array sensors can sense the formation of supported phospholipid bilayers on their surface and that they can monitor changes in mechanical properties of lipid bilayers. Supported lipid bilayers were formed on top of microfabricated cantilevers by vesicle fusion. The formation of bilayers led to a bending of the cantilevers of 70-590 nm comparable to a surface stress of 27-224 mN/m. Physisorption of bilayers of DOPC and other bilayers on the silicon oxide surface of cantilevers led to a tensile bending of about 70 nm whereas formation of chemisorbed bilayers of mixed thiolated (DPPTE) and non-thiolated lipids (DOPC) on the gold side of cantilevers led to a compressive bending of nearly 600 nm which depended on the ratio of DPPTE to DOPC. First results on bending of bilayer-covered cantilevers due to their interaction with the pore-forming peptide melittin are shown. The results demonstrate that cantilever sensors with immobilized bilayers can be used as model systems to investigate mechanical properties of cellular membranes and may be used for screening of membrane processes involving modification, lateral expansion, or contraction of membranes.     

Electrochemical and microscopic characteristics of thiolipid layers as simple models of cell membranes

The thiolipid 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) has been proposed as a component of a simple model of cell membranes, which can be used for the studies of the interactions with drugs and other biologically important species. Depending on the deposition technique, Langmuir-Blodgett method or self-assembly, the obtained model membranes exhibit differences in the organization and properties, as shown by electrochemical techniques. The surface concentration and area per molecule of DPPTE model membranes were determined using chronocoulometry, which gives more reliable results than the widely used reductive desorption method. The mean surface concentration of self-assembled DPPTE monolayer deposited on gold electrode is equal to 4.52 × 10(-10) mol·cm(-2), which corresponds to the area per molecule of 36.7 ?(2). Moreover, model membranes prepared by means of LB method tend to be less compact than self-assembled DPPTE monolayers. Adsorption/desorption behavior of the DPPTE molecules on Au(111) was also visualized by EC-STM method. At the beginning of the process at negative potentials, the physisorbed molecules formed a flat-lying adlayer. Changing the potential in the positive direction resulted in the formation of Au-S bonds, and as a consequence the upstanding phase with higher packing density was observed. The thickness of such a layer determined by atomic force microscopy method is equal to 2.08 nm and corresponds to that of a monomolecular film.     

Investigation of interaction of Leu-enkephalin with lipid membranes

Enkephalins are peptides with morphine-like activity. To achieve their biological function, they must be transported from an aqueous phase to the lipid-rich environment of their membrane bound receptor proteins. In our study, zeta potential (ZP) method was used to detect the association of Leu-enkephalin and Leu-enkephalinamide with phospholipid liposomes constituted from egg-phosphatidylcholine (EPC), dioleoyl-phosphatidylethanolamine (DOPE), cholesterol (Chol), sphingomyelin (SM) as well as soybean phospholipid (SBPL). Transfer of the peptides over lipid membranes was examined by electrophysiology technique (ET) and fluorescence spectroscopy (FS), and further confirmed using 4-fluoro-7-nitrobenzofurazan (NBD-F) labeled Leu-enkephalin (NBD-F-enkephalin) with confocal laser scanning microscopy method (CLSM). Results of zeta potential showed that enkephalinamide associated with lipid membranes and gradually saturated on the membranes either hydrophobically or electrostatically or both. Data from electrophysiology technique indicated that Leu-enkephalin could cause transmembrane currents, suggesting the transfer of peptides across lipid membranes. Transfer examined by fluorescence spectroscopy implied that it could be separated into three steps, adsorption, transportation and desorption, which was afterward reaffirmed by confocal laser scanning microscopy. Transfer efficiencies of enkephalin across SBPL, EPC/DOPE, EPC/DOPE/SM, EPC/SM and EPC/Chol lipid bilayer membranes were evaluated with ET and CLSM experiments. Results showed that the addition of either sphingomyelin or cholesterol, or negatively charged lipid in lipid membrane composition could lower the transfer efficiency.     

Hydrophobic/superhydrophobic oxidized metal surfaces showing negligible contact angle hysteresis

The blood coagulation system relies on lipid membrane constituents to act as regulators of the coagulation process upon vascular trauma, and in particular the 2D configuration of the lipid membranes is known to efficiently catalyze enzymatic activity of blood coagulation factors. This work demonstrates a new application of a recently developed methodology to study blood coagulation at lipid membrane interfaces with the use of imaging technology. Lipid membranes with varied net charges were formed on silica supports by systematically using different combinations of lipids where neutral phosphocholine (PC) lipids were mixed with phospholipids having either positively charged ethylphosphocholine (EPC), or negatively charged phosphatidylserine (PS) headgroups. Coagulation imaging demonstrated that negatively charged SiO(2) and membrane surfaces exposing PS (obtained from liposomes containing 30% of PS) had coagulation times which were significantly shorter than those for plain PC membranes and EPC exposing membrane surfaces (obtained from liposomes containing 30% of EPC). Coagulation times decreased non-linearly with increasing negative surface charge for lipid membranes. A threshold value for shorter coagulation times was observed below a PS content of ～6%. We conclude that the lipid membranes on solid support studied with the imaging setup as presented in this study offers a flexible and non-expensive solution for coagulation studies at biological membranes. It will be interesting to extend the present study towards examining coagulation on more complex lipid-based model systems.     

Plasmonic Resonant Nanoantennas Induce Changes in the Shape and the Intensity of Infrared Spectra of Phospholipids

Surface enhanced infrared absorption spectroscopic studies (SEIRAS) as a technique to study biological molecules in extremely low concentrations is greatly evolving. In order to use the technique for identification of the structure and interactions of such biological molecules, it is necessary to identify the effects of the plasmonic electric-field enhancement on the spectral signature. In this study the spectral properties of 1,2-Dipalmitoyl-sn-glycero-3 phosphothioethanol (DPPTE) phospholipid immobilized on gold nanoantennas, specifically designed to enhance the vibrational fingerprints of lipid molecules were studied. An AFM study demonstrates an organization of the DPPTE phospholipid in bilayers on the nanoantenna structure. The spectral data were compared to SEIRAS active gold surfaces based on nanoparticles, plain gold and plain substrate (Si) for different temperatures. The shape of the infrared signals, the peak positions and their relative intensities were found to be sensitive to the type of surface and the presence of an enhancement. The strongest shifts in position and intensity were seen for the nanoantennas, and a smaller effect was seen for the DPPTE immobilized on gold nanoparticles. This information is crucial for interpretation of data obtained for biological molecules measured on such structures, for future application in nanodevices for biologically or medically relevant samples.     

Micro-BLMs on highly ordered porous silicon substrates: rupture process and lateral mobility

In a recent paper, we hypothesized that the continuous increase in membrane conductance observed for nano-BLMs is the result of an independent rupturing of single membranes or membrane patches covering the pores of the porous material. To prove this hypothesis, we prepared micro-BLMs on porous silicon substrates with a pore size of 7 mum. The upper surface of the silicon substrate was coated with a gold layer, followed by the chemisorption of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) and subsequent addition of a droplet of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) dissolved in n-decane. The lipid membranes were fluorescently labeled and investigated by means of fluorescence microscopy and impedance spectroscopy. Impedance spectroscopy revealed the formation of pore-suspending bilayers with high membrane resistance. Increases in membrane capacitance and membrane conductance were observed. This increase in membrane conductance could be unambiguously related to the individual rupturing of membranes suspending the pores of the porous material as visualized by means of fluorescence microscopy. Moreover, by fluorescence recovery after photobleaching experiments, we investigated the lateral mobility of the lipids within the micro-BLMs leading to a mean effective diffusion coefficient of Deff = (14 +/- 1) microm2/s.     

Templated assembly of biomembranes on silica microspheres using bacteriorhodopsin conjugates as structural anchors

Membrane proteins are some of the most sophisticated molecules found in nature. These molecules have extraordinary recognition properties; hence, they represent a vast source of specialized materials with potential uses in sensing and screening applications. However, the strict requirement of the native lipid environment to preserve their structure and functionality presents an impediment in building biofunctional materials from these molecules. In general, the purification protocols remove the native lipid support structures found in the cellular environment that stabilize the membrane proteins. Furthermore, the membrane protein structure is often highly complex, typified by large, multisubunit complexes that not only span the lipid bilayer but also contain large (>2 nm) cytoplasmic and extracellular domains that protrude from the membrane. The present study is focused on using a biomimetic approach to build a stable, fluid microenvironment to be used to incorporate larger membrane proteins of interest into a tether-supported lipid bilayer membrane adequately spaced above a substrate passivated to liposome fusion and nonspecific adsorption. Our aim is to reintroduce the supporting structures of the native cell membrane using self-assembled supramolecular complexes constructed on microspheres in an artificial cytoskeleton motif. Central to our architecture is to utilize bacteriorhodopsin (bR), a transmembrane protein, as a biomembrane anchoring molecule to be tethered to surfaces of interest as a sparse structural element in the design. Compared to a typical lipid tether, which inserts into one leaflet of the lipid bilayer, bR anchoring provides an over 8-fold greater hydrophobic surface area in contact with the bilayer. In the work presented here, the silica microsphere surface was biofunctionalized with streptavidin to make it a suitable supporting interface. This was achieved by self-assembly of (p-aminophenyl)trimethoxysilane on the silica surface followed by subsequent conjugation of biotin-PEG3400 (PEG = poly(ethylene glycol) and PEG2000 for further passivation and the binding of streptavidin. We have conjugated bR with biotin-PEG3400 through amine-based coupling to use it as a tether. The biotin-PEG-bR conjugate was further labeled with Texas Red to facilitate localization via fluorescence imaging. Confocal microscopy was utilized to analyze the microsphere surface at different stages of surface modification by employing fluorescent staining techniques. Sparely tethered supported lipid bilayer membranes were constructed successfully on streptavidin-functionalized silica particles (5 mum) using a detergent-based method in which tethered bR nucleates self-assembly of the bilayer membrane. The fluidity of the supported membranes was analyzed using fluorescence recovery after photobleaching in confocal imaging detection mode. The phospholipid diffusion coefficients obtained from these studies indicated that nativelike fluidity was achieved in the tether-supported membranes, thus providing a prospective microenvironment for insertion of membrane proteins of interest.     

Parallel-oriented fibrogenesis of a beta-sheet forming peptide on supported lipid bilayers

Peptide self-assembly on substrates is currently an intensively studied topic that provides a promising strategy for fabrication of soft materials and is also important for revealing the surface chemistry of amyloidogenic proteins that aggregate on cell membranes. We investigated the fibrogenesis of a beta-sheet forming peptide Abeta(26-35) on supported lipid bilayers (SLBs) by in situ atomic force microscopy (AFM), circular dichroism (CD), and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The results show that the Abeta(26-35) nanofilaments' growth is oriented to a specific direction and formed a highly ordered, large-scale, parallel-oriented surface pattern on membranes. The parallel-oriented fibrogenesis of Abeta(26-35) was able to occur on different lipid membranes rather than on solid substrates. It implies that the parallel-oriented fibrogenesis was associated with the distinct properties of lipid membranes, such as the fluid nature of lipid molecules on membranes. The membrane fluidity may allow the peptide assemblies to float at the water-membrane interface and easily orient to an energetically favorable state. These results provide an insight into the surface chemistry of peptide self-assembly on lipid membranes and highlight a possible way to fabricate supramolecular architectures on the surface of soft materials.     

Gas permeability of phosphatidylcholine impregnated in a porous cellulose nitrate membrane

Egg-yolk phosphatidylcholine (EPC) and dipalmitoyl phosphatidylcholine (DPPC) were impregnated in a porous cellulose nitrate membrane, and permeabilities of oxygen, nitrogen, and carbon dioxide through these composite-type membranes were measured. Gases are suggested to permeate across the membrane by an activated flow of the solution—diffusion type through the impregnated lipid matrix. The EPC-filled membrane shows higher permeability and smaller activation energy of permeation compared to the DPPCfilled membrane, which is ascribed to the higher mobility of fatty acid chains of EPC compared to those of DPPC.     

Self-assembly of gold nanorods induced by intermolecular interactions of surface-anchored lipids

Surface-modified gold nanorods (Au NRs) with 1,2-dipalmitoyl- sn-glycero-3-phosphothioethanol (DPPTE) were synthesized, and their self-assembled structures on a silicon substrate were observed using a scanning electron microscope (SEM). The Au NR-DPPTE complex formed characteristic one- and two-dimensional self-assemblies induced by intermolecular interactions of surface-anchored lipids via simple drying process. The interparticle distance between neighboring NRs was uniform at around 5.0 nm, which was consistent with the thickness of the lipid bilayer. Furthermore, we observed the anisotropic configurations of the NR complex, preferentially oriented in a lateral or perpendicular fashion, in a two-dimensional assembled structure dependent on the interfacial hydrophilicity or hydrophobicity of the silicon surface.     

Evaluation of the membrane-binding properties of the proximal region of the angiotensin II receptor (AT1A) carboxyl terminus by surface plasmon resonance.

The proximal region of the angiotensin II receptor (AT1A) carboxyl-terminus (known as helix VIII) is important for receptor function. In this study, we used surface plasmon resonance (SPR) to examine the interaction of helix VIII-derived peptides with three model lipid membranes. The membrane-binding properties of these synthetic peptides, as well as a series of peptide analogues with modified amino acid sequences, could be explained by both amino acid sequence and kinetic binding data by SPR. The helix VIII peptides showed a higher affinity for lipid membranes that contained negatively charged phospholipid, rather than zwitterionic phospholipid. The findings of an SPR study may be useful for estimating the cooperative binding of intracellular receptor domains with G proteins and the components of the lipid bilayer.     

Functional Tethered Bilayer Lipid Membranes on Aluminum Oxide

Tethered bilayer lipid membranes are established as well‐suited model membrane systems adaptable to different surfaces, for example, gold and silicon. These solid supported membranes are highly flexible in their tethering and lipid parts and can thus be optimized for functional incorporation of membrane proteins. The excellent sealing properties of the tethered membranes allow incorporated ion‐channel proteins to be investigated. Preparation of ultrasmooth aluminum oxide by sputtering and synthesis of new tethering lipids with phosphonic acid anchor groups enable formation of an electrically sealing membrane on this surface. This process is monitored by electrochemical impedance spectroscopy and by surface plasmon resonance spectroscopy. High sealing performance of the membrane and functional incorporation of the ion carrier valinomycin are demonstrated.     

Proton transport into a tethered bilayer lipid membrane

Tethered bilayer lipid membranes (tBLMs) are increasingly used to study biological membranes, membrane proteins and a variety of related topics. A tBLM is formed by binding a lipid bilayer to a metal surface (usually gold) via a hydrophilic tether (usually an ethyleneoxy chain). In this report we present an electrochemical study on ubiquinone in a tBLM which has provided insights into the properties of this hydrophilic layer, which has a very limited capability of storing and releasing protons. It is concluded that the often observed decrease in tBLM resistance upon addition of ionophores (or protonophores) could be due to the penetration of ions (or protons) into the membrane rather than transport through the membrane.     

Whole-body rocking motion of a fusion peptide in lipid bilayers from size-dispersed 15N NMR relaxation

Biological membranes present a highly fluid environment, and integration of proteins within such membranes is itself highly dynamic: proteins diffuse laterally within the plane of the membrane and rotationally about the normal vector of this plane. We demonstrate that whole-body motions of proteins within a lipid bilayer can be determined from NMR (15)N relaxation rates collected for different-sized bicelles. The importance of membrane integration and interaction is particularly acute for proteins and peptides that function on the membrane itself, as is the case for pore-forming and fusion-inducing proteins. For the influenza hemagglutinin fusion peptide, which lies on the surface of membranes and catalyzes the fusion of membranes and vesicles, we found large-amplitude, rigid-body wobbling motions on the nanosecond time scale relative to the lipid bilayer. This behavior complements prior analyses where data were commonly interpreted in terms of a static oblique angle of insertion for the fusion peptide with respect to the membrane. Quantitative disentanglement of the relative motions of two interacting objects by systematic variation of the size of one is applicable to a wide range of systems beyond protein-membrane interactions.     

Surface miscibility of EPC/DOTAP/DOPE in binary and ternary mixed monolayers

Surface pressure (π)-molecular area (A) curves were used to characterize the packing of pseudo-ternary mixed Langmuir monolayers of egg phosphatidylcholine (EPC), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and L-α-dioleoyl phosphatidylethanolamine (DOPE). This pseudo-ternary mixture EPC/DOPE/DOTAP has been successfully employed in liposome formulations designed for DNA non-viral vectors. Pseudo-binary mixtures were also studied as a control. Miscibility behavior was inferred from π-A curves applying the additivity rule by calculating the excess free energy of mixture (ΔG(Exc)). The interaction between the lipids was also deduced from the surface compressional modulus (C(s)(-1)). The deviation from ideality shows dependence on the lipid polar head type and monolayer composition. For lower DOPE concentrations, the forces are predominantly attractive. However, if the monolayer is DOPE rich, the DOTAP presence disturbs the PE-PE intermolecular interaction and the net interaction is then repulsive. The ternary monolayer EPC/DOPE/DOTAP presented itself in two configurations, modulated by the DOPE content, in a similar behavior to the DOPE/DOTAP monolayers. These results contribute to the understanding of the lipid interactions and packing in self-assembled systems associated with the in vitro and in vivo stability of liposomes.     

Supported lipid bilayers at skeletonized surfaces for the study of transmembrane proteins

Skeletonized zirconium phosphonate surfaces are used to support planar lipid bilayers and are shown to be viable substrates for studying transmembrane proteins. The skeletonized surfaces provide space between the bilayer and the solid support to enable protein insertion and avoid denaturation. The skeletonized zirconium octadecylphosphonate surfaces were prepared using Langmuir-Blodgett techniques by mixing octadecanol with octadecylphosphonic acid. After zirconation of the transferred monolayer, rinsing the coating with organic solvent removes the octadecanol, leaving holes in the film ranging from ～50 to ～500 nm in diameter, depending on the octadecanol content. Upon subsequent deposition of a lipid bilayer, either by vesicle fusion or by Langmuir-Blodgett/Langmuir-Schaefer techniques, the lipid assemblies span the holes providing reservoirs beneath the bilayer. The viability of the supported bilayers as model membranes for transmembrane proteins was demonstrated by examining two approaches for incorporating the proteins. The BK channel protein inserts directly into a preformed bilayer on the skeletonized surface, in contrast to a bilayer on a nonskeletonized film, for which the protein associates only weakly. As a second approach, the integrin α(5)β(1) was reconstituted in lipid vesicles, and its inclusion in supported bilayers on the skeletonized surface was achieved by vesicle fusion. The integrin retains its ability to recognize the extracellular matrix protein fibronectin when supported on the skeletonized film, again in contrast to the response if the bilayer is supported on a nonskeletonized film.     

Effects of 3FTx Protein Fraction from Naja ashei Venom on the Model and Native Membranes: Recognition and Implications for the Mechanisms of Toxicity

Three-finger toxins are naturally occurring proteins in Elapidae snake venoms. Nowadays, they are gaining popularity because of their therapeutic potential. On the other hand, these proteins may cause undesirable reactions inside the body′s cells. A full assessment of the safety of Naja ashei venom components for human cell application is still unknown. The aim of the study was to determine the effect of the exogenous application of three-finger toxins on the cells of monocytes (U-937) and promyelocytes (HL-60), with particular emphasis on the modification of their membranes under the influence of various doses of 3FTx protein fraction (0–120 ng/mL). The fraction exhibiting the highest proportion of 3FTx proteins after size exclusion chromatography (SEC) separation was used in the experiments. The structural response of cell membranes was described on the basis of single-component and multi-component Langmuir monolayers that mimicked the native membranes. The results show that the mechanism of protein–lipid interactions depends on both the presence of lipid polar parts (especially zwitterionic type of lipids) and the degree of membrane saturation (the greatest-for unsaturated lipids). The biochemical indicators reflecting the tested cells (MDA, LDH, cell survival, induction of inflammation, LD50) proved the results that were obtained for the model.     

Multicomponent membranes on solid substrates: Interfaces for protein binding

Even though lipid membranes deposited on a solid support are now used for more than 20 years as a model system for cellular membranes, their potential has, as yet, not been fully exploited. Only in recent years, the composition of solid supported membranes became more and more complex, which is a prerequisite for the elucidation of biologically relevant protein adsorption processes. Multicomponent bilayers resemble the heterogeneity of the biological membrane, which is composed of hundreds of different lipids varying in their headgroup and acyl chain composition. The development of a multitude of elaborated surface sensitive techniques allows to study the phase behavior of these membranes and their interaction with proteins, some of which will be highlighted in this review.     

Incorporation of alpha-hemolysin in different tethered bilayer lipid membrane architectures

Tethered bilayer lipid membranes are stable solid supported model membrane systems. They can be used to investigate the incorporation and function of membrane proteins. In order to study ion translocation mediated via incorporated proteins, insulating membranes are necessary. The architecture of the membrane can have an important effect on both the electrical properties of the lipid bilayer as well as on the possibility to functionally host proteins. Alpha-hemolysin pores have been functionally incorporated into a tethered bilayer lipid membrane coupled to a gold electrode. The protein incorporation has been monitored optically and electrically and the influence of the molecular structure of the anchor lipids on the insertion properties has been investigated.     

Simultaneous removal of thiolated membrane proteins resulting in nanostructured lipid layers

Self-organization of membrane-embedded peptides and proteins causes the formation of lipid mesostructures in the membranes. One example is purple membranes (PM), which consist of lipids and bacteriorhodopsin (BR) as the only protein component. The BRs form a hexagonal crystalline lattice. A complementary structure is formed by the lipids. Employing BR and PM as an example, we report a method where major parts of the mesoscopic self-assembled protein structures can be extracted from the lipid bilayer membrane. A complementary lipid nanostructure remains on the substrate. To remove such a large number of thiolated proteins simultaneously by applying a mechanical force, they are first reacted at physiological conditions with gold nanoparticles, and then a thin gold film is sputtered onto them that fuses with the gold nanoparticles forming a uniform layer, which finally can be lifted off. In this step, all of the previously gold-labeled proteins are pulled out of the membrane simultaneously. A stable lipid nanostructure is obtained on the mica substrate. Its stability is due to either binding of the lipids to the substrate through ionic bonds or to enough residual proteins to stabilize the lipid nanostructure against reorganization. This method may be applied easily and efficiently wherever thiolated proteins or peptides are employed as self-assembling and structure-inducing units in lipid membranes.