Oriented immobilization and electron transfer to the cytochrome c oxidase

Direct electron transfer to cytochrome c oxidase (CcO) is investigated as a function of packing density of the surface layer. This is varied by the surface concentration of chelator molecules when the enzyme is immobilized on the electrode using the his-tag technology. Chelator molecules with a terminal nitrilotriacetic acid group are synthesized ex situ in contrast to in situ synthesis used in a previous work. Self-assembled monolayers of the chelator mixed at different mole fractions with a dilution molecule are prepared to bind the CcO after complex formation with Ni²⁺ ions. The CcO, which is immobilized in the solubilized form, is then reconstituted into a protein-tethered bilayer lipid membrane (ptBLM). Varying the mixing ratio of chelator to dilution molecules enabled us to control the packing density of CcO residing in the ptBLM. Subtle differences in the architecture of the protein/lipid layers revealed by surface-enhanced IR absorption spectroscopy are considered to be essential for an effective electron transfer. Cyclic voltammograms are measured under anaerobic conditions at different scan rates and analyzed by means of a model which describes the transfer of four electrons to CcO in the ptBLM. The rate constants thus obtained show a marked dependence on the packing density.     

A novel method to fabricate patterned bilayer lipid membranes

We introduce a new method for forming tethered bilayer lipid membranes on surfaces patterned using a photocleavable self-assembled monolayer (SAM). A SAM terminated with a hydrophobic fluorocarbon residue was bound to a gold surface through a link containing a photocleavable ortho-nitrobenzyl moiety. Hydrophilic regions were produced by irradiation with soft UV (365 nm) through a photomask. The patterned surface was characterized by scanning electron microscopy and electrochemical impedance spectroscopy. Tethered bilayer lipid membranes with well-defined bilayer and monolayer regions were then formed by exposure to egg PC vesicles. The membranes had resistance and capacitance values of 0.52 MOmega.cm2 and 0.83 microF.cm-2, respectively.     

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.     

Fabrication of highly insulating tethered bilayer lipid membrane using yeast cell membrane fractions for measuring ion channel activity

A tethered bilayer lipid membrane (tBLM) was fabricated on a gold electrode using 1,2-dipalmitoyl-sn-glycero-phosphothioethanol as a tethering lipid and the membrane fractions of Saccharomyces pombe yeast cells to deposit the upper leaflet. The membrane fractions were characterized using transmission electron microscopy and dynamic light scattering and found to be similar in size to small unilamellar vesicles of synthetic lipids. The dynamics of membrane-fraction deposition and rupture on the tethering-lipid layer were measured using quartz crystal microgravimetry. The electrochemical properties of the resulting tBLM were characterized using electrical impedance spectroscopy and cyclic voltammetry. The tBLM's electrical resistance was greater than 1 MOmegacm(2), suggesting a defect-free membrane. The suitability of tBLM produced using membrane fractions for measuring ion-channel activities was shown by a decrease in membrane resistance from 1.6 to 0.43 MOmegacm(2) following addition of gramicidin. The use of membrane fractions to form high-quality tBLM on gold electrodes suggests a new approach to characterize membrane proteins, in which the upper leaflet of the tBLM is deposited, and overexpressed membrane proteins are incorporated, in a single step. This approach would be especially useful for proteins whose activity is lost or altered during extraction, purification, and reconstitution, or whose activities are strongly influenced by the lipid composition of the bilayer.     

Surface response methodology for the study of supported membrane formation

We report on the investigations of the formation of the tethered lipid bilayer by vesicle deposition on amine-functionalized surfaces. The tethered bilayer was created by the deposition of egg-PC vesicles containing 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly-(ethyleneglycol)-N-hydroxysuccinimide as anchoring molecules on an amine-coated surface. This approach is an easy route for the formation of a biomimetic-supported membrane. A Doelhert experimental design was applied to determine the conditions leading to the formation of a continuous and defect-free tethered bilayer on different surfaces (gold and glass). Doehlert designs allow modeling of the experimental responses by second-order polynomial equations as a function of experimental factors. Four factors expected to influence bilayer formation were studied: the lipid concentration in the vesicle suspension, the mass percentage of anchoring molecules in the vesicles, the contact time between the vesicles and the surface, and the resting time of the membrane after buffer rinse. The optimization of the membrane preparation parameters was achieved by monitoring lipid assembly formation using surface plasmon resonance spectroscopy on gold and by fluorescence recovery after photobleaching on glass. Three characteristic responses were systematically measured: the bilayer thickness, the lipid diffusion coefficient, and the lipid mobile fraction. The simultaneous inspection of the three characteristics revealed that a restricted experimental domain leads to properties that are in accordance with a bilayer presence. The factors of this domain are a lipid concentration from 0.1 to 1 mg/mL, 4-8% of anchoring molecules in the vesicles, 1-4 h of contact time between vesicles and surface, and 21-24 h of resting time after buffer rinse. Under these conditions, a membrane having a lipid mass per surface between 545 +/- 5 and 590 +/- 10 ng/cm2, a diffusion coefficient of between 2.5 +/- 0.3 x 10(-8) and 3.60 +/- 0.5 x 10(-8) cm2/s, and a mobile fraction between 94 +/- 2 and 99 +/- 1% was formed. These findings were confirmed by atomic force microscopy observations, which showed the presence of a continuous and homogeneous bilayer in the determined experimental domain. This formation procedure presents many advantages; it provides an easily obtainable biomimetic membrane model for proteins studies and offers a versatile tethered bilayer because it can be adapted easily to various types of supports.     

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.     

Incorporation of Na(+),K(+)-ATP-ase into the thiolipid biomimetic assemblies via the fusion of proteoliposomes

The black lipid membranes (BLMs) are artificial membrane systems that have been widely used in the study of different biological processes. In this paper the planar bilayer lipid membranes have been used to study the behavior of thiolipid molecules-dipalmitoyl-phosphatidyl-ethanolamine-mercaptopropionamide (DPPE-MPA) and cholesteryl 3-mercaptopropionate (Chs-MPA)-as compared to classical BLM made of natural lipids. We present our experiments on black thiolipid bilayer (BTM) formation from a thiolipid solution and basic results of pump currents generated by sodium-potassium pump-Na(+),K(+)-ATP-ase-introduced to such bilayer systems via proteoliposome adsorption with subsequent fusion. Our results imply that no substantial difference exists between BLMs formed from classical lipids and those made from thiolipids used in this study. The same thiolipid molecules were subsequently used for the formation of covalently bound, tethered bilayer lipid membranes (t-BLMs) on polycrystalline gold electrodes. Similarly, as in the case of BLMs, we took advantage of proteoliposome adsorption/fusion to obtain a t-BLM system with reconstituted enzyme. The vesicle fusion on hydrophobic or hydrophilic substrates is one of the main ways to obtain a bilayer system with incorporated biological species. In this paper we present also our preliminary results of electrochemical experiments using rapid solution exchange technique on such t-BLMs systems and their comparison with painted solid supported membranes (SSMs) and BLMs. We have also followed the process of vesicles fusion onto thiolipid monolayer by means of in situ atomic force microscopy in tapping mode (TM-AFM). On the basis of these experiments, we conclude that DPPE-MPA and Chs-MPA molecules used in our experiments preserve lipid properties, allowing for at least partial reconstitution of Na(+),K(+)-ATP-ase into such t-BLMs. On the other hand, the relatively compact organization on polycrystalline gold and the hydrophobic nature of the first monolayer of tethered thiolipids slows down the proteoliposome fusion onto such monolayers and consequently hinders the protein insertion. However, this effect can be overcome by mechanical stimulus that facilitates proteoliposome delamination onto the self-assembled monolayer.     

Orientational control of the physiological reaction of cytochrome c oxidase tethered to a gold electrode

The physiological reaction of a membrane protein is reconstituted on a solid-supported electrode by orientational control via the position of an affinity tag. Recombinant cytochrome c oxidase (CcO) from Rhodobacter sphaeroides is immobilized on a chemically modified gold surface via the affinity of a histidine tag (His-tag) to a nickel chelating nitrilotriacetic acid surface. Control of the orientation is achieved by the adsorption of CcO through the His-tag engineered into the two opposite sites of the membrane protein surface. After reconstitution into a lipid layer, the functionality of this enzyme film electrode is probed by surface-enhanced infrared absorption spectroscopy and cyclic voltammetry. We demonstrate that cytochrome c (Cc) binds and initiates the catalytic reaction of CcO only when the latter is orientated with subunit II facing the bulk aqueous phase while Cc does not interact with the oppositely orientated CcO. We infer from the observed catalytic dioxygen reduction at potentials below 240 mV (vs a normal hydrogen electrode) that reduced Cc mediates electron input into CcO in a way similar to the physiological pathway. The quantitative analysis of the IR spectra indicates the presence of an inactive population of Cc bound to CcO at equal amounts as the redox-active population. This methodological approach demonstrates that the orientation of the membrane protein can be controlled depending on the position of the affinity tag. The approach is considered to be of general applicability as the introduction of affinity tags is routine in current biochemistry.     

Oriented attachment and membrane reconstitution of His-tagged cytochrome c oxidase to a gold electrode: in situ monitoring by surface-enhanced infrared absorption spectroscopy

A novel concept is introduced for the oriented incorporation of membrane proteins into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent was immobilized on a chemically modified gold surface via the affinity of its histidine-tag to a nickel-chelating nitrilo-triacetic acid (NTA) surface. The oriented protein monolayer was reconstituted into the lipid environment by detergent substitution. The individual steps of the surface modification, including (1) chemical modification of the gold support, (2) adsorption of the protein, and (3) reconstitution of the lipid bilayer, were followed in situ by means of surface-enhanced infrared absorption spectroscopy (SEIRAS) and accompanied by normal-mode analysis. The high surface sensitivity of SEIRAS allows for the identification of each chemical reaction process within the monolayer at the molecular level. Finally, full functionality of the surface-tethered cytochrome c oxidase was demonstrated by cyclic voltammetry after binding of the natural electron donor cytochrome c.     

Immobilization of acetylcholinesterase in lipid membranes deposited on self-assembled monolayers

Human red blood cell acetylcholinesterase was incorporated into planar lipid membranes deposited on alkanethiol self-assembled monolayers (SAMs) on gold substrates. Activity of the protein in the membrane was detected with a standard photometric assay and was determined to be similar to the protein in detergent solution or incorporated in lipid vesicles. Monolayer and bilayer lipid membranes were generated by fusing liposomes to hydrophobic and hydrophilic SAMs, respectively. Liposomes were formed by the injection method using the lipid dimyristoylphosphatidylcholine (DMPC). The formation of alkanethiol SAMs and lipid monolayers on SAMs was confirmed by sessile drop goniometry, ellipsometry, and electrochemical impedance spectroscopy. In this work, we report acetylcholinesterase immobilization in lipid membranes deposited on SAMs formed on the gold surface and compare its activity to enzyme in solution.     

Redox enzymes in tethered membranes

An electrode surface is presented that enables the characterization of redox-active membrane enzymes in a native-like environment. An ubiquinol oxidase from Escherichia coli, cytochrome bo(3) (cbo(3)), has been co-immobilized into tethered bilayer lipid membranes (tBLMs). The tBLM is formed on gold surfaces functionalized with cholesterol tethers which insert into the lower leaflet of the membrane. The planar membrane architecture is formed by self-assembly of proteoliposomes, and its structure is characterized by surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), and tapping-mode atomic force microscopy (TM-AFM). The functionality of cbo(3) is investigated by cyclic voltammetry (CV) and is confirmed by the catalytic reduction of oxygen. Interfacial electron transfer to cbo(3) is mediated by the membrane-localized ubiquinol-8, the physiological electron donor of cbo(3). Enzyme coverages observed with TM-AFM and CV coincide (2-8.5 fmol.cm(-)(2)), indicating that most-if not all-cbo(3) on the surface is catalytically active and thus retains its integrity during immobilization.     

In situ formation and characterization of poly(ethylene glycol)-supported lipid bilayers on gold surfaces

Inclusion of a polymer cushion between a lipid bilayer membrane and a solid surface has been suggested as a means to provide a soft, deformable layer that will allow for transmembrane protein insertion and mobility. In this study, mobile, tethered lipid bilayers were formed on a poly(ethylene glycol) (PEG) support via a two-step adsorption process. The PEG films were prepared by coadsorbing a heterofunctional, telechelic PEG lipopolymer (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000-N-[3-(2-(pyridyldithio)propionate]) (DSPE-PEG-PDP) and a nonlipid functionalized PEG-PDP from an ethanol/water mixture, as described in a previous paper (Munro, J. C.; Frank, C. W. Langmuir 2004, 20, 3339-3349). Then a two-step lipid adsorption strategy was used. First, lipids were adsorbed onto the PEG support from a hexane solution. Second, vesicles were adsorbed and fused on the surface to create a bilayer in an aqueous environment. Fluorescence recovery after photobleaching experiments show that this process results in mobile bilayers with diffusion coefficients on the order of 2 microm2/s. The mobility of the bilayers is decreased slightly by increasing the density of tethered lipids. The formation of bilayers, and not multilayer structures, is also confirmed by surface plasmon resonance, which was used to determine in situ film thickness, and by fluorimetry, which was used to determine quantitatively the fluorescence intensity for each 18 by 18 mm sample. Unfortunately, fluorescence microscopy also shows that there are large defects on the samples, which limits the utility of this system.     

Microfluidic fabrication of addressable tethered lipid bilayer arrays and optimization using SPR with silane-derivatized nanoglassy substrates

We report the microfluidic fabrication of robust and fluid tethered bilayer arrays within a poly(dimethylsiloxane) (PDMS) chip, and demonstrate its addressability and biosensing by incorporating the GM1 receptor into the bilayer framework for detection of cholera toxin. Rapid optimization of the experimental conditions is achieved by using nanoglassified surfaces in combination with surface plasmon resonance. The ultrathin glassy film on gold mimics glass surfaces employed in microfluidics, allowing real-time monitoring of multiple assembly steps and therefore permitting rapid prototyping of microfluidic arrays. The tethered bilayer array utilizes a covalently immobilized biotinylated protein for generation of well-defined capture zones where a streptavidin link is employed for the immobilization of biotinylated vesicles. Fusion of captured vesicles is accomplished using a concentrated PEG solution, and the lateral diffusion of the tethered bilayer membrane is characterized by fluorescence recovery after photobleaching methods. The tethered membrane arrays demonstrate marked stability and high mobility, which provide an ideal host environment for membrane-associated proteins and open new avenues for high-throughput analysis of these proteins.     

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.     

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.     

Direct electrochemical interaction between a modified gold electrode and a bacterial membrane extract

A novel electrochemical approach is described for redox-active membrane proteins. A total membrane extract (in the form of vesicles) of Bacillus subtilis is tethered onto gold surfaces modified with cholesterol based thiols. The membrane vesicles remain intact on the surface and do not rupture or fuse to form a planar bilayer. Oxidation/reduction signals are obtained of the natural co-enzyme, menaquinone-7, located in the membrane. The membrane protein, succinate menaquinone oxidoreductase (SQR), remains in the vesicles and is able to reduce fumarate using menaquinone as mediator. The catalysis of the reverse reaction (oxidation of succinate), which is the natural catalytic function of SQR, is almost absent with menaquinone. However, adding the co-enzyme ubiquinone, which has a reduction potential that is about 0.2 V higher, restores the succinate oxidation activity.     

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.     

Formation and finite element analysis of tethered bilayer lipid structures

Rapid solvent exchange of an ethanolic solution of diphytanoyl phosphatidylcholine (DPhyPC) in the presence of a mixed self-assembled monolayer (SAM) [thiolipid/β-mercaptoethanol (βME) (3/7 mol/mol) on Au] shows a transition from densely packed tethered bilayer lipid membranes [(dp)tBLMs], to loosely packed tethered bilayer lipid membranes [(lp)tBLMs], and tethered bilayer liposome nanoparticles (tBLNs) with decreasing DPhyPC concentration. The tethered lipidic constructs in the aqueous medium were analyzed by atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). Finite element analysis (FEA) was applied to interpret spectral EIS features without referring to equivalent circuit modeling. Using structural data obtained earlier from neutron reflectometry and dielectric constants of lipid bilayers, we reproduced experimentally observed features of the electrochemical impedance (EI) spectra of complex surface constructs involving small pinhole defects, large membrane-free patches, and bound liposomes. We demonstrated by FEA that highly insulating (dp)tBLMs with low-defect density exhibit EI spectra in the shape of a perfect semicircle with or without low-frequency upward "tails" in the Cole-Cole representation. Such EI spectra were observed at DPhyPC concentrations of >5 × 10(-3) mol L(-1). While AFM was not able to visualize very small lateral defects in such films, EI spectra unambiguously signaled their presence by increased low frequency "tails". Using FEA we demonstrate that films with large diameter visible defects (>25 nm by AFM) produce EI spectral features consisting of two semicircles of comparable size. Such films were typically obtained at DPhyPC concentrations of <5 × 10(-3) mol L(-1). At DPhyPC concentrations of <1.0 × 10(-3) mol L(-1) the planar bilayer structures were replaced by ellipsoidal liposomes with diameters ranging from 50 to 500 nm as observed in AFM images. Despite the distinct surface morphology change, the EI curves exhibited two semicircle spectral features typical for the large size defects in planar tBLMs. FEA revealed that, to account for these EI features for bound liposome systems (50-500 nm diameter), one needs to assume much lower tBLM conductivities of the submembrane space, which separates the electrode surface and the phospholipid bilayer. Alternatively, FEA indicates that such features may also be observed on composite surfaces containing both bound liposomes and patches of planar bilayers. Triple semicircular features, observed in some of the experimental EI curves, were attributed to an increased complexity of the real tBLMs. The modeling demonstrated that such features are typical for heterogeneous tBLM surfaces containing large patches of different defectiveness levels. By integrating AFM, EIS, and FEA data, our work provides diagnostic criteria allowing the precise characterization of the properties and the morphology of surface supported bilayer systems.     

On the origin of the polar order of T4 lysozyme on planar model surfaces

Site directed spin labeling is used to investigate the origin of the macroscopic alignment of T4 lysozyme vectorially tethered to planar biomimetic surfaces. T4 lysozyme was adsorbed to a quartz-supported dioleoylphosphatidylcholine (DOPC) bilayer by selective binding of the histidine-tagged protein to functionalized headgroups (1,2-dioleoyl-sn-glycero-3-[[N(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl], DOGS NTA) of the bilayer. This results in a polar oriented ensemble of proteins on the surface, which gives rise to angular-dependent electron paramagnetic resonance (EPR) spectra. In order to reveal the mechanism of the protein alignment, the influence of protein coverage on the order of the molecules was addressed. Along the lines described previously for a full monolayer (Jacobsen, et al. Biophys. J. 2005, 88, 4351), the polar orientation of the molecules was inferred from an analysis of the EPR line shape using the stochastic Liouville equation (SLE) approach developed by Freed and co-workers. The simulations reveal that the orientation of the protein is strongly determined by lateral protein-protein interactions. In comparison to the lipid bilayer, a fusion protein of T4 lysozyme (T4L) with Annexin XII was investigated, where the two-dimensional crystallization of Annexin XII on a dioleoylphosphatidylserine (DOPS) bilayer provides a surface layer of regularly anchored T4L molecules. For this system, it is found that the interaction between T4L and Annexin plays a more important role for understanding the structure in the adsorbed state.     

Designing a Useful Lipid Raft Model Membrane for Electrochemical and Surface Analytical Studies

A model biomimetic system for the study of protein reconstitution or drug interactions should include lipid rafts in the mixed lipid monolayer, since they are usually the domains embedding membrane proteins and peptides. Four model lipid films composed of three components: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (Chol) and sphingomyelin (SM) mixed in different molar ratios were proposed and investigated using surface pressure measurements and thermodynamic analysis of the monolayers at the air–water interface and imaged by Brewster angle microscopy. The ternary monolayers were transferred from the air–water onto the gold electrodes to form bilayer films and were studied for the first time by electrochemical methods: alternative current voltammetry and electrochemical impedance spectroscopy and imaged by atomic force microscopy. In excess of DOPC, the ternary systems remained too liquid for the raft region to be stable, while in the excess of cholesterol the layers were too solid. The layers with SM in excess lead to the formation of Chol:SM complexes but the amount of the fluid matrix was very low. The equimolar content of the three components lead to the formation of a stable and well-organized assembly with well-developed raft microdomains of larger thickness, surrounded by the more fluid part of the bilayer. The latter is proposed as a convenient raft model membrane for further physicochemical studies of interactions with drugs or pollutants or incorporation of membrane proteins.