Membrane Biophysics

The bulk structure of biological membranes consists of a bilayer of amphipathic lipids. According to the fluid mosaic model proposed by Singer and Nicholson, the glycerophospholipid bilayer is a two-dimensional fluid construct that allows the lateral movement of membrane components. Different types of lateral interactions among membrane components can take place, giving rise to multiple levels of lateral order that lead to highly organized structures. Early observations suggested that some of the lipid components of biological membranes may play active roles in the creation of these levels of order. In the late 1980s, a diverse series of experimental findings collectively gave rise to the lipid raft hypothesis. Lipid rafts were originally defined as membrane domains, i.e., ordered structures created as a consequence of the lateral segregation of sphingolipids and differing from the surrounding membrane in their molecular composition and properties. This definition was subsequently modified to introduce the notion that lipid rafts correspond to membrane areas stabilized by the presence of cholesterol within a liquid-ordered phase. During the past two decades, the concept of lipid rafts has become extremely popular among cell biologists, and these structures have been suggested to be involved in a great variety of cellular functions and biological events. During the same period, however, some groups presented experimental evidence that appeared to contradict the basic tenets that underlie the lipid raft concept. The concept is currently being re-defined, with greater consistency regarding the true nature and role of lipid rafts. In this article we will review the concepts, criticisms, and the novel confirmatory findings relating to the lipid raft hypothesis.

ARISING FROM S.A. Shelby et al. Nature Chemical Biology https://doi.org/10.1038/s41589-023-01268-8 (2023)

Phase separation has been a popular framework for interpreting molecular organization in membranes since the 1970s1-3. However, it is typically invoked without evidence for phase coexistence. In a recent article, Shelby et al. present a correlation between B cell receptor (BCR) colocalization with fluorescent probes in live-cell and in vitro systems, which they state “directly and conclusively establishes phase separation as a functional organizing principle in cell plasma membranes”4. This verdict assumes causation from correlation while neglecting simpler explanations. The rationale for comparing isolated vesicles with live cells is precisely that phase separation is not directly observed in the latter. If the authors argue that phases are routinely induced by BCR clusters in live cells, as might be tentatively inferred from the paper’s title, conclusions, and illustrations, they are contradicting the well-documented idea that membrane proteins are surrounded by heterogeneous lipid distributions5.

Advances in fluorescence microscopy have enabled high-resolution tracking of individual biomolecules in living cells. However, accurate estimation of diffusion parameters from single-particle trajectories remains challenging due to static and dynamic localization errors inherent in these measurements. While previous studies have characterized how such errors affect mean-squared displacement (MSD) analysis, practical guidelines for minimizing them during data acquisition and correcting them during analysis are still lacking. Here, we combine theoretical modeling and simulations to evaluate how exposure time and sampling rate influence the accuracy of MSD-based inference under fractional Brownian motion (FBM), a canonical model of anomalous diffusion. We demonstrate that decoupling exposure and sampling times enables escape from the error-prone regime, thus improving inference accuracy, and that incorporating an offset in nonlinear MSD fitting substantially improves the estimation of the anomalous diffusion exponent. We validate this framework using trajectories of cytoplasmic particles in Escherichia coli, recovering consistent diffusion parameters across multiple data sets. Our findings offer practical strategies to improve both experimental design and data analysis in single-particle tracking of live or synthetic systems.

ABSTRACTMany, but not all, organisms use quinones to conserve energy in their electron transport chains. Fermentative bacteria and methane-producing archaea (methanogens) do not produce quinones but have devised other ways to generate ATP. Methanophenazine (MPh) is a unique membrane electron carrier found inMethanosarcinaspecies that plays the same role as quinones in the electron transport chain. To extend the analogy between quinones and MPh, we compared the MPh pool sizes between two well-studiedMethanosarcinaspecies,Methanosarcina acetivoransC2A andMethanosarcina barkeriFusaro, to the quinone pool size in the bacteriumEscherichia coli. We found the quantity of MPh per cell increases as cultures transition from exponential growth to stationary phase, and absolute quantities of MPh were 3-fold higher inM. acetivoransthan inM. barkeri. The concentration of MPh suggests the cell membrane ofM. acetivorans, but not ofM. barkeri, is electrically quantized as if it were a single conduct...

Cellular membranes are highly organized structures with multiple and multi-dimensional levels of order where lipid components are active players. The lipid role is especially evident in rafts, where lipid-driven collective interaction dictates the local structure of a membrane. However, lipids play as well other roles in many aspects of membrane mechanics and function. In this review, we would like to re-focus the attention of the readers on the importance of gangliosides in organizing the fine structure of cellular membranes, in lateral and transverse directions. Important biological events are likely to be affected such as the dynamic control of the shape of specialized plasma membrane areas and of the intracellular organelles, the in-and outward budding and fusion of membrane vesicles, the physical and functional coupling of the outer and the inner plasma membrane leaflet, involved in the transduction of signals across the membrane.

Translational motion of neurotransmitter receptors is key for determining receptor number at the synapse and hence, synaptic efficacy. We combine live-cell STORM superresolution microscopy of nicotinic acetylcholine receptor (nAChR) with single-particle tracking, mean-squared displacement (MSD), turning angle, ergodicity, and clustering analyses to characterize the lateral motion of individual molecules and their collective behaviour. nAChR diffusion is highly heterogeneous: subdiffusive, Brownian and, less frequently, superdiffusive. At the single-track level, free walks are transiently interrupted by ms-long confinement sojourns occurring in nanodomains of ~36 nm radius. Cholesterol modulates the time and the area spent in confinement. Turning angle analysis reveals anticorrelated steps with time-lag dependence, in good agreement with the permeable fence model. At the ensemble level, nanocluster assembly occurs in second-long bursts separated by periods of cluster disassembly. Thu...

Biological membranes constitute a critical component in all living cells. In addition to providing a conducive environment to a wide range of cellular processes, including transport and signaling, mounting evidence has established active participation of specific lipids in modulating membrane protein function through various mechanisms. Understanding lipid-protein interactions underlying these mechanisms at a sufficiently high resolution has proven extremely challenging, partly due to the semi-fluid nature of the membrane. In order to address this challenge computationally, multiple methods have been developed, including an alternative membrane representation termed highly mobile membrane mimetic (HMMM) in which lateral lipid diffusion has been significantly enhanced without compromising atomic details. The model allows for efficient sampling of lipid-protein interactions at atomic resolution, thereby significantly enhancing the effectiveness of molecular dynamics simulations in cap...

The concept of transient nanometric domains known as lipid rafts has brought interest to reassess the validity of the Singer-Nicholson model of a fluid bilayer for cell membranes. However, this new view is still insufficient to explain the cellular control of surface lipid diversity or membrane deformability. During the past decade, the hypothesis that some lipids form large (submicrometric/ mesoscale vs nanometric rafts) and stable (> min vs sec) membrane domains has emerged, largely based on indirect methods. Morphological evidence for stable submicrometric lipid domains, wellaccepted for artificial and highly specialized biological membranes, was further reported for a variety of living cells from prokaryotes to yeast and mammalian cells. However, results remained questioned based on limitations of available fluorescent tools, use of poor lipid fixatives, and imaging artifacts due to non-resolved membrane projections. In this review, we will discuss recent evidence generated using powerful and innovative approaches such as lipid-specific toxin fragments that support the existence of submicrometric domains. We will integrate documented mechanisms involved in the formation and maintenance of these domains, and provide a perspective on their relevance on membrane deformability and regulation of membrane protein distribution.

The nuclear envelope segregates the nucleoplasm from the cytoplasm and is a key feature of eukaryotic cells. Nuclear envelope architecture is comprised of two concentric membrane shells which fuse at multiple sites and yet maintain a uniform separation of 30-50 nm over the rest of the membrane. Studies have revealed the roles for numerous nuclear proteins in forming and maintaining the architecture of the nuclear envelope. However, there is a lack of consensus on the fundamental forces and physical mechanisms that establish the geometry. The objective of this review is to discuss recent findings in the context of membrane mechanics in an effort to define open questions and possible answers.

Supported planar phospholipid membranes are used in a range of biophysical measurements, typically for characterizing protein–membrane interactions. Liposome deposition is the most common method to create such membranes. The ability of liposomes to fuse into a lamellar membrane during deposition is strongly dependent on the surface chemistry; some important substrate materials such as oxidized gold do not promote liposome fusion. Circumventing this determinism poses an enduring challenge to membrane biophysics. Here, the authors show that the effect of surface chemistry can be overcome by using osmotic stress. Reproducible single bilayer coverage was achieved on oxidized gold surface from liposomes of a variety of lipid compositions, as demonstrated by quartz crystal microbalance measurements and confirmed via fluorescence microscopy imaging. The continuity of the deposit was confirmed by fluorescence recovery after photobleaching. Using mixtures of di-myristoyl and di-palmitoyl lip...

Biological membranes are highly dynamic in their ability to orchestrate vital mechanisms including cellular protection, organelle compartmentalization, cellular biomechanics, nutrient transport, molecular/enzymatic recognition, and membrane fusion. Controlling lipid composition of different membranes allows cells to regulate their membrane characteristics, thus modifying their physical properties to permit specific protein interactions and drive structural function (membrane deformation facilitates vesicle budding and fusion) and signal transduction. Yet, how lipids control protein structure and function is still poorly understood and needs systematic investigation. In this review, we explore different in vitro membrane models and summarize our current understanding of the interplay between membrane biophysical properties and lipid-protein interaction, taken as example few proteins involved in muscular activity (dystrophin), digestion and Legionella pneumophila effector protein DrrA. The monolayer model with its movable barriers aims to mimic any membrane deformation while surface pressure modulation imitates lipid packing and membrane curvature changes. It is frequently used to investigate peripheral protein binding to the lipid headgroups. Examples of how lipid lateral pressure modifies protein interaction and organization within the membrane are presented using various biophysical techniques. Interestingly, the shear elasticity and surface viscosity of the monolayer will increase upon specific protein(s) binding, supporting the importance of such mechanical link for membrane stability. The lipid bilayer models such as vesicles are not only used to investigate direct protein binding based on the lipid nature, but more importantly to assess how local membrane curvature (vesicles with different size) influence the binding properties of a protein. Also, supported lipid bilayer model has been used widely to characterize diffusion law of lipids within the bilayer and/or protein/biomolecule binding and diffusion on the membrane. These membrane models continue to elucidate important advances regarding the dynamic properties harmonizing lipid-protein interaction.

The role of the Warburg effect in cancer remains to be elucidated with a resurgence in research efforts over the past decade. Why a cancer cell would prefer to use energy inefficient glycolysis, leading to an alteration of pH both inside and outside of the cell, remains to be uncovered. The development of MDR represents a major challenge in the treatment of cancer and it is explained, so far, by the over expression of drug transporters such as the well-known and archetypal P-glycoprotein (Pgp). However, controversies exist regarding the function of Pgp in multi-drug resistance. We suggest here that Pgp-mediated MDR relies fundamentally on pH alterations mediated by the Warburg effect. Furthermore, we propose that the use of proton pump and/or transporters inhibitors (PPIs/PTIs) in cancer are key to controlling both MDR, i.e. sensitize tumors to antineoplastic agents, and drug-related adverse effects.

Examining the fundamental structure and processes of living cells at the nanoscale poses a unique analytical challenge, as cells are dynamic, chemically diverse, and fragile. A case in point is the cell membrane, which is too small to be seen directly with optical microscopy and provides little observational contrast for other methods. As a consequence, nanoscale characterization of the membrane has been performed ex vivo or in the presence of exogenous labels used to enhance contrast and impart specificity. Here, we introduce an isotopic labeling strategy in the gram-positive bacterium Bacillus subtilis to investigate the nanoscale structure and organization of its plasma membrane in vivo. Through genetic and chemical manipulation of the organism, we labeled the cell and its membrane independently with specific amounts of hydrogen (H) and deuterium (D). These isotopes have different neutron scattering properties without altering the chemical composition of the cells. From neutron s...

Depression is one of the most common psychiatric diseases in the population. Agomelatine is a novel antidepressant drug with melatonin receptor agonistic and serotonin 5-HT2C antagonistic properties. Furthermore, being a melatonergic drug, agomelatine has the potential of being used in therapeutic applications like melatonin as an antioxidant, anti-inflammatory and antiapoptotic drug. The action mechanism of agomelatine on the membrane structure has not been clarified yet. In the present study, we aimed to investigate the interaction of agomelatine with model membranes of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylgylcerol (DPPG) by Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). We found that agomelatine interacts with the head group in such a manner that it destabilizes the membrane architecture to a large extent. Thus, agomelatine causes alterations in the order, packing and dynamics of the DPPC and DPPG model memb...

Membrane bending is an extensively studied problem from both modeling and experimental perspectives because of the wide implications of curvature generation in cell biology. Many of the curvature generating aspects in membranes can be attributed to interactions between proteins and membranes. These interactions include protein diffusion and formation of aggregates due to protein-protein interactions in the plane of the membrane. Recently, we developed a model that couples the in-plane flow of lipids and diffusion of proteins with the out-of-plane bending of the membrane. Building on this work, here, we focus on the role of explicit aggregation of proteins on the surface of the membrane in the presence of membrane bending and diffusion. We develop a comprehensive framework that includes lipid flow, membrane bending, the entropy of protein distribution, along with an explicit aggregation potential and derive the governing equations for the coupled system. We compare this framework to the Cahn-Hillard formalism to predict the regimes in which the proteins form patterns on the membrane. We demonstrate the utility of this model using numerical simulations to predict how aggregation and diffusion, when coupled with curvature generation, can alter the landscape of membrane-protein interactions.

A theoretical solvation model ofpeptides and proteins that mimics the heterogeneous membranewater system was proposed. Our approach is based on the combined use of atomic parameters of solvation for water and hydrocarbons, which approximates the hydrated polar groups and acyl chains of lipids, respectively. This model was tested in simulations of several peptides: a nonpolar 20-mer polyleucine, a hydrophobic peptide with terminal polar groups, and a strongly amphiphilic peptide. The conformational space of the peptides in the presence of the membrane was studied by the Monte Carlo method. Unlike a polar solvent and vacuum, the membrane-like environment was shown to stabilize the tx-helical conformation: low-energy structures have a helicity index of 100% in all cases. At the same time, the energetically most favorable orientations of the peptides relative to the membrane depend on their hydrophobic properties: nonpolar polyleucine is entirely immersed in the bilayer and the hydrophobic peptide with polar groups at the termini adopts a transbilayer orientation, whereas the amphiphilic peptide lies at the interface parallel to the membrane plane. The results of the simulations agree well with the available experimental data for these systems. In the following communications of this series, we plan to describe applications of the solvation model to membrane-bound proteins and peptides with biologically important functional activities.

Biological membranes are highly dynamic in their ability to orchestrate vital mechanisms including cellular protection, organelle compartmentalization, cellular biomechanics, nutrient transport, molecular/enzymatic recognition, and membrane fusion. Controlling lipid composition of different membranes allows cells to regulate their membrane characteristics, thus modifying their physical properties to permit specific protein interactions and drive structural function (membrane deformation facilitates vesicle budding and fusion) and signal transduction. Yet, how lipids control protein structure and function is still poorly understood and needs systematic investigation. In this review, we explore different in vitro membrane models and summarize our current understanding of the interplay between membrane biophysical properties and lipid-protein interaction, taken as example few proteins involved in muscular activity (dystrophin), digestion and Legionella pneumophila effector protein DrrA. The monolayer model with its movable barriers aims to mimic any membrane deformation while surface pressure modulation imitates lipid packing and membrane curvature changes. It is frequently used to investigate peripheral protein binding to the lipid headgroups. Examples of how lipid lateral pressure modifies protein interaction and organization within the membrane are presented using various biophysical techniques. Interestingly, the shear elasticity and surface viscosity of the monolayer will increase upon specific protein(s) binding, supporting the importance of such mechanical link for membrane stability. The lipid bilayer models such as vesicles are not only used to investigate direct protein binding based on the lipid nature, but more importantly to assess how local membrane curvature (vesicles with different size) influence the binding properties of a protein. Also, supported lipid bilayer model has been used widely to characterize diffusion law of lipids within the bilayer and/or protein/biomolecule binding and diffusion on the membrane. These membrane models continue to elucidate important advances regarding the dynamic properties harmonizing lipid-protein interaction.

Biological membranes are highly dynamic in their ability to orchestrate vital mechanisms including cellular protection, organelle compartmentalization, cellular biomechanics, nutrient transport, molecular/enzymatic recognition, and membrane fusion. Controlling lipid composition of different membranes allows cells to regulate their membrane characteristics, thus modifying their physical properties to permit specific protein interactions and drive structural function (membrane deformation facilitates vesicle budding and fusion) and signal transduction. Yet, how lipids control protein structure and function is still poorly understood and needs systematic investigation. In this review, we explore different in vitro membrane models and summarize our current understanding of the interplay between membrane biophysical properties and lipid-protein interaction, taken as example few proteins involved in muscular activity (dystrophin), digestion and Legionella pneumophila effector protein DrrA. The monolayer model with its movable barriers aims to mimic any membrane deformation while surface pressure modulation imitates lipid packing and membrane curvature changes. It is frequently used to investigate peripheral protein binding to the lipid headgroups. Examples of how lipid lateral pressure modifies protein interaction and organization within the membrane are presented using various biophysical techniques. Interestingly, the shear elasticity and surface viscosity of the monolayer will increase upon specific protein(s) binding, supporting the importance of such mechanical link for membrane stability. The lipid bilayer models such as vesicles are not only used to investigate direct protein binding based on the lipid nature, but more importantly to assess how local membrane curvature (vesicles with different size) influence the binding properties of a protein. Also, supported lipid bilayer model has been used widely to characterize diffusion law of lipids within the bilayer and/or protein/biomolecule binding and diffusion on the membrane. These membrane models continue to elucidate important advances regarding the dynamic properties harmonizing lipid-protein interaction.

High-resolution structural information on membrane proteins is essential for understanding cell biology and for structure-based design of new medical drugs and drug delivery strategies. X-ray diffraction (XRD) can provide Ångstrom-level information about the structure of membrane proteins. Ideally protein structures should be solved in environments as close to the original biological context as possible. However, it is virtually impossible to crystallize proteins within the complex environment of a biological membrane. Instead, membrane proteins are typically transferred from their native membrane environment into detergent micelles, chemically stabilized and crystallized, all of which can compromise the conformation. This makes it imperative to develop alternative highresolution techniques which are compatible with biological conditions. Here, we describe how a combination of surface-sensitive vibrational spectroscopy in model membranes and molecular dynamics simulations can account for the native membrane environment. We observe the structure of glycerol facilitator channel (GlpF), an aquaporin membrane channel finely tuned to selectively transport water and glycerol molecules across the membrane barrier. We find subtle but significant differences between the XRD structure and the inferred in situ structure of GlpF. SIGNIFICANCE: Proteins at the surfaces and interfaces of cells play important roles in biology. While methods to determine the structure of proteins with high resolution within crystals and in solution, it has been experimentally challenging to track membrane protein folding, motion and action within hydrated lipid bilayers. We show that a combination of crystallography, surface spectroscopy and computer simulations can elucidate the structure of a large membrane protein, aquaporin, when the methods are linked through theoretical modelling. Starting with the known structure of aquaporin in its crystalline form, we follow the structural relaxation into the interfacial structure within a membrane environment.

Understanding the structure of proteins at surfaces is key in fields such as biomaterials research, biosensor design, membrane biophysics, and drug design. A particularly important factor is the orientation of proteins when bound to a particular surface. The orientation of the active site of enzymes or protein sensors and the availability of binding pockets within membrane proteins are important design parameters for engineers developing new sensors, surfaces, and drugs. Recently developed methods to probe protein orientation, including immunoessays and mass spectrometry, either lack structural resolution or require harsh experimental conditions. We here report a new method to track the absolute orientation of interfacial proteins using phase-resolved sum frequency generation spectroscopy in combination with molecular dynamics simulations and theoretical spectral calculations. As a model system we have determined the orientation of a helical lysine-leucine peptide at the air−water interface. The data show that the absolute orientation of the helix can be reliably determined even for orientations almost parallel to the surface.

Lipid membranes, particularly under nonequilibrium conditions, have recently been investigated ever more vigorously because of their relevance in the biological context. We survey our recent approaches to the theoretical study of lipid bilayers that are perturbed in different ways. Self-organization phenomena involving curvature and/or composition spatiotemporal organization are investigated in membrane systems subjected to externally induced chemical reactions, transversal mass transport and insertion of proteins. The outcomes of these studies are expected to be applicable to different curvature and lateral organization phenomena in synthetic lipid bilayers and also in plasmatic cell membranes.

The curvature sensitive localization of proteins on membranes is vital for many cell biological processes. Coarse-grained models are routinely employed to study the curvature sensing phenomena and membrane morphology at the length scale of few micrometers. Two prevalent phenomenological models exist for modeling experimental observations of curvature sensing, (1) the spontaneous curvature model and (2) the curvature mismatch model, which differ in their treatment of the change in elastic energy due to the binding of proteins on the membrane. In this work, the prediction of sensing and generation behaviour, by these two models, are investigated using analytical calculations as well as Dynamic Triangulation Monte Carlo simulations of quasi-spherical vesicles. While the spontaneous curvature model yields a monotonically decreasing sensing curve as a function of vesicle radius, the curvature mismatch model results in a non-monotonic sensing curve. We highlight the main differences in the interpretation of the protein-related parameters in the two models. We further propose that the spontaneous curvature model is appropriate for modeling peripheral proteins employing the hydrophobic insertion mechanism, with minimal modification of membrane rigidity, while the curvature mismatch model is appropriate for modeling curvature generation using scaffolding mechanism where there is significant stiffening of the membrane due to protein binding.

The curvature sensitive localization of proteins on membranes is vital for many cell biological processes. Coarse-grained models are routinely employed to study the curvature sensing phenomena and membrane morphology at the length scale of few micrometers. Two prevalent phenomenological models exist for modeling experimental observations of curvature sensing, (1) the spontaneous curvature model and (2) the curvature mismatch model, which differ in their treatment of the change in elastic energy due to the binding of proteins on the membrane. In this work, the prediction of sensing and generation behaviour, by these two models, are investigated using analytical calculations as well as Dynamic Triangulation Monte Carlo simulations of quasi-spherical vesicles. While the spontaneous curvature model yields a monotonically decreasing sensing curve as a function of vesicle radius, the curvature mismatch model results in a non-monotonic sensing curve. We highlight the main differences in the interpretation of the protein-related parameters in the two models. We further propose that the spontaneous curvature model is appropriate for modeling peripheral proteins employing the hydrophobic insertion mechanism, with minimal modification of membrane rigidity, while the curvature mismatch model is appropriate for modeling curvature generation using scaffolding mechanism where there is significant stiffening of the membrane due to protein binding.

Research investigating lipid membrane curvature generation and sensing is a rapidly developing frontier in membrane physical chemistry and biophysics. The fast recent progress is based on the discovery of a plethora of proteins involved in coupling membrane shape to cellular membrane function, the design of new quantitative experimental techniques to study aspects of membrane curvature, and the development of analytical theories and simulation techniques that allow a mechanistic interpretation of quantitative measurements. The present review first provides an overview of important classes of membrane proteins for which function is coupled to membrane curvature. We then survey several mechanisms that are assumed to underlie membrane curvature sensing and generation. Finally, we discuss relatively simple thermodynamic/mechanical models that allow quantitative interpretation of experimental observations.

Cell-penetrating peptides (CPPs) are short peptides that can translocate and transport cargoes into the intracellular milieu by crossing biological membranes. The mode of interaction and internalization of cell-penetrating peptides has long been controversial. While their interaction with anionic membranes is quite well understood, the insertion and behavior of CPPs in zwitterionic membranes, a major lipid component of eukaryotic cell membranes, is poorly studied. Herein, we investigated the membrane insertion of RW16 into zwitterionic membranes, a versatile CPP that also presents antibacterial and antitumor activities. Using complementary approaches, including NMR spectroscopy, fluorescence spectroscopy, circular dichroism, and molecular dynamic simulations, we determined the high-resolution structure of RW16 and measured its membrane insertion and orientation properties into zwitterionic membranes. Altogether, these results contribute to explaining the versatile properties of this...

We investigate the Poisson ratio n of fluid lipid bilayers, i.e., the question how area strains compare to the changes in membrane thickness (or, equivalently, volume) that accompany them. We first examine existing experimental results on the area-and volume compressibility of lipid membranes. Analyzing them within the framework of linear elasticity theory for homogeneous thin fluid sheets leads us to conclude that lipid membrane deformations are to a very good approximation volume-preserving, with a Poisson ratio that is likely about 3% smaller than the common soft matter limit n ¼ 1 2. These results are fully consistent with atomistic simulations of a DOPC membrane at varying amount of applied lateral stress, for which we instead deduce n by directly comparing area-and volume strains. To assess the problematic assumption of transverse homogeneity, we also define a depth-resolved Poisson ratio n(z) and determine it through a refined analysis of the same set of simulations. We find that throughout the membrane's thickness, n(z) is close to the value derived assuming homogeneity, with only minor variations of borderline statistical significance.

One of the many aspects of membrane biophysics dealt with in this Faraday Discussion regards the material moduli that describe energies at a supramolecular level. This introductory lecture first critically reviews differences in reported numerical values of the bending modulus K C , which is a central property for the biologically important flexibility of membranes. It is speculated that there may be a reason that the shape analysis method tends to give larger values of K C than the micromechanical manipulation method or the more recent X-ray method that agree very well with each other. Another theme of membrane biophysics is the use of simulations to provide exquisite detail of structures and processes. This lecture critically reviews the application of atomic level simulations to the quantitative structure of simple single component lipid bilayers and diagnostics are introduced to evaluate simulations. Another theme of this Faraday Discussion was lateral heterogeneity in biomembranes with many different lipids. Coarse grained simulations and analytical theories promise to synergistically enhance experimental studies when their interaction parameters are tuned to agree with experimental data, such as the slopes of experimental tie lines in ternary phase diagrams. Finally, attention is called to contributions that add relevant biological molecules to bilayers and to contributions that study the exciting shape changes and different nonbilayer structures with different lipids.

Values of area per lipid A ranging from 56 to 72 A2 have been reported from essentially the same SCD data from DPPC in the La phase. The differences are due primarily to three separate binary choices in interpretation. It is argued that one particular combination is best; this yields A = 62 ± 2 A2 for DPPC at 500C. Each preceding interpretation agrees with at least one of the three present choices and disagrees with at least one.

Despite favorable advancements in therapy cancer is still not curative in many cases, which is often due to inadequate specificity for tumor cells. In this study derivatives of a short cationic peptide derived from the human host defense peptide lactoferricin were optimized in their selective toxicity towards cancer cells. We proved that the target of these peptides is the negatively charged membrane lipid phosphatidylserine (PS), specifically exposed on the surface of cancer cells. We have studied the membrane interaction of three peptides namely LF11-322, its N-acyl derivative 6-methyloctanoyl-LF11-322 and its retro repeat derivative R(etro)-DIM-P-LF11-322 with liposomes mimicking cancerous and non-cancerous cell membranes composed of PS and phosphatidylcholine (PC), respectively. Calorimetric and permeability studies showed that N-acylation and even more the repeat derivative of LF11-322 leads to strongly improved interaction with the cancer mimic PS, whereas only the N-acyl deri...

A method for determining permeability of phospholipid bilayer based on the osmotic swelling of micrometer-sized giant unilamellar vesicles (GUVs) is presented as an alternative to the two established techniques, dynamic light scattering on liposome suspension, and electrical measurements on planar lipid bilayers. In the described technique, an individual GUV is transferred using a micropipette from a sucrose/glucose solution into an isomolar solution containing the solute under investigation. Throughout the experiment, vesicle cross-section is monitored and recorded using a digital camera mounted on a phase-contrast microscope. Using a least-squares procedure for circle fitting, vesicle radius R is computed from the recorded images of vesicle cross-section. Two methods for determining membrane permeability from the obtained R(t) dependence are described: the first one uses the slope of R(t) for a spherical GUV, and the second one the R(t) dependence around the transition point at which a flaccid vesicle transforms into a spherical one. We demonstrate that both methods give consistent estimates for membrane permeability.

A model of vesicle electrodeformation is described which obtains a parametrized vesicle shape by minimizing the sum of the membrane bending energy and the energy due to the electric field. Both the vesicle membrane and the aqueous media inside and outside the vesicle are treated as leaky dielectrics, and the vesicle itself is modelled as a nearly spherical shape enclosed within a thin membrane. It is demonstrated (a) that the model achieves a good quantitative agreement with the experimentally determined prolate-to-oblate transition frequencies in the kHz range, and (b) that the model can explain a phase diagram of shapes of giant phospholipid vesicles with respect to two parameters: the frequency of the applied AC electric field and the ratio of the electrical conductivities of the aqueous media inside and outside the vesicle, explored in a recent paper (S. Aranda et al., Biophys. J. 95:L19-L21, 2008). A possible use of the frequency-dependent shape transitions of phospholipid vesicles in conductometry of microliter samples is discussed. Keywords electrodeformation • giant phospholipid vesicle • leaky dielectric • membrane bending energy • vesicle shape PACS 87.16.D-• 41.20.Cv 1 Introduction An increasing awareness of the effects of the electromagnetic field on biological samples [1], as well as its use in biotechnology [2] has instigated numerous studies

With recent advances in X-ray crystallography of membrane proteins promising many new highresolution structures, MD simulations become increasingly valuable for understanding membrane protein function, as they can unleash dynamic behavior concealed in the static structures. Dramatic increase in computational power in synergy with more efficient computational methodologies allows one today to carry out molecular dynamics simulations of any structurally known membrane protein in its native environment, covering the time scale of up to 0.1 μsec. At the frontier of membrane protein simulations are ion channels, aquaporins, passive and active transporters, and bioenergetic proteins. In this review we summarize recent developments in this rapidly evolving field.

Supported lipid bilayers have proven effective as model membranes for investigating biophysical processes and in development of sensor and array technologies. The ability to modify lipid bilayers after their formation and in situ could greatly advance membrane technologies, but is difficult via current state-of-the-art technologies. Here we demonstrate a novel method that allows the controlled post-formation processing and modification of complex supported lipid bilayer arrangements, under aqueous conditions. We exploit the destabilization effect of lipopolysaccharide, an amphiphilic biomolecule, interacting with lipid bilayers to generate voids that can be backfilled to introduce desired membrane components. We further demonstrate that when used in combination with a single, traditional soft lithography process, it is possible to generate hierarchically-organized membrane domains and microscale 2-D array patterns of domains. Significantly, this technique can be used to repeatedly m...

Membrane proteins play crucial roles in a range of biological processes. High resolution structures provide insights into the functional mechanisms of membrane proteins, but detailed biophysical characterization of membrane proteins is difficult. Complementary to experimental techniques, molecular dynamics simulations is a powerful tool in providing more complete description of the dynamics and energetics of membrane proteins with high spatial-temporal resolution. In this chapter, we provide a survey of the current methods and technique issues for setting up and running simulations of membrane proteins. The recent progress in applying simulations to understanding various biophysical properties of membrane proteins is outlined.

Membrane proteins are unique, in that they can function properly only when they are bound to cellular membranes in a distinct manner. Therefore, positioning of membrane proteins with respect to the membrane is required in addition to the three-dimensional structures in order to understand their detailed molecular mechanisms. Atomic-resolution structures of membrane proteins that have been determined to date provide the atom coordinates in arbitrary coordinate systems with no relation to the membrane and therefore provide little or no information on how the protein would interact with the membrane. This is especially true for peripheral membrane proteins, because they, unlike integral proteins, are devoid of well-defined hydrophobic transmembrane domains. Here, we present a novel technique for determination of the configuration of a protein-membrane complex that involves protein ligation, segmental isotope labeling, polarized infrared spectroscopy, membrane depthdependent fluorescence quenching, and analytical geometry algorithms. We have applied this approach to determine the structure of a membranebound phospholipase A 2. Our results provide an unprecedented structure of a membrane-bound protein in which the z-coordinate of each atom is the distance from the membrane center and therefore allows precise location of each amino acid relative to the membrane. Given the functional significance of the orientation and location of membrane-bound proteins with respect to the membrane, we propose to specify this structural feature as the "quinary" structure of membrane proteins.

The shape transformations of lipid vesicles induced by the adhesion to a flat surface is investigated. We perform the calculations within the framework of the Helfrich spontaneous curvature model. The calculations were performed for a few values of the reduced volume and the spontaneous curvature. The range of stability for different shapes (oblate, prolate, and stomatocyte) of adhered vesicles is determined. New physical phenomena such as budding induced by the adhesion of vesicles are reported.

Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.

Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.

Background: The mechanism behind diabetes-associated membrane damage by islet amyloid polypeptide (IAPP) is poorly understood. Results: IAPP induces and senses membrane curvature under conditions associated with membrane damage and binds to mitochondrial cristae in vivo. Conclusion: IAPP is a membrane-remodeling and curvature-sensing protein. Significance: Aberrant membrane remodeling could inform disruption of membrane integrity in diabetes and perhaps other amyloid diseases.

Background: The mechanism behind diabetes-associated membrane damage by islet amyloid polypeptide (IAPP) is poorly understood. Results: IAPP induces and senses membrane curvature under conditions associated with membrane damage and binds to mitochondrial cristae in vivo. Conclusion: IAPP is a membrane-remodeling and curvature-sensing protein. Significance: Aberrant membrane remodeling could inform disruption of membrane integrity in diabetes and perhaps other amyloid diseases.