Academic literature on the topic 'Lipid-bilayer insertion'

DNA–lipid complexes are of biomedical importance as delivery vectors for gene therapy. To gain insight into the interactions of DNA with zwitterionic and cationic (dimyristoyltrimethylammonium propane (DMTAP)) lipids, we have used coarse-grained molecular dynamics simulations to study the self-assembly of DPPC and DPPC/DMTAP lipid bilayers in the presence of a DNA dodecamer. We observed the spontaneous formation of lipid bilayers from initial systems containing randomly placed lipids, water–counterions and DNA. In both the DPPC and DPPC/DMTAP simulations, the DNA molecule is located at the water–lipid headgroup interface, lying approximately parallel to the plane of the bilayer. We have also calculated the potential of mean force for transferring a DNA dodecamer through a DPPC/DMTAP bilayer. A high energetic barrier to DNA insertion into the hydrophobic core of the bilayer is observed. The DNA adopts a transmembrane orientation only in this region. Local bilayer deformation in the vicinity of the DNA molecule is observed, largely as a result of the DNA–DMTAP headgroup attraction.

To advance mechanistic understanding of membrane-associated peptide folding and insertion, we have studied the kinetics of three single tryptophan pHLIP (pH-Low Insertion Peptide) variants, where tryptophan residues are located near the N terminus, near the middle, and near the inserting C-terminal end of the pHLIP transmembrane helix. Single-tryptophan pHLIP variants allowed us to probe different parts of the peptide in the pathways of peptide insertion into the lipid bilayer (triggered by a pH drop) and peptide exit from the bilayer (triggered by a rise in pH). By using pH jumps of different magnitudes, we slowed down the processes and established the intermediates that helped us to understand the principles of insertion and exit. The obtained results should also aid the applications in medicine that are now entering the clinic.

Molecular-dynamics simulations covering 30ns of both a natural and a synthetic antimicrobial peptide in the presence of a zwitterionic lipid bilayer were performed. In both simulations, copies of the peptides were placed in an α-helical conformation on either side of the bilayer about 10Å (1Å = 0.1nm) from the interface, with either the hydrophobic or the positively charged face of the helix directed toward the bilayer surface. The degree of peptide—lipid interaction was dependent on the starting configuration: surface binding and subsequent penetration of the bilayer was observed for the hydrophobically oriented peptides, while the charge-oriented peptides demonstrated at most partial surface binding. Aromatic residues near the N-termini of the peptides appear to play an important role in driving peptide—lipid interactions. A correlation between the extent of peptide—lipid interactions and helical stability was observed in the simulations. Insertion of the peptides into the bilayer caused a dramatic increase in the lateral area per lipid and decrease in the bilayer thickness, resulting in substantial disordering of the lipid chains. Results from the simulations are consistent with early stages of proposed mechanisms for the lytic activity of antimicrobial peptides. In addition to these ‘free’ simulations, 25ns simulations were carried out with the peptides constrained at three different distances relative to the bilayer interface. The constraint forces are in agreement with the extent of peptide—bilayer insertion observed in the free simulations.

The pH low-insertion peptide (pHLIP) serves as a model system for peptide insertion and folding across a lipid bilayer. It has three general states: (I) soluble in water or (II) bound to the surface of a lipid bilayer as an unstructured monomer, and (III) inserted across the bilayer as a monomeric α-helix. We used fluorescence spectroscopy and isothermal titration calorimetry to study the interactions of pHLIP with a palmitoyloleoylphosphatidylcholine (POPC) lipid bilayer and to calculate the transition energies between states. We found that the Gibbs free energy of binding to a POPC surface at low pHLIP concentration (state I–state II transition) at 37°C is approximately −7 kcal/mol near neutral pH and that the free energy of insertion and folding across a lipid bilayer at low pH (state II–state III transition) is nearly −2 kcal/mol. We discuss a number of related thermodynamic parameters from our measurements. Besides its fundamental interest as a model system for the study of membrane protein folding, pHLIP has utility as an agent to target diseased tissues and translocate molecules through the membrane into the cytoplasm of cells in environments with elevated levels of extracellular acidity, as in cancer and inflammation. The results give the amount of energy that might be used to move cargo molecules across a membrane.

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.

ABSTRACT The 1,024-amino-acid acylated hemolysin of Escherichia coli subverts host cell functions and causes cell lysis. Both activities require insertion of the toxin into target mammalian cell membranes. To identify directly the principal toxin sequences dictating membrane binding and insertion, we assayed the lipid bilayer interaction of native protoxin, stably active toxin, and recombinant peptides. Binding was assessed by flotation of protein-liposome mixtures through density gradients, and insertion was assessed by labeling with a photoactivatable probe incorporated into the target lipid bilayer. Both the active acylated hemolysin and the inactive unacylated protoxin were able to bind and also insert. Ca2+binding, which is required for toxin activity, did not influence the in vitro interaction with liposomes. Three overlapping large peptides were expressed separately. A C-terminal peptide including residues 601 to 1024 did not interact in either assay. An internal peptide spanning residues 496 to 831, including the two acylation sites, bound to phospholipid vesicles and showed a low level of insertion-dependent labeling. In vitro acylation had no effect on the bilayer interaction of either this peptide or the full-length protoxin. An N-terminal peptide comprising residues 1 to 520 also bound to phospholipid vesicles and showed strong insertion-dependent labeling, ca. 5- to 25-fold that of the internal peptide. Generation of five smaller peptides from the N-terminal region identified the principal determinant of lipid insertion as the hydrophobic sequence encompassing residues 177 to 411, which is conserved among hemolysin-related toxins.

Vibrio parahaemolyticus secretes thermostable direct hemolysin (TDH), a major virulence factor. Earlier studies report that TDH is a pore-forming toxin. However, the characteristics of pores formed by TDH in the lipid bilayer, which is permeable to small ions, remain to be elucidated. Ion channel-like activities were observed in lipid bilayers containing TDH. Three types of conductance were identified. All the channels displayed relatively low ion selectivity, and similar ion permeability. The Cl− channel inhibitors, DIDS, glybenclamide, and NPPB, did not affect the channel activity of pores formed by TDH. R7, a mutant toxin of TDH, also forms pores with channel-like activity in lipid bilayers. The ion permeability of these channels is similar to that of TDH. R7 binds cultured cells and liposomes to a lower extent, compared to TDH. R7 does not display significant hemolytic activity and cell cytotoxicity, possibly owing to the difficulty of insertion into lipid membranes. Once R7 is assembled within lipid membranes, it may assume the same structure as TDH. The authors propose that the single glycine at position 62, substituted with serine in the R7 mutant toxin, plays an important role in TDH insertion into the lipid bilayer.

Knowledge about the insertion and stabilization of membrane proteins is a key step towards understanding their function and enabling membrane protein design. Transmembrane helices are normally quite hydrophobic to insert efficiently, but there are many exceptions with unfavorable polar or titratable residues. Since evolutionary conserved these amino acids are likely of paramount functional importance, e.g. the four arginines in the S4 voltage sensor helix of voltage-gated ion channels. This has lead to vivid discussion about their conformation, protonation state and cost of insertion. To address such questions, the main focus of this thesis has been membrane protein solvation in lipid bilayers, evaluated using molecular dynamics simulations methods. A main result is that polar and charged amino acids tend to deform the bilayer by pulling water/head-groups into the hydrophobic core to keep their hydrogen bonds paired, thus demonstrating the adaptiveness of the membrane to allow specific and quite complex solvation. In addition, this retained hydration suggests that the solvation cost is mainly due to entropy, not enthalpy loss. To further quantify solvation properties, free energy profiles were calculated for all amino acids in pure bilayers, with shapes correlating well with experimental in vivo values but with higher magnitudes. Additional profiles were calculated for different protonation states of the titratable amino acids, varying lipid composition and with transmembrane helices present in the bilayer. While the two first both influence solvation properties, the latter seems to be a critical aspect. When the protein fraction in the models resemble biological membranes, the solvation cost drops significantly - even to values compatible with experiment. In conclusion, by using simulation based methods I have been able to provide atomic scale explanations to experimental results, and in particular present a hypothesis for how the solvation of charged groups occurs.

L'objectif de l'approche des simulations moléculaires interactive (Interactive Molecular Simulations - IMS) est d'observer en direct la dynamique conformationnelle d'une simulation moléculaire en cours. Le retour visuel instantané permet un suivi instructif ainsi que l'observation des changements structurels imposés par la manipulation de l'IMS par l'utilisateur. J'ai mené une étude approfondie des connaissances pour rassembler et synthétiser l'ensemble des recherches qui ont développé l'IMS. La dynamique moléculaire interactive (Interactive Molecular Dynamics - IMD) est l'un des premiers protocoles IMS qui a posé les bases du développement de cette approche. Mon laboratoire de thèse s'est inspirée de celle-ci pour développer le moteur de simulation BioSpring basé sur le modèle de réseaux élastique. Ce modèle permet de simuler la flexibilité de grands ensembles biomoléculaires et ainsi potentiellement révéler des changements à longue échelle de temps qui ne seraient pas facilement saisis par la dynamique moléculaire. Ce moteur de simulation ainsi que le logiciel de visualisation UnityMol, développé par le biais du moteur de jeu Unity3D, et liés par l'interface de communication MDDriver ont été étendus pour les faire converger vers une suite logicielle complète. Le but est de fournir à un expérimentateur, qu'il soit expert ou profane, une boîte à outils complète pour modéliser, afficher et contrôler interactivement l'ensemble des paramètres d'une simulation. L'implémentation particulière d'un tel protocole, basé sur une communication formalisée et extensible entre les différents composants, a été pensée pour pouvoir facilement intégrer de nouvelles possibilités de manipulation interactive et des jeux de données expérimentales qui s'ajouteront aux contraintes imposées à la simulation. L'utilisateur peut donc manipuler la molécule d'intérêt sous le contrôle des propriétés biophysiques intégrés dans le modèle simulé, tout en ayant la possibilité de piloter à la volée les paramètres de simulation. Aussi, un des objectifs initiaux de cette thèse était d'intégrer la gestion des contraintes d'interaction ambigües du logiciel d'amarrage biomoléculaire HADDOCK directement dans UnityMol, rendant possible l'utilisation de ces mêmes contraintes à une variété de moteurs de simulations. Un axe principal de ces recherches était de développer un algorithme de positionnement rapide et interactif de protéines dans des membranes implicite tiré d'un modèle appelé Integrative Membrane Protein and Lipid Association Method (IMPALA) développée par l'équipe de Robert Brasseur en 1998. La première étape consistait à effectuer une recherche approfondie des conditions dans lesquelles les expériences ont été réalisées à l'époque, afin de vérifier la méthode et de valider notre propre implémentation. Nous verrons qu'elle ouvre des questions intéressantes sur la manière dont on peut reproduire les expériences scientifiques. L'étape finale qui conclue cette thèse était le développement d'une nouvelle méthode universelle d'interaction lipide-protéine, UNILIPID, qui est un modèle d'incorporation interactif de protéines dans les membranes implicites. Elle est indépendante de l'échelle de représentation, peut être appliquée à des niveaux tout atomes, gros-grains jusqu'au niveau d'un grain par acide aminé. La représentation de la dernière version Martini3[6] ainsi qu'une méthode d'échantillonnage Monte-Carlo et de simulation de dynamique des corps rigides ont été spécialement intégrés à la méthode, en plus de divers outils de préparation de systèmes. En outre, UNILIPID est une approche versatile qui reproduit précisément des termes d'hydrophobicité expérimentaux pour chaque acide aminé. En plus de membranes implicites simples, je décrirai une implémentation analytique de membranes doubles ainsi qu'une généralisation à des membranes de forme arbitraire, toutes deux s'appuyant sur des applications inédites
The goal of Interactive Molecular Simulations (IMS) is to observe the conformational dynamics of a molecular simulation in real-time. Instant visual feedback enables informative monitoring and observation of structural changes imposed by the user's manipulation of the IMS. I conducted an in-depth study of knowledge to gather and synthesize all the research that has developed IMS. Interactive Molecular Dynamics (IMD) is one of the first IMS protocols that laid the foundation for the development of this approach. My thesis laboratory was inspired by IMD to develop the BioSpring simulation engine based on the elastic network model. This model allows for the simulation of the flexibility of large biomolecular ensembles, potentially revealing long-timescale changes that would not be easily captured by molecular dynamics. This simulation engine, along with the UnityMol visualization software, developed through the Unity3D game engine, and linked by the MDDriver communication interface, has been extended to converge towards a complete software suite. The goal is to provide an experimenter, whether an expert or novice, with a complete toolbox for modeling, displaying, and interactively controlling all parameters of a simulation. The particular implementation of such a protocol, based on formalized and extensible communication between the different components, was designed to easily integrate new possibilities for interactive manipulation and sets of experimental data that will be added to the restraints imposed on the simulation. Therefore, the user can manipulate the molecule of interest under the control of biophysical properties integrated into the simulated model, while also having the ability to dynamically adjust simulation parameters. Furthermore, one of the initial objectives of this thesis was to integrate the management of ambiguous interaction constraints from the HADDOCK biomolecular docking software directly into UnityMol, making it possible to use these same restraints with a variety of simulation engines. A primary focus of this research was to develop a fast and interactive protein positioning algorithm in implicit membranes using a model called the Integrative Membrane Protein and Lipid Association Method (IMPALA), developed by Robert Brasseur's team in 1998. The first step was to conduct an in-depth search of the conditions under which the experiments were performed at the time to verify the method and validate our own implementation. We will see that this opens up interesting questions about how scientific experiments can be reproduced. The final step that concluded this thesis was the development of a new universal lipid-protein interaction method, UNILIPID, which is an interactive protein incorporation model in implicit membranes. It is independent of the representation scale and can be applied at the all-atom, coarse-grain, or grain-by-grain level. The latest Martini3 representation, as well as a Monte Carlo sampling method and rigid body dynamics simulation, have been specially integrated into the method, in addition to various system preparation tools. Furthermore, UNILIPID is a versatile approach that precisely reproduces experimental hydrophobicity terms for each amino acid. In addition to simple implicit membranes, I will describe an analytical implementation of double membranes as well as a generalization to arbitrarily shaped membranes, both of which rely on novel applications

Many signaling mechanisms in living cells occur at biological boundaries via cell surface receptors and membrane proteins embedded in lipid bilayers. The coordination of actions of sensory and motor neurons in the nervous system represents one example of many that heavily depends on lipid membrane bound receptor mediated signaling. As a result, chemical and biological toxins that disrupt these neural signals can have severe physiological effects, including paralysis and death. Botulinum neurotoxin Type A (BoNT/A) is a proteolytic toxin that inserts through vesicle membranes and cleaves membrane receptors involved with synaptic acetylcholine uptake and nervous system signal conduction. In this work, we investigate the use of a Bioinspired liquid-supported interface bilayer for studying the insertion of BoNT/A toxin molecules into synthetic lipid bilayers. DPhPC lipid bilayers are formed using the regulated attachment method (RAM), as developed by Sarles and Leo, and we perform current measurements on membranes exposed to BoNT/A toxin to characterize activity of toxin interacting with the synthetic bilayer. Control tests without toxin present are also presented. The results of these tests show an increase in the magnitude of current through the bilayer when the toxin is included. We interpret these initial results to mean that incorporation of BoNT/A toxin at a high concentration in an interface bilayer increases the permeability of the membrane as a result of toxin molecules spanning the thickness of the bilayer.

One of the most important biological components is lipid nanobio membrane. The lipid membranes of alive cells and their mechanical properties play an important role in biophysical investigations. Some proteins affect the shape and properties of the nanobio membrane while interacting with it. In this study a multiscale approach is experienced: first a 100ns all atom (fine-grained) molecular dynamics simulation is done to investigate the binding of CTX A3, a protein from snake venom, to a phosphatidylcholine lipid bilayer, second, a 5 micro seconds coarse-grained molecular dynamics simulation is carried out to compute the pressure tensor, lateral pressure, surface tension, and first moment of lateral pressure. Our simulations reveal that the insertion of CTX A3 into one monolayer results in an asymmetrical change in the lateral pressure and distribution of surface tension of the individual bilayer leaflets. The relative variation in the surface tension of the two monolayers as a result of a change in the contribution of the various intermolecular forces may be expressed morphologically and lead to deformation of the lipid membrane.

Our research focuses on creating smart materials that utilize synthetic cell membranes assembled at liquid interfaces for autonomic sensing, actuation, and energy conversion. Unlike single membrane assemblies, systems featuring many membranes have the potential to offer multi-functionality, greater transduction sensitivity, and even emergent behaviors in response to environmental stimuli, similar to living tissue, which utilizes networks of highly packed cells to accomplish tasks. Here, we present for the first time a novel microfluidic platform capable of generating a stream of alternating droplet compositions, i.e. A-B-A-B, and sequentially capturing these droplets in precise locations to enable the spontaneous formation of synthetic lipid bilayers between droplets of different compositions (i.e. A and B) in an enclosed substrate. This platform preserves a key feature of the droplet interface bilayer (DIB) method, which allows asymmetric conditions within and across the membrane to be prescribed by simply using droplets containing different species. In this work, we demonstrate the ability to assemble bilayers consisting of asymmetric lipid compositions and, separately, show that alternating droplets containing the same lipid type can also be used to control the direction of ion channel insertion. In the first study, A and B droplet types contain liposomes comprised of different lipid types, which are used to establish an asymmetric composition of the leaflets that make up the lipid bilayer. This asymmetry results in a dc, non-zero membrane potential, which we measure via membrane capacitance versus bias voltage. In the second study, alamethicin peptides are included in only one of the droplet types, which enable voltage-dependent insertion to occur only at one polarity. Cyclic voltammetry measurements are performed to confirm the direction of insertion of alamethicin channels in bilayers. Also, these results show the ability to perform simultaneously electrical measurements on multiple DIB, which increases the experimental capacity and efficiency of a microfluidic approach. The ability to produce alternating droplets in a high throughput manner with electrical access provides a system to investigate the effects of lipid asymmetry on the function of membrane proteins in a controlled model system.

Molecular models of lipid bilayers have ignored the interface of two monolayers of nanobiomembranes in detail by now, however in this paper a new physical model is proposed based on variation of surface tension in the interface of two monolayers of membrane. Experimental results have shown that some peptides and proteins like antimicrobial peptides and cytotoxins are able to change the shape of — or in some cases to destroy — the bilayer membrane during insertion to external monolayer. All interfaces in nanobiomembrane are liquid-liquid type. In this paper appropriate ensembles to simulate liquid/liquid interfaces are presented with special focus on proper ones for surface tension analysis.

Recent research in our group has shown that artificial cell membranes formed at the base of a hair-like structure can be used to sense air flow in a manner similar to the mechanotransduction processes found in mammalian hair cells. Our previous work demonstrated that a single artificial hair cell can be formed in an open substrate. However, that study also motivated the need to develop fully-encapsulated devices that feature arrays of hair-cells. Since the transduction element in this concept is an artificial cell membrane, or lipid bilayer, this work investigates two parallel substrate designs for providing encapsulation and a method for forming arrays of bilayers. In one effort, a flexible substrate with internal compartments for hosting the biomolecules and mating cap are constructed and experimentally characterized. The regulated attachment method (RAM) is used to form interface bilayers within the sealed device. Capacitance measurements of the sealed interface bilayer show that the sealing cap slightly compresses the bottom insert and reduces the size of the enclosed bilayer. Single channel measurements of alamethicin peptides further verify that the sealed device, which is also leak-proof under water, can be used to detect the insertion and gating activity of transmembrane proteins in the membrane. The second effort pursued herein is the fabrication and initial testing of a method to form arrays of interface bilayers by using anchored hydrogel pads that support curved aqueous lenses in oil. In this fashion, the configuration of the array does not require manipulating droplets, but instead depends on the arrangement of the built-in gels used to support the aqueous lenses. As with RAM, mechanical force is used to promote contact of adjacent aqueous lenses held in the flexible substrate. Initial tests show that gel-supported lenses can be used for forming multiple lipid bilayers within the device and that these interfaces can be interrogated individually or collectively using an electrode switching circuit.

Memristors are solid-state devices that exhibit voltage-controlled conductance. This tunable functionality enables the implementation of biologically-inspired synaptic functions in solid-state neuromorphic computing systems. However, while memristors are meant to emulate an intricate signal transduction process performed by soft biomolecular structures, they are commonly constructed from silicon- or polymer-based materials. As a result, the volatility, intricate design, and high-energy resistance switching in memristive devices, usually, leads to energy consumption in memristors that is several orders of magnitude higher than in natural synapses. Additionally, solid-state memristors fail to achieve the coupled dynamics and selectivity of synaptic ion exchange that are believed to be necessary for initiating both short- and long-term potentiation (STP and LTP) in neural synapses, as well as paired-pulse facilitation (PPF) in the presynaptic terminal. LTP is a phenomenon mostly responsible for driving synaptic learning and memory, features that enable signal transduction between neurons to be history-dependent and adaptable. In contrast, current memristive devices rely on engineered external programming parameters to imitate LTP. Because of these fundamental differences, we believe a biomolecular approach offers untapped potential for constructing synapse-like systems. Here, we report on a synthetic biomembrane system with biomolecule-regulated (alamethicin) variable ion conductance that emulates vital operational principals of biological synapse. The proposed system consists of a synthetic droplet interface bilayer (DIB) assembled at the conjoining interface of two monolayer-encased aqueous droplets in oil. The droplets contain voltage-activated alamethicin (Alm) peptides, capable of creating conductive pathways for ion transport through the impermeable lipid membrane. The insertion of the peptides and formation of transmembrane ion channels is achieved at externally applied potentials higher than ∼70 m V. Just like in biological synapses, where the incorporation of additional receptors is responsible for changing the synaptic weight (i.e. conductance), we demonstrate that the weight of our synaptic mimic may be changed by controlling the number of alamethicin ion channels created in a synthetic lipid membrane. More alamethicin peptides are incorporated by increasing the post-threshold external potential, thus leading to higher conductance levels for ion transport. The current-voltage responses of the alamethicin-based synapse also exhibit significant “pinched” hysteresis — a characteristic of memristors that is fundamental to mimicking synapse plasticity. We demonstrate the system’s capability of exhibiting STP/PPF behaviors in response to high-frequency 50 ms, 150 mV voltage pulses. We also present and discuss an analytical model for an alamethicin-based memristor, classifying that later as a “generic memristor”.