Coarse-grained transmembrane proteins: hydrophobic matching, aggregation, and their effect on fusion

Molecular transport between organelles is predominantly governed by vesicle fission and fusion. Unlike experimental vesicles, the fused vesicles in molecular dynamics simulations do not become spherical readily, because the lipid and water distribution is inappropriate for the fused state and spontaneous amendment is slow. Here, we study the hypothesis that enhanced transport across the membrane of water, lipids, or both is required to produce spherical vesicles. This is done by adding several kinds of model proteins to fusing vesicles. The results show that equilibration of both water and lipid content is a requirement for spherical vesicles. In addition, the effect of these transmembrane proteins is studied in bilayers and vesicles, including investigations into hydrophobic matching and aggregation. Our simulations show that the level of aggregation does not only depend on hydrophobic mismatch, but also on protein shape. Additionally, one of the proteins promotes fusion by inducing pore formation. Incorporation of these proteins allows even flat membranes to fuse spontaneously. Moreover, we encountered a novel spontaneous vesicle enlargement mechanism we call the engulfing lobe, which may explain how lipids added to a vesicle solution are quickly incorporated into the inner monolayer.     

Nanotube-vesicle networks with functionalized membranes and interiors

We describe nanotube-vesicle networks with reconstituted membrane protein from cells and with interior activity defined by an injection of microparticles or molecular probes. The functionality of a membrane protein after reconstitution was verified by single-channel ion conductance measurements in excised inside-out patches from the vesicle membranes. The distribution of protein, determined by fluorescence detection, in the network membrane was homogeneous and could diffuse via a nanotube connecting two vesicles. We also show how injecting small unilamellar protein-containing vesicles can differentiate the contents of individual containers in a network. The combination of membrane activity and interior activity was demonstrated by ionophore-assisted accumulation, and internal Calcium Green-mediated detection, of Ca2+ within a single network container. This system can model a variety of biological functions and complex biological multicompartment structures and might serve as a platform for constructing complex sensor and computational devices.     

Probing the interplay between amyloidogenic proteins and membranes using lipid monolayers and bilayers

Many degenerative diseases such as Alzheimer's and Parkinson's involve proteins that have a tendency to misfold and aggregate eventually forming amyloid fibers. This review describes the use of monolayers, bilayers, supported membranes, and vesicles as model systems that have helped elucidate the mechanisms and consequences of the interactions between amyloidogenic proteins and membranes. These are twofold: membranes favor the formation of amyloid structures and these induce damage in those membranes. We describe studies that show how interfaces, especially charged ones, favor amyloidogenic protein aggregation by several means. First, surfaces increase the effective protein concentration reducing a three-dimensional system to a two-dimensional one. Second, charged surfaces allow electrostatic interactions with the protein. Anionic lipids as well as rafts, rich in cholesterol and gangliosides, prove to play an especially important role. Finally, these amphipathic systems also offer a hydrophobic environment favoring conformational changes, oligomerization, and eventual formation of mature fibers. In addition, we examine several models for membrane permeabilization: protein pores, leakage induced by extraction of lipids, chaotic pores, and membrane tension, presenting illustrative examples of experimental evidence in support of these models. The picture that emerges from recent work is one where more than one mechanism is in play. Which mechanism prevails depends on the protein, its aggregation state, and the lipid environment in which the interactions occur.     

In Situ Vesicle Formation by Native Chemical Ligation

Phospholipid vesicles are of intense fundamental and practical interest, yet methods for their de novo generation from reactive precursors are limited. A non‐enzymatic and chemoselective method to spontaneously generate phospholipid membranes from water‐soluble starting materials would be a powerful tool for generating vesicles and studying lipid membranes. Here we describe the use of native chemical ligation (NCL) to rapidly prepare phospholipids spontaneously from thioesters. While NCL is one of the most popular tools for synthesizing proteins and nucleic acids, to our knowledge this is the first example of using NCL to generate phospholipids de novo. The lipids are capable of in situ synthesis and self‐assembly into vesicles that can grow to several microns in diameter. The selectivity of the NCL reaction makes in situ membrane formation compatible with biological materials such as proteins. This work expands the application of NCL to the formation of phospholipid membranes.     

Distance Measurement on an Endogenous Membrane Transporter in E. coli Cells and Native Membranes Using EPR Spectroscopy

Membrane proteins may be influenced by the environment, and they may be unstable in detergents or fail to crystallize. As a result, approaches to characterize structures in a native environment are highly desirable. Here, we report a novel general strategy for precise distance measurements on outer membrane proteins in whole Escherichia coli cells and isolated outer membranes. The cobalamin transporter BtuB was overexpressed and spin‐labeled in whole cells and outer membranes and interspin distances were measured to a spin‐labeled cobalamin using pulse EPR spectroscopy. A comparative analysis of the data reveals a similar interspin distance between whole cells, outer membranes, and synthetic vesicles. This approach provides an elegant way to study conformational changes or protein–protein/ligand interactions at surface‐exposed sites of membrane protein complexes in whole cells and native membranes, and provides a method to validate outer membrane protein structures in their native environment.     

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.     

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.     

Efficient Synthesis of Methanesulphonate-Derived Lipid Chains for Attachment of Proteins to Lipid Membranes

We have developed an easy and flexible synthetic methodology to obtain lipid chains containing methanothiosulfonate terminal groups with the aim to attach them to natural proteins as functional groups. There are many proteins found in nature that are modified by lipids, and this is a key part of their function. For example, the prion protein is attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor, and this protein is thought to be the causative agent in diseases such as bovine spongiform encephalopathy (BSE; “mad cow disease”) and the human equivalent Creutzfeldt–Jakob disease. However, production of large amounts of protein in bacteria results in proteins that lack these lipid modifications. The lipid chains containing methanothiosulfonate terminal groups that we have synthesized here can be attached to these proteins through the thiol contained in the side chain of the cysteine residue, which can be incorporated into the protein sequence at the desired position.     

Effect of tetracaine on model and erythrocyte membranes by DSC and EPR

Summary DSC and EPR experiments were performed on human erythrocyte membranes and DPPC vesicles in order to study the effect of the anaesthetic drug tetracaine on structure and dynamics of the lipid region. Experiments using spin label technique showed that tetracaine induced fluidity changes of the lipid region in the environment of the fatty acid probe molecules incorporated into the membranes in the vicinity of the lipid-water interface. Similarly to EPR observations, DSC measurements reported decrease of the main melting and the pretransition temperature in comparison to control DPPC vesicles, which is the sign of destabilisation of the structure in the head group region of the lipids. Similar effect was observed in the case of erythrocytes where the protein conformation was also controlled in the presence of drug. A separated membrane melting with well distinguished membrane protein phase transition was found that was affected significantly by tetracaine. These results suggest that tetracaine is able to modify not only the internal dynamics of erythrocyte membranes and produce destabilisation of the lipid structure, but the protein system as well. These might lead to further damage of the biological functions.     

Molecular dynamics simulations of rhodopsin in different one-component lipid bilayers

Four 20 ns molecular dynamic simulations of rhodopsin embedded in different one-component lipid bilayers have been carried out to ascertain the importance of membrane lipids on the protein structure. Specifically, dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), palmitoyl oleoyl phosphatidylcholine (POPC), and palmitoyl linoleyl phosphatidylcholine (PLPC) lipid bilayers have been considered for the present work. The results reported here provide information on the hydrophobic matching between the protein and the bilayer and about the differential effects of the protein on the thickness of the different membranes. Furthermore, a careful analysis of the individual protein-lipid interactions permits the identification of residues that exhibit permanent interactions with atoms of the lipid environment that may putatively act as hooks of the protein to the membrane. The analysis of the trajectories also provides information about the effect of the bilayer on the protein structure, including secondary structural elements, salt bridges, and rigid-body motions.     

Stimulus Dependent Redistribution of Membrane Raft Cholesterol in Human Platelets

Cell membranes provide a requisite dynamic interface to facilitate communication between the extracellular environment and the intracellular milieu. These membranes contain proteins that span and/or are loosely associated with the lipid bilayer. The organization of lipids and proteins components into membrane micro-domains provides a temporal and spatial signaling platform for communication. Recently, cholesterol and sphingomyelin enriched membrane micro-domains known as lipid rafts have been implicated in cell signaling events. In these studies we have advanced our hypothesis that stimulus dependent rearrangement of cholesterol into and out of membrane rafts provides a unique lipid–mediated regulatory mechanism. Using fluorescent derivatives of cholesterol, we have shown that membrane raft associated cholesterol was altered in response to collagen-induced platelet aggregatory stimulation. Collagen stimulation resulted in a rapid redistribution of cholesterol from the outer to the inner membrane monolayer. The reorganization of the outer membrane monolayer resulted in a concomitant increase in outer monolayer fluidity. These studies are the first to show that membrane cholesterol was released from the exchangeable membrane raft pool in response to physiological stimuli.     

Asymmetric ABC-triblock copolymer membranes induce a directed insertion of membrane proteins

Asymmetric molecules and materials provide an important basis for the organization and function of biological systems. It is well known that, for example, the inner and outer leaflets of biological membranes are strictly asymmetric with respect to lipid composition and distribution. This plays a crucial role for many membrane-related processes like carrier-mediated transport or insertion and orientation of integral membrane proteins. Most artificial membrane systems are, however, symmetric with respect to their midplane and membrane proteins are incorporated with random orientation. Here we describe a new approach to induce a directed insertion of membrane proteins into asymmetric membranes formed by amphiphilic ABC triblock copolymers with two chemically different water-soluble blocks A and C. In a comparative study we have reconstituted His-tag labeled Aquaporin 0 in lipid, ABA block copolymer, and ABC block copolymer vesicles. Immunolabeling, colorimetric, and fluorescence studies clearly show that a preferential orientation of the protein is only observed in the asymmetric ABC triblock copolymer membranes.     

Lipid/protein interactions and the membrane/water interfacial region

Despite the importance of lipid/protein interactions in the folding, assembly, stability, and function of membrane proteins, information at an atomic level on how such proteins interact with the lipids that surround them remains sparse. The dynamics and flexible nature of the protein/bilayer interaction make it difficult to study, for example, by crystallographic means. However, based on recent progress in molecular simulations of membranes it is possible to address this problem computationally. This communication reports one of the first attempts to use multiple ns molecular simulations to establish a qualitative picture of the intermolecular interactions between the lipids of a bilayer and two topologically different membrane proteins for which a high resolution (2 A or better) X-ray structure is available.     

In Vitro Synthesis of the E. coli Sec Translocon from DNA

Difficulties in constructing complex lipid/protein membranes have severely limited the development of functional artificial cells endowed with vital membrane‐related functions. The Sec translocon membrane channel, which mediates the insertion of membrane proteins into the plasma membrane, was constructed in the membrane of lipid vesicles through in vitro expression of its component proteins. The components of the Sec translocon were synthesized from their respective genes in the presence of liposomes, thereby bringing about a functional complex. The synthesized E. coli Sec translocon mediated the membrane translocation of single‐ and multi‐span membrane proteins. The successful translocation of a functional peptidase into the liposome lumen further confirmed the proper insertion of the translocon complex. Our results demonstrate the feasible construction of artificial cells, the membranes of which can be functionalized by directly decoding genetic information into membrane functions.     

Pyrenesulfonyl azide as a fluorescent label for the study of protein-lipid boundaries of acetylcholine receptors in membranes

Acetylcholine receptor (AcChR) enriched membrane fragments from Torpedo californica electroplax were labeled by in situ photogenerated nitrenes from a hydrophobic fluorescent probe, pyrene-1-sulfonyl azide. Preferential photolabeling of membrane proteins, mainly AcChR, has been achieved and there is a pronounced exposure of the 48,000 and 55,000 molecular weight subunits of AcChR to the lipid environment of the membrane core. Covalent attachment of the photogenerated fluorescence probe does not perturb the alpha-neurotoxins' binding properties of membrane-bound AcChR or the desensitization kinetics induced by prolonged exposures to cholinergic agonists. Non-covalent photoproducts can be conveniently removed from labeled membrane preparations by exchange into lipid vesicles prepared from electroplax membrane lipids. Fluorescence features of model pyrene sulfonyl amide derivatives, such as fine vibrational structure of emission spectra of fluorescence lifetimes, are highly sensitive to the solvent milieu. The covalently bound probe shows similar fluorescence properties in situ. PySA photoproducts have great potential to spectroscopically monitor neurotransmitter induced events on selected AcChR subunits exposed to the hydrophobic environment of membranes.     

Electrophoretic separation method for membrane pore‐forming proteins in multilayer lipid membranes

In this paper, we report on a novel electrophoretic separation and analysis method for membrane pore‐forming proteins in multilayer lipid membranes (MLMs) in order to overcome the problems related to current separation and analysis methods of membrane proteins, and to obtain a high‐performance separation method on the basis of specific properties of the lipid membranes. We constructed MLMs, and subsequently characterized membrane pore‐forming protein behavior in MLMs. Through the use of these MLMs, we were able to successfully separate and analyze membrane pore‐forming proteins in MLMs. To the best of our knowledge, this research is the first example of membrane pore‐forming protein separation in lipid membranes. Our method can be expected to be applied for the separation and analysis of other membrane proteins including intrinsic membrane proteins and to result in high‐performance by utilizing the specific properties of lipid membranes.     

beta-Barrel membrane bacterial proteins: structure, function, assembly and interaction with lipids

Membrane proteins, although constituting about one-third of all proteins encoded by the genomes of living organisms, are still strongly underrepresented in the database of 3D protein structures, which reflects the big challenge presented by this class of proteins. Structural biologists, by employing electron and x-ray approaches, are continuously revealing new and fundamental insights into the structure, function, assembly and interaction with lipids of membrane proteins. To date, two structural motifs, alpha-helices and beta-sheets, have been found in membrane proteins and interestingly these two structural motives correlate with the location: while alpha-helical bundles are most often found in the receptors and ion channels of plasma and endoplasmic reticulum membranes, beta-barrels are restricted to the outer membrane of Gram-negative bacteria and in the mitochondrial membrane, and represent the structural motif used by several microbial toxins to form cytotoxic transmembrane channels. The beta-barrel, while being a rigid and stable motif is a versatile scaffold, having a wide variation in the size of the barrel, in the mechanism to open or close the gate and to impose selectivity on substrates. Even if the number of x-ray structures of integral membrane proteins has greatly increased in recent years, only a few of them provide information at a molecular level on how proteins interact with lipids that surround them in the membrane. The detailed mechanism of protein lipid interactions is of fundamental importance for understanding membrane protein folding, membrane adsorption, insertion and function in lipid bilayers. Both specific and unspecific interactions with lipids may participate in protein folding and assembly.     

Interaction of cationic lipid vesicles with model cell membranes--as determined by neutron reflectivity

Transfection of cells by DNA (for the purposes of gene therapy) can be effectively engineered through the use of cationic lipid/DNA "lipoplexes", although the transfection efficiency of these lipoplexes is sensitive to the neutral "helper" lipid included. Here, neutron reflectivity has been used to investigate the role of the helper lipid present during the interaction of cationic lipid vesicles with model cell membranes. Dimethyldioctadecylammonium bromide (DDAB) vesicles were formed with two different helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and cholesterol, and the interaction of these vesicles with a supported phospholipid bilayer was determined. DOPE-containing vesicles were found to interact faster with the membrane than those containing cholesterol, and vesicles containing either of the neutral helper lipids were found to interact faster than when DDAB alone was present. The interaction between the vesicles and the membrane was characterized by an exchange of lipid between the membrane and the lipid aggregates in solution; the deposition of vesicle bilayers on the surface of the membrane was not apparent.     

Conformation and Topology of Diacylglycerol Kinase in E.coli Membranes Revealed by Solid‐state NMR Spectroscopy

Solid‐state NMR is a powerful tool for studying membrane proteins in a native‐like lipid environment. 3D magic angle spinning (MAS) NMR was employed to characterize the structure of E.coli diacylglycerol kinase (DAGK) reconstituted into its native E.coli lipid membranes. The secondary structure and topology of DAGK revealed by solid‐state NMR are different from those determined by solution‐state NMR and X‐ray crystallography. This study provides a good example for demonstrating the influence of membrane environments on the structure of membrane proteins.