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.     

Directed assembly of surface-supported bilayers with transmembrane helices

The lateral assembly of transmembrane (TM) helices gives rise to membrane proteins with complex folds, which play important roles in biochemical processes. Therefore, the assembly of surface-supported bilayers containing TM helices is the first step toward the development of functional biomembrane mimetics. Here we report novel directed assembly of surface-supported lipid bilayers with laterally mobile TM helices. The TM helices were incorporated into lipid monolayers at the air/water interface, and the monolayers were then transferred onto glass substrates using Langmuir-Blodgett (LB) deposition. Finally, bilayers were assembled using lipid vesicle fusion on top of the LB monolayers. The novelty is the incorporation of the peptides into the monolayer at the first step of bilayer assembly, which allows control over the peptide concentration and orientation. The transmembrane orientation of the peptides was confirmed using oriented circular dichroism (OCD), lateral mobility was assessed using fluorescence recovery after photobleaching (FRAP), and diffusion coefficients were determined using a novel boundary profile evolution (BPE) method. The described directed-assembly approach can be used to develop versatile bilayer platforms for studying membrane proteins interactions in native bilayer environments.     

Nanoporous microbead supported bilayers: stability, physical characterization, and incorporation of functional transmembrane proteins

The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.     

A novel approach to the micropatterning of proteins using dewetting of polymer bilayers

The ability to control protein and cell positioning on a microscopic scale is crucial in many biomedical and bioengineering applications, such as tissue engineering and the development of biosensors. We propose here a novel, simple, and versatile method for the micropatterning of proteins. Micropatterned substrates are produced by the dewetting of a metastable polymer film on top of another polymer film. Selective adsorption, or micropatterning, of proteins can be achieved on such substrates by choosing pairs of polymers which differ in protein affinity. In this study, patterns were produced in bilayers of poly(methylmethacrylate) (PMMA) and polystyrene (PS), and of PMMA and octadecyltrichlorosilane (OTS). Fluorescence microscopy and atomic force microscopy (AFM) provide evidence that model proteins adsorb preferentially on isolated bio-adhesive (PS and OTS) micropatches in a protein-resistant (PMMA) matrix. "Inverse" protein patterns, containing non-adhesive (PMMA) islands in a protein-adhesive (PS) matrix can also be produced. Such micropatterned substrates could potentially be used in the development of biosensors and bioassays, and in the study of cell growth and motility.     

Mechanisms of the interaction of alpha-helical transmembrane peptides with phospholipid bilayers

The synthetic peptide acetyl-K(2)-G-L(24)-K(2)-A-amide (P(24)) and its analogs have been successfully utilized as models of the hydrophobic transmembrane alpha-helical segments of integral membrane proteins. The central polyleucine region of these peptides was designed to form a maximally stable, very hydrophobic alpha-helix which will partition strongly into the hydrophobic environment of the lipid bilayer core, while the dilysine caps were designed to anchor the ends of these peptides to the polar surface of the lipid bilayer and to inhibit the lateral aggregation of these peptides. Moreover, the normally positively charged N-terminus and the negatively charged C-terminus have both been blocked in order to provide a symmetrical tetracationic peptide, which will more faithfully mimic the transbilayer region of natural membrane proteins and preclude favorable electrostatic interactions. In fact, P(24) adopts a very stable alpha-helical conformation and transbilayer orientation in lipid model membranes. The results of our recent studies of the interaction of this family of alpha-helical transmembrane peptides with phospholipid bilayers are summarized here.     

Interaction of polyacrylic acid with lipid bilayers: effect of polymer mass

Polyanion‐coated lipid vesicles are proposed to have an appreciable potential for drug delivery because of their ability to control the permeability of lipid bilayers by environmental parameters such as pH and temperature. However, details of the interaction of this class of polymers with lipids and their mechanisms of induced permeability are still being debated. In this work, we applied ¹H NOESY to study details of the interaction of polyacrylic acid (PAA) fractions of molecular weights 5 and 240 kDa with dimyristoylphosphatidylcholine vesicles. We showed that PAA of two different molecular masses modifies lipid bilayers increasing disorder and probability of close contact between polar and hydrophobic groups. PAA molecules adsorb near the interface of lipid bilayers but do not penetrate into the hydrophobic core of the bilayer and, thus, cannot participate in formation of transbilayer channels, proposed in earlier works. Increasing the molecular mass of PAA from 5 kDa to 240 kDa does not change the effect of PAA on the bilayer, although PAA240 forms a more compact structure (either intra‐molecular or inter‐molecular) and interacts more strongly with interface lipid protons. Copyright © 2013 John Wiley & Sons, Ltd.     

Molecular-scale structure in fluid-gel patterned bilayers: stability of interfaces and transmembrane distribution

Variations in two-dimensional membrane structures on the molecular length scale are considered to have an effect on the mechanisms by which living cell membranes maintain their functionality. We created a molecular model of a patterned bilayer to asses the static and dynamic variations of membrane lateral and transbilayer distribution in two-component lipid bilayers on the molecular level. We study DSPC (distearoylphosphatidylcholine) nanometer domains in a fluid DLPC (dilauroylphosphatidylcholine) background. The system exhibits coexisting fluid and gel phases and is studied on a microsecond time scale. We characterize three different kinds of patterns: symmetric domains, asymmetric domains, and symmetric-asymmetric domains. Preferred bilayer configurations on the nanoscale are those that minimize the hydrophobic mismatch. We find nanoscale patterns to be dynamic structures with mainly lateral and rotational diffusion affecting their stability on the microsecond time scale.     

Membrane potential and electrostatics of phospholipid bilayers with asymmetric transmembrane distribution of anionic lipids

It is well-established that native plasma membranes are characterized by an asymmetric distribution of charged (anionic) lipids across the membrane. To clarify how the asymmetry can affect membrane electrostatics, we have performed extensive atomic-scale molecular dynamics simulations of asymmetric lipid membranes composed of zwitterionic (phosphatidylcholine (PC) or phosphatidylethanolamine (PE)) and anionic (phosphatidylserine (PS)) leaflets. It turns out that the asymmetry in transmembrane distribution of anionic lipids gives rise to a nonzero potential difference between the two sides of the membrane. This potential arises from the difference in surface charges of the two leaflets. The magnitude of the intrinsic membrane potential was found to be 238 mV and 198 mV for PS/PC and PS/PE membranes, respectively. Remarkably, this potential is of the same sign as the membrane potential in cells. Our findings, being in reasonable agreement with available experimental data, lend support to the idea that the transmembrane lipid asymmetry typical of most living cells contributes to the membrane potential.     

beta-Barrel transmembrane proteins: Geometric modelling, detection of transmembrane region, and structural properties

The location of the membrane lipid bilayer relative to a transmembrane protein structure is important in protein engineering. Since it is not present on the determined structures, it is essential to automatically define the membrane embedded protein region in order to test mutation effects or to design potential drugs. beta-Barrel transmembrane proteins, present in nature as outer membrane proteins (OMPs), comprise one of the two transmembrane protein fold classes. Lately, the number of their determined structures has increased and this enables the implementation and evaluation of structure-based annotation methods and their more comprehensive study. In this paper, we propose two new algorithms for (i) the geometric modelling of beta-barrels and (ii) the detection of the transmembrane region of a beta-barrel transmembrane protein. The geometric modelling algorithm combines a non-linear least square minimization method and a genetic algorithm in order to find the characteristics (axis, radius) of a shape with axial symmetry which best models a beta-barrel. The transmembrane region is detected by profiling the external residues of the beta-barrel along its axis in terms of hydrophobicity and existence of aromatic and charged residues. TbB-Tool implements these algorithms and is available in . A non-redundant set of 22 OMPs is used in order to evaluate the algorithms implemented and the results are very satisfying. In addition, we quantify the abundance of all amino acids and the average hydrophobicity for external and internal beta-stranded residues along the axis of beta-barrel, thus confirming and extending other researchers' results.     

Anomalous diffusion of oligomerized transmembrane proteins

Transmembrane proteins frequently form (transient) oligomers on biomembranes, e.g., while participating in protein sorting and signaling events. Using coarse-grained membrane simulations we show here that transmembrane proteins show a subdiffusive motion on short time scales when being part of a linear oligomer, i.e., a flexible polymer, embedded in a two-dimensional membrane. Our results are in agreement with previous experimental observations. They further indicate that polymers of transmembrane proteins are well described by predictions from Rouse theory in two dimensions even in the presence of hydrodynamic interactions.     

Supported lipid bilayers on spacious and pH-responsive polymer cushions with varied hydrophilicity

We report the successful formation of supported multicomponent lipid bilayer membranes (sLBMs) on polymer cushions consisting of a set of alternating maleic acid copolymers. The formation of sLBMs was triggered by a transient reduction of the electrostatic repulsion between the polymer cushions and the lipid vesicles by lowering the solution's pH to 4. Upon formation, the stability of the sLBMs was not affected by subsequent variations of the environmental pH. The degree of hydrophilicity and swelling of the anionic polymer cushions was found to determine both the kinetics of the membrane formation and the mobility of the lipid bilayer with lipid diffusion coefficients in the range from 0.26 to 2.6 microm2s(-1). The introduced polymer cushion system is concluded to provide a versatile base for the integration of active transmembrane proteins in sLBMs.     

Analysis of transmembrane proteins from eukaryotic cells

The topography and properties of plasma membrane proteins from mouse L-929 cells are studied by comparing their availability for enzymatic labeling on the external and internal surfaces of the membrane. In order to study the internal surface, phagolysosomes are prepared from cells after they ingest latex particles. The plasma membrane surrounding these seem to have an "inside-out" orientation. The sugars of the membrane glycoproteins in intact phagolysosomes are not available for interaction with lectins or available for periodate-borotritide labeling. A comparison of the lectin-binding proteins labeled by lactoperoxidase-catalyzed iodination on the external cell surface with those labeled on the internal cell surface suggests that a variety of plasma membrane glycoproteins span the lipid bilayer. Using two-dimensional gel electrophoresis it has been shown that selected proteins are labeled at both the internal and external faces of the plasma membrane. Analysis of the 2-D gel electrophoregrams reveals that there are two distinct prominent proteins at 60,000 and 100,000 daltons which are enzymatically iodinated from both sides of the membrane. The partial hydrolysis of the 100,000 dalton protein reveals that different peptides are iodinated when the iodination is performed on intact cells or on the phagolysosomes. These proteins are extensively phosphorylated in cells incubated with inorganic 32P. We conclude that the phagolysosome is probably oriented in an "inside-out" configuration and that this membrane preparation can be used to study the topographic organization of membrane proteins. The use of oriented membranes, selective labeling of proteins, and affinity separation of proteins in combination with gel electrophoresis to define the position and properties of proteins is discussed.     

Light-induced damage to the retina: role of rhodopsin chromophore revisited

The presence of the regenerable visual pigment rhodopsin has been shown to be primarily responsible for the acute photodamage to the retina. The photoexcitation of rhodopsin leads to isomerization of its chromophore 11-cis-retinal to all-trans-retinal (ATR). ATR is a potent photosensitizer and its role in mediating photodamage has been suspected for over two decades. However, there was lack of experimental evidence that free ATR exists in the retina in sufficient concentrations to impose a risk of photosensitized damage. Identification in the retina of a retinal dimer and a pyridinium bisretinoid, so called A2E, and determination of its biosynthetic pathway indicate that substantial amounts of ATR do accumulate in the retina. Both light damage and A2E accumulation are facilitated under conditions where efficient retinoid cycle operates. Efficient retinoid cycle leads to rapid regeneration of rhodopsin, which may result in ATR release from the opsin "exit site" before its enzymatic reduction to all-trans-retinol. Here we discuss photodamage to the retina where ATR could play a role as the main toxic and/or phototoxic agent. Moreover, we discuss secondary products of (photo)toxic properties accumulating within retinal lipofuscin as a result of ATR accumulation.     

Vesicle deformations by clusters of transmembrane proteins

We carry out a coarse-grained molecular dynamics simulation of phospholipid vesicles with transmembrane proteins. We measure the mean and Gaussian curvatures of our protein-embedded vesicles and quantitatively show how protein clusters change the shapes of their host vesicles. The effects of depletion force and vesiculation on protein clustering are also investigated. By increasing the protein concentration, clusters are fragmented to smaller bundles, which are then redistributed to form more symmetric structures corresponding to lower bending energies. Big clusters and highly aspherical vesicles cannot be formed when the fraction of protein to lipid molecules is large.     

Patterns of the ferrocyanide-iodate-sulfite reaction revisited: the role of immobilized carboxylic functions

We experimentally demonstrate that the standing lamella reaction-diffusion patterns initially observed 17 years ago in a gel-filled open spatial reactor operated with the ferrocyanide-iodate-sulfite bistable reaction requires an upper critical concentration of low-mobility species with weak acid functional groups, a parameter that was overlooked at the time and had made observations difficult to reproduce. The present approach enables the control of the space scale separation between activatory and inhibitory processes. It makes the wealth of exotic pattern dynamics observed earlier easier to reproduce and understand. This contribution should considerably revive the interest in this reaction and boost the search for the control of reaction-diffusion patterns in other bistable systems.     

Prediction of transmembrane proteins based on the continuous wavelet transform

A novel method based on continuous wavelet transform (CWT) for predicting the number and location of helices in membrane proteins is presented. Two bacteria proteins are chosen as examples to describe the prediction of transmembrane helices (HTM) by using this method. Selections of an appropriate dilation and hydrophobicity data types are discussed in the text. The results indicate that CWT is a promising approach for the prediction of HTM.     

Scanning near-field IR microscopy of proteins in lipid bilayers

We use infrared near-field microscopy to chemically map the morphology of biological matrices. The investigated sample is built up from surface-tethered membrane proteins (cytochrome c oxidase) reconstituted in a lipid bilayer. We have carried out infrared near-field measurements in the frequency range between 1600 and 1800 cm(-1). By simultaneously recording the topography and chemical fingerprint of the protein-tethered lipid bilayer with a lateral resolution of 80 nm × 80 nm, we were able to probe locally the chemical signature of this membrane and to provide a local map of its surface morphology.     

Short gold nanorod growth revisited: the critical role of the bromide counterion

A one-step, surfactant-assisted, seed-mediated method has been utilized for the growth of short gold nanorods with reasonable yield by modifying an established synthesis protocol. Among the various parameters that influence nanorod growth, the impact of the bromide counterion has been closely scrutinized. During this study it has been shown that, irrespective of its origin, the bromide counterion [cetyltrimethylammonium bromide (CTAB) or NaBr] plays a crucial role in the formation of nanorods in the sense that there is a critical [Br(-)]/[Au(3+)] ratio (around 200) to achieve nanorods with a maximum aspect ratio. Beyond this value, bromide can be considered as a poisoning agent unless shorter nanorods are required. The use of AgNO(3) helps in symmetry breaking for gold nanorod growth, whereas the bromide counterion controls the growth kinetics by selective adsorption on the facets of the growth direction. Thus, a proper balance between bromide ions and gold cations is also one of the necessary parameters for controlling the size of the gold nanorods; this has been discussed thoroughly. The results have been discussed based on their absorption spectra and finally shape evolution has been confirmed by TEM. Due to their efficient absorption in the near-IR region, these short nanorods were used in photothermal imaging of living COS-7 cells with improved signal-to-background ratios.     

Synthetic and natural polycationic polymer nanoparticles interact selectively with fluid-phase domains of DMPC lipid bilayers

Polycationic polymers are known to disrupt lipid bilayers. In this letter, we report the dependence of this disruption on the lipid structural phase. DMPC bilayers are exposed to two polycationic polymeric nanoparticles, PAMAM dendrimers and MSI-78. We find that regions of the bilayer that are in the gel phase are unaffected by the presence of polymers, whereas the liquid phase is disrupted.