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The interactions of viral fusion peptides from influenza (E4K and Ac-E4K) and human immunodeficiency virus (gp41 and Ac-gp41) with planar lipid bilayers and monolayers was investigated herein. A combination of surface-sensitive techniques, including quartz crystal microbalance with dissipation (QCM-D), Langmuir-Blodgett area-pressure isotherms with Micro-Brewster angle microscopy, and neutron reflectometry, was employed. Differences in the interactions of the viral fusion peptides with lipid bilayers featuring ordered and disordered phases, as well as lipid rafts, were revealed. The HIV fusion peptide (gp41) exhibited strong binding to the DOPC/DOPS bilayer, comprising a liquid disordered phase, with neutron reflectometry (NR) showing interaction with the bilayer's headgroup area. Conversely, negligible binding was observed with lipid bilayers in a liquid ordered phase. Notably, the influenza peptide (E4K) demonstrated slower binding kinetics with DOPC/DOPS bilayers and distinct interactions compared to gp41, as observed through QCM-D. This suggests different mechanisms of interaction with the lipid bilayers: one peptide interacts more within the headgroup region, while the other is more involved in transmembrane interactions. These findings hold implications for understanding viral fusion mechanisms and developing antimicrobials and antivirals targeting membrane interactions. The differential binding behaviours of the viral fusion peptides underscore the importance of considering membrane composition and properties in therapeutic strategy design., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

At present, consumers increasingly favored the natural food preservatives with fewer side-effects on health. The green tea catechins and black tea theaflavins attracted considerable interest, and their antibacterial effects were extensively reported in the literature. Epicatechin (EC), a green tea catechin without a gallate moiety, showed no bactericidal activity, whereas the theaflavin (TF), also lacking a gallate moiety, exhibited potent bactericidal activity, and the antibacterial effects of green tea catechins and black tea theaflavins were closely correlated with their abilities to disrupt the bacterial cell membrane. In our present study, the mechanisms of membrane interaction modes and behaviors of TF and EC were explored by molecular dynamics simulations. It was demonstrated that TF exhibited markedly stronger affinity for the POPG bilayer compared to EC. Additionally, the hydrophobic interactions of tropolone/catechol rings with the acyl chain part could significantly contribute to the penetration of TF into the POPG bilayer. It was also found that the resorcinol/pyran rings were the key functional groups in TF for forming hydrogen bonds with the POPG bilayer. We believed that the findings from our current study could offer useful insights to better understand the stronger antibacterial effects of TF compared to EC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The widely used Laurdan probe has two conformers, resulting in different optical properties when embedded in a lipid bilayer membrane, as demonstrated by our previous simulations. Up to now, the two conformers' optical responses have, however, not been investigated when the temperature and the phase of the membrane change. Since Laurdan is known to be both a molecular rotor and a solvatochromic probe, it is subject to a profound interaction with both neighboring lipids and water molecules. In the current study, molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics calculations are performed for a DPPC membrane at eight temperatures between 270K and 320K, while the position, orientation, fluorescence lifetime and fluorescence anisotropy of the embedded probes are monitored. The importance of both conformers is proven through a stringent comparison with experiments, which corroborates the theoretical findings. It is seen that for Conf-I, the excited state lifetime is longer than the relaxation of the environment, while for Conf-II, the surroundings are not yet adapted when the probe returns to the ground state. Throughout the temperature range, the lifetime and anisotropy decay curves can be used to identify the different membrane phases. The current work might, therefore, be of importance for biomedical studies on diseases, which are associated with cell membrane transformations.

Transmembrane peptides play important roles in many biological processes by interacting with lipid membranes. This study investigates how the transmembrane domain of the influenza A virus M2 protein, M2TM, affects the structure and mechanics of model lipid bilayers. Atomic force microscopy (AFM) imaging revealed small decreases in bilayer thickness with increasing peptide concentrations. AFM-based force spectroscopy experiments complemented by theoretical model analysis demonstrated significant decreases in bilayer's Young's modulus (E) and lateral area compressibility modulus (K A ). This suggests that M2TM disrupts the cohesive interactions between neighboring lipid molecules, leading to a decrease in both the bilayer's resistance to indentation (E) and its ability to resist lateral compression/expansion (K A ). The large decreases in bilayer elastic parameters (i.e., E and K A ) contrast with small changes in bilayer thickness, implying that bilayer mechanics are not solely dictated by bilayer thickness in the presence of transmembrane peptides. The observed significant reduction in bilayer mechanical properties suggests a softening effect on the bilayer, potentially facilitating membrane curvature generation, a crucial step for M2-mediated viral budding. In parallel, our Raman spectroscopy revealed small but statistically significant changes in hydrocarbon chain vibrational dynamics, indicative of minor disordering in lipid chain conformation. Our findings provide useful insights into the complex interplay between transmembrane peptides and lipid bilayers, highlighting the significance of peptide-lipid interactions in modulating membrane structure, mechanics, and molecular dynamics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Biosensors play an important role in numerous research fields. Quartz crystal microbalances with dissipation monitoring (QCM-Ds) are sensitive devices, and binding events can be observed in real-time. In combination with aptamers, they have great potential for selective and label-free detection of various targets. In this study, an alternative surface functionalization for a QCM-D-based aptasensor was developed, which mimics an artificial cell membrane and thus creates a physiologically close environment for the binding of the target to the sensor. Vesicle spreading was used to form a supported lipid bilayer (SLB) of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphethanolamine-N-(cap biotinyl) (biotin-PE). The SLB was then coated with streptavidin followed by applying a biotinylated aptamer against thrombin. SLB formation was investigated in terms of temperature and composition. Temperatures of 25 °C and below led to incomplete SLB formation, whereas a full bilayer was built at higher temperatures. We observed only a small influence of the content of biotinylated lipids in the mixture on the further binding of streptavidin. The functionalization of the sensor surface with the thrombin aptamer and the subsequent thrombin binding were investigated at different concentrations. The sensor could be reconstituted by incubation with a 5 M urea solution, which resulted in the release of the thrombin from the sensor surface. Thereafter, it was possible to rebind thrombin. Thrombin in spiked samples of human serum was successfully detected. The developed system can be easily applied to other target analytes using the desired aptamers.

Artificial lipid bilayers have revolutionized biochemical and biophysical research by providing a versatile interface to study aspects of cell membranes and membrane-bound processes in a controlled environment. Artificial bilayers also play a central role in numerous biosensing applications, form the foundational interface for liposomal drug delivery, and provide a vital structure for the development of synthetic cells. But unlike the envelope in many living cells, artificial bilayers can be mechanically fragile. Here, we develop prototype scaffolds for artificial bilayers made from multiple chemically linked tiers of actin filaments that can be bonded to lipid headgroups. We call the interlinked and layered assembly a multiple minimal actin cortex (multi-MAC). Construction of multi-MACs has the potential to significantly increase the bilayer's resistance to applied stress while retaining many desirable physical and chemical properties that are characteristic of lipid bilayers. Furthermore, the linking chemistry of multi-MACs is generalizable and can be applied almost anywhere lipid bilayers are important. This work describes a filament-by-filament approach to multi-MAC assembly that produces distinct 2D and 3D architectures. The nature of the structure depends on a combination of the underlying chemical conditions. Using fluorescence imaging techniques in model planar bilayers, we explore how multi-MACs vary with electrostatic charge, assembly time, ionic strength, and type of chemical linker. We also assess how the presence of a multi-MAC alters the underlying lateral diffusion of lipids and investigate the ability of multi-MACs to withstand exposure to shear stress.

To improve the oral absorption and anticancer efficacy of the BCS-IV drug etoposide (ETO), oral nanocrystal-loaded lipid carriers (Lipo@NCs) were developed in this study by modifying the BCS-IV drug nanocrystal with the lipid bilayer. The ETO-Lipo@NCs were prepared by the thin film hydration high-pressure homogenization method, and the core of positively charged ETO nanocrystals was prepared by the sonoprecipitation-high pressure homogenization method. The optimized ETO-Lipo@NCs were spherical particles with an average particle size of 220.3 ± 14.2 nm and a zeta potential of -9.95 ± 0.81 mV, respectively. The successful coating of a lipid bilayer on the surface of nanocrystals in ETO-Lipo@NCs was confirmed by several characterization methods. Compared to nanocrystals, the release rate and degree of Lipo@NCs in SIF were significantly decreased, indicating that the lipid bilayer can effectively prevent the rapid dissolution of core nanocrystals. ETO-Lipo@NCs demonstrated a significant improvement in the intestinal permeability and absorption of ETO in a single intestinal perfusion experiment. In the cells, ETO-Lipo@NCs showed enhanced cellular uptake and transepithelial transport compared with ETO nanocrystals. Pharmacokinetic analysis indicated that ETO-Lipo@NCs had a longer plasma half-life than ETO solution, and the oral bioavailability of ETO-Lipo@NCs was 1.96- and 10.92-fold higher than that of ETO nanocrystals and ETO coarse crystals, respectively. Moreover, the ETO-Lipo@NCs orally dosed at 10 mg/kg exhibited an excellent inhibitory effect against tumors in a subcutaneous Lewis lung carcinoma (LLC) xenograft model compared with other preparations. These results indicate that the Lipo@NCs formulation has an oral absorption-promoting effect of the BCS-IV drug ETO, which could warrant further application in the oral delivery of other poorly bioavailable drugs.

The human immunodeficiency virus type 1 (HIV-1) matrix protein contains a highly basic region, MA-HBR, crucial for various stages of viral replication. To elucidate the interactions between the polybasic peptide MA-HBR and lipid bilayers, we employed liquid-based atomic force microscopy (AFM) imaging and force spectroscopy on lipid bilayers of differing compositions. In 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, AFM imaging revealed the formation of annulus-shaped protrusions upon exposure to the polybasic peptide, accompanied by distinctive mechanical responses characterized by enhanced bilayer puncture forces. Importantly, our AFM-based force spectroscopy measurements unveiled that MA-HBR induces interleaflet decoupling within the cohesive bilayer organization. This is evidenced by a force discontinuity observed within the bilayer's elastic deformation regime. In POPC/cholesterol bilayers, MA-HBR caused similar yet smaller annular protrusions, demonstrating an intriguing interplay with cholesterol-rich membranes. In contrast, in bilayers containing anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) lipids, MA-HBR induced unique annular protrusions, granular nanoparticles, and nanotubules, showcasing its distinctive effects in anionic lipid-enriched environments. Notably, our force spectroscopy data revealed that anionic POPS lipids weakened interleaflet adhesion within the bilayer, resulting in interleaflet decoupling, which potentially contributes to the specific bilayer perturbations induced by MA-HBR. Collectively, our findings highlight the remarkable variations in how the polybasic peptide, MA-HBR, interacts with lipid bilayers of differing compositions, shedding light on its role in host membrane restructuring during HIV-1 infection., (© 2023. European Biophysical Societies' Association.)

The lateral stress profile of a lipid bilayer constitutes a valuable link between molecular simulation and mesoscopic elastic theory. Even though it is frequently calculated in simulations, its statistical precision (or that of observables derived from it) is often left unspecified. This omission can be problematic, as uncertainties are prerequisite to assessing statistical significance. In this chapter, we provide a comprehensive yet accessible overview of the statistical error analysis for the lateral stress profile. We detail two relatively simple but powerful techniques for generating error bars: block-averaging and bootstrapping. Combining these methods allows us to reliably estimate uncertainties, even in the presence of both temporal and spatial correlations, which are ubiquitous in simulation data. We illustrate these techniques with simple examples like stress moments, but also more complex observables such as the location of stress profile extrema and the monolayer neutral surface., (Copyright © 2024. Published by Elsevier Inc.)

Macrophages (MAs), which play vital roles in human immune responses and lipid metabolisms, are implicated in the development and progression of atherosclerosis, a major contributor to cardiovascular diseases. Specifically, the abnormal lipid metabolism of oxidized low-density lipids (oxLDLs) in MAs is believed to be a crucial factor. However, the precise mechanism by which the MA membrane contributes to this altered lipid metabolism remains unclear. Lipidomic studies have revealed significant differences in membrane composition between various MA phenotypes. This study serves to provide and characterize complex realistic computational models for naïve (M0) and Kdo2-lipid A-activated (M1) state MA. Analyses of surface area per lipid (SA/lip), area compressibility modulus (K A ), carbon‑hydrogen order parameter (S CH ), electron density profile (EDP), tilt angles, two-dimension radial distribution functions (2D RDFs), mean squared displacement (MSD), hydrogen bonds (H-bonds), lipid clustering, and lipid wobble were conducted for both models. Results indicate that the M1 state MA membrane is more tightly packed, with increased chain order across lipid species, and forms PSM-DOPG-CHOL and PSM-SLPC-CHOL clusters. Importantly, the bilayer thicknesses reported for the models are in good agreement with experimental data for the thicknesses of transmembrane regions for MA integral proteins. These findings validate the described models as physiologically accurate for future computational studies of MA membranes and their residing proteins., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jeffery Klauda reports financial support was provided by National Science Foundation., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Tamoxifen as an antiestrogen is successfully applied for the clinical treatment of breast cancer in pre- and post-menopausal women. Due to the side effects related to the oral administration of Tamoxifen (such as deep vein thrombosis, pulmonary embolism, hot flushes, ocular disturbances and some types of cancer), liposomal drug delivery is recommended for taking this drug. Drug encapsulation in a liposomal or lipid drug delivery system improves the pharmacokinetic and pharmacodynamic properties. In this regard, we carried out 200-ns molecular dynamics (MD) simulations for three systems (pure DPPC and neutral and protonated Tamoxifen-loaded DPPC). Here, DPPC is a model lipid bilayer to provide us with conditions like liposomal drug delivery systems to investigate the interactions between Tamoxifen and DPPC lipid bilayers and to estimate the preferred location and orientation of the drug molecule inside the bilayer membrane. Properties such as area per lipid, membrane thickness, lateral diffusion coefficient, order parameters and mass density, were surveyed. With insertion of neutral and protonated Tamoxifen inside the DPPC lipid bilayers, area per lipid and membrane thickness increased slightly. Also, Tamoxifen induce ordering of the hydrocarbon chains in DPPC bilayer. Analysis of MD trajectories shows that neutral Tamoxifen is predominantly found in the hydrophobic tail region, whereas protonated Tamoxifen is located at the lipid-water interface (polar region of DPPC lipid bilayers)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

A promising new class of biosensors leverages the sensing mechanisms of living cells by incorporating native transmembrane proteins into biomimetic membranes. Conducting polymers (CPs) can further improve the detection of electrochemical signals from these biological recognition elements through their low electrical impedance. Supported lipid bilayers (SLBs) on CPs mimic the structure and biology of the cell membrane to enable such sensing, but their extrapolation to new target analytes and healthcare applications has been difficult due to their poor stability and limited membrane properties. Blending native phospholipids with synthetic block copolymers to create a hybrid SLB (HSLB) may address these challenges by allowing for the tuning of chemical and physical properties during membrane design. We establish the first example of HSLBs on a CP device and show that polymer incorporation enhances bilayer resilience and thus offers important benefits toward bio-hybrid bioelectronics for sensing applications. Importantly, HSLBs outperform traditional phospholipid bilayers in stability by exhibiting strong electrical sealing after exposure to physiologically relevant enzymes that cause phospholipid hydrolysis and membrane degradation. We investigate the impact of HSLB composition on membranes and device performance and demonstrate the ability to finely adjust the lateral diffusivity of HSLBs with modest changes in block copolymer content through a large compositional range. The inclusion of the block copolymer into the bilayer does not disrupt electrical sealing on CP electrodes, an essential metric for electrochemical sensors, or the insertion of a representative transmembrane protein. This work interfacing tunable and stable HSLBs with CPs paves the way for future bioinspired sensors that combine the exciting developments from both bioelectronics and synthetic biology.

Membrane proteins are attractive targets for drug discovery due to their crucial roles in various biological processes. Studying the binding poses of amphipathic molecules to membrane proteins is essential for understanding the functions of membrane proteins and docking simulations can facilitate the screening of protein-ligand complexes at low computational costs. However, identifying docking poses for a ligand in non-aqueous environments such as lipid bilayers can be challenging. To address this issue, we propose a new docking score called logP-corrected membrane docking (LoCoMock) score. To screen putative protein-ligand complexes embedded in a membrane, the LoCoMock score considers the affinity between a target ligand and the membrane. It combines the docking score of the protein-ligand complex with the logP of the target ligand. In demonstrations using several model ligands, the LoCoMock score screened more putative complexes than the conventional docking score. As extended docking, the LoCoMock score makes it possible to screen membrane proteins more effectively as drug targets than the conventional docking., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Mechanical forces modify the cell membrane potential by opening mechanosensitive ion channels. We report the design and construction of a lipid bilayer tensiometer to study channels that respond to lateral membrane tension, [Formula: see text] , in the range 0.2 to 1.4 [Formula: see text] (0.8 to 5.7 [Formula: see text] ). The instrument consists of a black-lipid-membrane bilayer, a custom-built microscope, and a high-resolution manometer. Values of [Formula: see text] are obtained from the determination of the bilayer curvature as a function of applied pressure by means of the Young-Laplace equation. We demonstrate that [Formula: see text] can be determined by calculating the bilayer radius of curvature from fluorescence microscopy imaging or from measurements of the bilayer's electrical capacitance, both yielding similar results. Using electrical capacitance, we show that the mechanosensitive potassium channel TRAAK responds to [Formula: see text] , not curvature. TRAAK channel open probability increases as [Formula: see text] is increased from 0.2 to 1.4 [Formula: see text] but open probability never reaches 0.5. Thus, TRAAK opens over a wide range of [Formula: see text] , but with a tension sensitivity about one-fifth that of the bacterial mechanosensitive channel MscL.

Hypothesis: The attractive interaction between a cationic surfactant monolayer at the air-water interface and vesicles, incorporating anionic lipids, is sufficient to drive the adsorption and deformation of the vesicles. Osmotic rupture of the vesicles produces a continuous lipid bilayer beneath the monolayer., Experimental: Specular neutron reflectivity has been measured from the surface of a purpose-built laminar flow trough, which allows for rapid adsorption of vesicles, the changes in salt concentration required for osmotic rupture of the adsorbed vesicles into a bilayer, and for neutron contrast variation of the sub-phase without disturbing the monolayer., Findings: The neutron reflectivity profiles measured after vesicle addition are consistent with the adsorption and flattening of the vesicles beneath the monolayer. An increase in the buffer salt concentration results in further flattening and fusion of the adsorbed vesicles, which are ruptured by a subsequent decrease in the salt concentration. This process results in a continuous, high coverage, bilayer suspended 11 Åbeneath the monolayer. As the bilayer is not constrained by a solid substrate, this new mimetic is well-suited to studying the structure of lipid bilayers that include transmembrane proteins., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The ubiquitous nanoplastics are now considered emergent pollutants in environments. Bioaccumulation of nanoplastics is an important indicator of their hazard. In this work, molecular dynamics were used to study the uptake of five nanoplastics (polyvinyl chloride (PVC), polystyrene (PS), polylactic acid (PLA), polypropylene (PP), and polyethylene terephthalate (PET)) onto DPPC (dipalmitoylphosphatidylcholine) bilayers. Results suggest that nanoplastics became compact after they were deposited in the human body. For PET, PLA, and PS nanoplastics, a free energy barrier of 4-22 kcal mol -1 needed to be overcome to transfer these polymers from the interface region to the center of the DPPC bilayer. Besides, the free energy difference of PVC and PP from the bulk H 2 O to the surface of DPPC was -18.67 kcal mol -1 and -25.94 kcal mol -1 , respectively. After uptake, the interaction between nanoplastics and lipid bilayer was dominated by the van der Waals rather than electrostatic interaction. Furthermore, the cytotoxicity of nanoplastics was also evaluated and it is reflected in their ability to decrease the thickness of the lipid bilayer. Overall, this work provides implications for understanding the bioaccumulation and toxicity of nanoplastic at the molecular level., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Electroporation is a cell-level phenomenon caused by an ionic imbalance in the membrane, being of great relevance in various fields of knowledge. A dependence of the pore formation kinetics on the environmental conditions (temperature and pressure) of the cell membrane has already been reported, but further clarification regarding how these variables affect the pore formation/resealing dynamics and the transport of molecules through the membrane is still lacking. The objective of the present study was to investigate the temperature (288-348 K) and pressure (1-5000 atm) effects on the electroporation kinetics using coarse-grained molecular dynamics simulations. Results shown that the time for pore formation and resealing increased with pressure and decreased with temperature, whereas the maximum pore radius increased with temperature and decreased with pressure. This behavior influenced the ion migration through the bilayer, and the higher ionic mobility was obtained in the 288 K/1000 atm simulations, i.e., a combination of low temperature and (not excessively) high pressure. These results were used to discuss some experimental observations regarding the extraction of intracellular compounds applying this technique. This study contributes to a better understanding of electroporation under different thermodynamic conditions and to an optimal selection of processing parameters in practical applications which exploit this phenomenon., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The influence and interaction of the ionizable amphiphilic drug amitriptyline hydrochloride (AMT) on a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) phospholipid bilayer supported on a silica surface have been investigated using a combination of neutron reflectometry and quartz crystal microbalance with dissipation monitoring. Adding AMT solutions with concentrations 3, 12, and 50 mM leaves the lipid bilayer mainly intact and we observe most of the AMT molecules attached to the head-group region of the outer bilayer leaflet. Virtually no AMT penetrates into the hydrophilic head-group region of the inner leaflet close to the silica surface. By adding 200 mM AMT solution, the lipid bilayer dissolved entirely, indicating a threshold concentration for the solubilization of the bilayer by AMT. The observed threshold concentration is consistent with the observation that various bilayer structures abruptly transform into mixed AMT-DOPC micelles beyond a certain AMT-DOPC composition. Based on our experimental observations, we suggest that the penetration of drug into the phospholipid bilayer, and subsequent solubilization of the membrane, follows a two-step mechanism with the outer leaflet being removed prior to the inner leaflet., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Lipid bilayer membranes are indispensable parts of cellular architecture. One of the integral properties of bilayer membranes is the environmental heterogeneity over a wide range of spatiotemporal scales. The environmental heterogeneity is a manifestation of the dynamic and compositional anisotropy in the plane of the membrane as well as along the bilayer normal. Fluorescence lifetime distribution analysis provides a spectroscopic tool to quantitatively characterize such heterogeneities. The review discusses recent applications of fluorescence lifetime distribution analysis utilizing the maximum entropy method to characterize horizontal and vertical heterogeneities in membranes., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Extraction of integral membrane proteins into detergents for structural and functional studies often leads to a strong loss in protein stability. The impact of the lipid bilayer on the thermodynamic stability of an integral membrane protein in comparison to its solubilized form in detergent was examined and compared for FhuA from Escherichia coli and for a mutant, FhuAΔ5-160, lacking the N-terminal cork domain. Urea-induced unfolding was monitored by fluorescence spectroscopy to determine the effective free energies [Formula: see text] of unfolding. To obtain enthalpic and entropic contributions of unfolding of FhuA, [Formula: see text] were determined at various temperatures. When solubilized in LDAO detergent, wt-FhuA and FhuAΔ5-160 unfolded in a single step. The 155-residue cork domain stabilized wt-FhuA by [Formula: see text]~ 40 kJ/mol. Reconstituted into lipid bilayers, wt-FhuA unfolded in two steps, while FhuAΔ5-160 unfolded in a single step, indicating an uncoupled unfolding of the cork domain. For FhuAΔ5-160 at 35 °C, [Formula: see text] increased from ~ 5 kJ/mol in LDAO micelles to about ~ 20 kJ/mol in lipid bilayers, while the temperature of unfolding increased from T M  ~ 49 °C in LDAO micelles to T M  ~ 75 °C in lipid bilayers. Enthalpies [Formula: see text]were much larger than free energies [Formula: see text], for FhuAΔ5-160 and for wt-FhuA, and compensated by a large gain of entropy upon unfolding. The gain in conformational entropy is expected to be similar for unfolding of FhuA from micelles or bilayers. The strongly increased T M and [Formula: see text] observed for the lipid bilayer-reconstituted FhuA in comparison to the LDAO-solubilized forms, therefore, very likely arise from a much-increased solvation entropy of FhuA in bilayers., (© 2022. The Author(s).)

The lipid second messenger diacylglycerol (DAG) is known for its involvement in many types of cellular signaling, especially as an endogenous agonist for protein kinase C (PKC). Evidence has emerged that the degree of saturation of the DAG molecules can affect PKC activation. DAG molecules with different acyl chain saturation have not only been observed to induce varying extents of PKC activation, but also to express selectivity towards different PKC isozymes. Both qualities are important for precise therapeutic activation of PKC; understanding DAG behavior at the molecular level in different environments has much potential in the development of drugs to target PKC. We used molecular dynamics simulations to study the behavior of two different unsaturated DAG species in lipid environments with varying degrees of unsaturation. We focus on phosphatidylethanolamine (PE) instead of phosphatidylcholine (PC) to more accurately model the relevant biomembranes. The effect of cholesterol (CHOL) on these systems was also explored. We found that both high level of unsaturation in the acyl chains of the DAG species and presence of CHOL in the surrounding membrane increase DAG molecule availability at the lipid-water interface. This can partially explain the previously observed differences in PKC activation strength and specificity, the complete mechanism is, however, likely to be more complex. Our simulations coupled with the current understanding of lipids highlight the need for more simulations of biologically accurate lipid environments in order to determine the correct correlations between molecular mechanisms and biological behavior when studying PKC activation., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Arenicin-3 is an amphipathic β-hairpin antimicrobial peptide that is produced by the lugworm Arenicola marina. In this study, we have investigated the mechanism of action of arenicin-3 and an optimized synthetic analogue, AA139, by studying their effects on lipid bilayer model membranes and Escherichia coli bacterial cells. The results show that simple amino acid changes can lead to subtle variations in their interaction with membranes and therefore alter their pre-clinical potency, selectivity and toxicity. While the mechanism of action of arenicin-3 is primarily dependent on universal membrane permeabilization, our data suggest that the analogue AA139 relies on more specific binding and insertion properties to elicit its improved antibacterial activity and lower toxicity, as exemplified by greater selectivity between lipid composition when inserting into model membranes i.e. the N-terminus of AA139 seems to insert deeper into lipid bilayers than arenicin-3 does, with a clear distinction between zwitterionic and negatively charged lipid bilayer vesicles, and AA139 demonstrates a cytoplasmic permeabilization dose response profile that is consistent with its greater antibacterial potency against E. coli cells compared to arenicin-3., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Many research efforts are devoted to improving the efficiency of chemotherapy. One of the aspects is to facilitate the transport of drugs across the cell membranes by attaching the therapeutics to a carrier molecule. The current study focuses on computational investigation of such a system with doxorubicin as the model drug, which is covalently bound to a cell-penetrating peptide. The correct description of its membrane translocation at the molecular level requires proper choice of the model membrane and of the simulation parameters. For the purpose, two phospholipid bilayers are built, one containing solely DPPC and another with mixed lipid content mimicking the composition of a human erythrocyte membrane. Atomistic molecular dynamics simulations are carried out in two types of periodic boundary conditions (2D and 3D PBC), in order to assess the effect of the periodicity dimensionality on the intermolecular interactions. The evolution of some basic characteristics of the bilayers and of the drug-peptide complex is tracked: mass density profiles, electrostatic potentials, lateral diffusion coefficients and areas per lipid, lipid-complex radial distribution functions, secondary structure of the peptide and orientation of the drug relative to the membrane. Thus, the influence of the periodic boundary conditions is quantified and it shows that the mixed system in 3D PBC is the most suitable for analysis of the translocation of the transporting moiety across cell membranes.

The micrometer-scale assembly of various DNA nanostructures is one of the major challenges for further progress in DNA nanotechnology. Programmed patterns of 1D and 2D DNA origami assembly using specific DNA strands and micrometer-sized lattice assembly using cross-shaped DNA origami were performed on a lipid bilayer surface. During the diffusion of DNA origami on the membrane surface, the formation of lattices and their rearrangement in real-time were observed using high-speed atomic force microscopy (HS-AFM). The formed lattices were used to further assemble DNA origami tiles into their cavities. Various patterns of lattice-tile complexes were created by changing the interactions between the lattice and tiles. For the control of the nanostructure formation, the photo-controlled assembly and disassembly of DNA origami were performed reversibly, and dynamic assembly and disassembly were observed on a lipid bilayer surface using HS-AFM. Using a lipid bilayer for DNA origami assembly, it is possible to perform a hierarchical assembly of multiple DNA origami nanostructures, such as the integration of functional components into a frame architecture.

Ibuprofen is a non-steroidal anti-inflammatory drug possessing analgesic and antipyretic activity. Electron paramagnetic resonance (EPR) spectroscopy could be applied to study its interaction with biological membranes and proteins if its spin-labeled analogs were synthesized. Here, a simple sequence of ibuprofen transformations-nitration, esterification, reduction, Sandmeyer reaction, Sonogashira cross-coupling, oxidation and saponification-was developed to attain this goal. The synthesis resulted in spin-labeled ibuprofen (ibuprofen-SL) in which the spin label TEMPOL is attached to the benzene ring. EPR spectra confirmed interaction of ibuprofen-SL with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers. Using 2 H electron spin echo envelope modulation (ESEEM) spectroscopy, ibuprofen-SL was found to be embedded into the hydrophobic bilayer interior.

The lateral pressure profile constitutes an important physical property of lipid bilayers, influencing the binding, insertion, and function of membrane-active peptides, such as antimicrobial peptides. In this study, we demonstrate that the lateral pressure profile can be manipulated using the peptides residing in different regions of the bilayer. A 19 F-labeled analogue of the amphiphilic peptide PGLa was used to probe the lateral pressure at different depths in the membrane. To evaluate the lateral pressure profile, we measured the orientation of this helical peptide with respect to the membrane using solid-state 19 F-NMR, which is indicative of its degree of insertion into the bilayer. Using this experimental approach, we observed that the depth of insertion of the probe peptide changed in the presence of additional peptides and, furthermore, correlated with their location in the membrane. In this way, we obtained a tool to manipulate, as well as to probe, the lateral pressure profile in membranes.

Oxidative stress plays an essential role in the regulation of vital processes in living organisms. Reactive oxygen species can react chemically with the constituents of the cells leading to irreversible damage. The first structure of the cell in contact with the environment that surrounds it is the membrane, which protects it and allows the exchange of substances. Some signals manifest when the components of a bilayer are undergoing oxidation, like an increase in the lipid area, decrease in the thickness of the bilayer, and exchange of the oxidized groups toward the bilayer surface. In this investigation, a molecular dynamics simulation was done on a set of Dioleoylphosphatidylcholine membranes with different percentage of oxidized lipids, in order to observe the effect of the oxidation degree on the membrane structure. It was found that, as higher the concentration of oxidized lipids is, the larger the damage of the membrane. This is reflected in the increase in the lipid area and the decrease in the thickness and membrane packing. Also, it was observed that hydrophobicity inside the membrane decreases as the oxidation percentage increases.Communicated by Ramaswamy H. Sarma.

Domain formation in "HLC" ternary lipid bilayers, comprising a high transition temperature (High-T m ) lipid, a Low-T m lipid, and cholesterol (Chol), has been extensively studied as raft-resembling systems. Recently, we reported the formation of submicron domains in an "LLC" lipid bilayer, encompassing Low-T m phosphatidylethanolamine (PE), Low-T m phosphatidylcholine (PC), and Chol. We hypothesized that the formation of this unique domain is driven by polyunsaturated PE. In this study, we explored the effects of the degree of PE unsaturation and the double bond distribution at the sn-position on the mechanism of formation and the composition of submicron domains. Supported lipid bilayers (SLBs), comprising PE with various degrees of unsaturation, monounsaturated PC (POPC), and Chol, were investigated using fluorescence microscopy, atomic force microscopy, and the force-distance curve measurement. The area fraction of submicron domains in PE+POPC+Chol-SLB increased with the PE concentration and degree of unsaturation of the PE acyl chain. The results indicated that the submicron domains were enriched with polyunsaturated PE and were in the liquid-disordered-like state, whereas their surrounding regions were in the liquid-ordered-like state. Segregation of polyunsaturated PE from the Chol-containing region generated submicron domains in the LLC lipid bilayer. We propose a mechanism for the formation of these submicron domains based on molecular interactions involving the hydrophobic and hydrophilic parts of the bilayer membrane., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Synthetic plastic oligomers can interact with the cells of living organisms by different ways. They can be intentionally administered to the human body as part of nanosized biomedical devices. They can be inhaled by exposed workers, during the production of multicomponent, polymer-based nanocomposites. They can leak out of food packaging. Most importantly, they can result from the degradation of plastic waste, and enter the food chain. A physicochemical characterization of the effects of synthetic polymers on the structure and dynamics of cell components is still lacking. Here, we combine a wide spectrum of experimental techniques (calorimetry, x-ray, and neutron scattering) with atomistic Molecular Dynamics simulations to study the interactions between short chains of polystyrene (25 monomers) and model lipid membranes (DPPC, in both gel and fluid phase). We find that doping doses of polystyrene oligomers alter the thermal properties of DPPC, stabilizing the fluid lipid phase. They perturb the membrane structure and dynamics, in a concentration-dependent fashion. Eventually, they modify the mechanical properties of DPPC, reducing its bending modulus in the fluid phase. Our results call for a systematic, interdisciplinary assessment of the mechanisms of interaction of synthetic, everyday use polymers with cell membranes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Inc.)

The membrane sculpting ability of BAR domains has been attributed to the intrinsic curvature of their banana-shaped dimeric structure. However, there is often a mismatch between this intrinsic curvature and the diameter of the membrane tubules generated. I-BAR domains are especially mysterious since they are almost flat but generate high negative membrane curvature. Here, we use atomistic implicit-solvent computer modeling to show that the membrane bending of the IRSp53 I-BAR domain is dictated by its higher oligomeric structure, whose curvature is completely unrelated to the intrinsic curvature of the dimer. Two other I-BARs give similar results, whereas a flat F-BAR sheet develops a concave membrane-binding interface, consistent with its observed positive membrane curvature generation. Laterally interacting helical spirals of I-BAR dimers on tube interiors are stable and have an enhanced binding energy that is sufficient for membrane bending to experimentally observed tubule diameters at a reasonable surface density., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely investigated in recent decades, the electrophysiological properties of pore-forming Archaerhodopsin (Arch), as studied in vitro, have remained largely unknown. Here, we formed unsupported bilayers between two channels of a microfluidic chip which enabled the simultaneous optical and electrical assessment of the bilayer in real time. Using a cell-free expression system, we recombinantly produced a GFP (green fluorescent protein) labelled as a variant of Arch-3. The label enabled us to follow the synthesis of Arch-3 and its incorporation into the bilayer by fluorescence microscopy when excited by blue light. Applying a green laser for excitation, we studied the electrophysiological properties of Arch-3 in the bilayer. The current signal obtained during excitation revealed distinct steps upwards and downwards, which we interpreted as the opening or closing of Arch-3 pores. From these steps, we estimated the pore radius to be 0.3 nm. In the cell-free extract, proteins can be modified simply by changing the DNA. In the future, this will enable us to study the photoelectrical properties of modified transmembrane protein constructs with ease. Our work, thus, represents a first step in studying signaling cascades in conjunction with coupled receptor proteins.

Human cell membranes consist of various lipids that are essential for their structure and function. It typically comprises phosphatidylcholine (POPC), phosphatidylethanolamine (POPE), phosphatidylserine (POPS), sphingomyelin (PSM), and cholesterol (CHL). Several experimental and computational techniques have been employed to characterize the composition of human cell membranes, however, CHL enriched membrane is still not clearly understood through these techniques. Molecular dynamics simulation results illustrated the biophysical properties of heterogeneous membranes based on the lipid composition as well as the concentration of lipids, exclusively for CHL and PSM. Herein, we have investigated the structure-function relationships of lipids comparatively to delineate the effect of heterogeneity on the biophysical properties of different membranes. It has been observed that the significant fraction of CHL (i.e., ~33% in ternary, ~25% in quaternary, and ~16% in senary type bilayers) in combination with other lipids introduced compactness, and increased the thickness of the membrane. The analysis of lipid mass density stated that the density of lipid head group, phosphate , and glycerol-ester in presence of CHL with or without PSM is an underlying reason for membrane ordering. Results also revealed that the presence of POPI and POPS are the reasons for an adequate drop in the ordering of lipid chain, particularly on POPE chain. The self-interaction of CHL, PSM, POPE and the interaction of CHL and POPC with POPE seem to determine the structure and function of the heterogeneous membrane. Our findings provide a qualitative understanding of the effect of membrane heterogeneity on the physiological properties of membranes. The structures inspected in this study would help to select the heterogeneous bilayer model to mimic the human cell membranes to analyse or characterize the membrane-associated phenomena., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The molecular details of the passive water flux across the hydrophobic membrane interior are still a matter of debate. One of the postulated mechanisms is the spontaneous, water-filled pore opening, which facilitates the hydrophilic connection between aqueous phases separated by the membrane. In the paper, we provide experimental evidence showing that the spontaneous lipid pore formation correlates with the membrane mechanics; hence, it depends on the composition of the lipid bilayer and the concentration of the osmotically active compound. Using liposomes as an experimental membrane model, osmotically induced water efflux was measured with the stopped-flow technique. Shapes of kinetic curves obtained at low osmotic pressure differences are interpreted in terms of two events: the lipid pore opening and water flow across the aqueous channel. The biological significance of the dependence of the lipid pore formation on the concentration difference of an osmotically active compound was illustrated by the demonstration that osmotically driven water flow can be accompanied by the dissipation of the pH gradient. The application of the Helfrich model to describe the probability of lipid pore opening was validated by demonstrating that the probability of pore opening correlates with the membrane bending rigidity. The correlation was determined by experimentally derived bending rigidity coefficients and probabilities of lipid pores opening.

Because of the negative side-effects of synthetic preservatives, the naturally-occurring polyphenols aroused intense interest of researchers. It has been suggested that chlorogenic acid (CA) and isochlorogenic acid A (iso-CAA) were good candidates to replace the synthetic preservatives. Moreover, the bactericidal activity of iso-CAA was stronger than CA, and the anti-bacterial activities of iso-CAA and CA were highly membrane-dependent. However, the mechanisms were still unclear. Therefore, in the present study, we investigated the mechanisms of the interactions between the two polyphenols and lipid bilayers through molecular dynamics simulations. The results revealed that iso-CAA could be inserted much deeper into POPG lipid bilayer than CA. We also found that hydrophobic interactions and hydrogen bonds both contributed to the insertion of iso-CAA into the POPG lipid bilayer, and the quinic acid moiety was the key structure in iso-CAA to form hydrogen bonds with POPG lipid bilayer. We believed that these findings would provide more useful information to explain the stronger bactericidal activity of iso-CAA than CA at the atomic level., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Lithium is regarded as a unique therapeutic agent for the management of bipolar disorder (BD). In efforts to explain the favourable effects of lithium in BD, a wide range of mechanisms was suggested. Among those, the effect of clinically relevant concentrations of lithium on the plasma membrane was extensively studied. However, the biophysical properties of brain membranes isolated from experimental animals exposed to acute, short-term and chronic lithium have not been performed to-date. In this study, we compared the biophysical parameters and level of lipid peroxidation in membranes isolated from forebrain cortex (FBC) of therapeutic lithium-treated and/or sleep-deprived rats. Lithium interaction with FBC membranes was characterized by appropriate fluorescent probes. DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulphonate) were used for characterization of the hydrophobic lipid core and Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) for the membrane-water interface. Lipid peroxidation was determined by immunoblot analysis of 4-HNE-(4-hydroxynonenal)-protein adducts. The organization of polar head-group region of FBC membranes, measured by Laurdan generalized polarization, was substantially altered by sleep deprivation and augmented by lithium treatment. Hydrophobic membrane interior characterized by steady-state anisotropy of DPH and TMA-DPH fluorescence was unchanged. Chronic lithium had a protective effect against peroxidative damage of membrane lipids in FBC. In summary, lithium administration at a therapeutic level and/or sleep deprivation as an animal model of mania resulted in changes in rat FBC membrane properties., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The thermodynamic (TD) properties of biological membranes play a central role for living systems. It has been suggested, for instance, that nonlinear pulses such as action potentials (APs) can only exist if the membrane state is in vicinity of a TD transition. Herein, two membrane properties in living systems - excitability and velocity - are analyzed for a broad spectrum of conditions (temperature (T), 3D-pressure (p) and pH-dependence). Based on experimental data from Characean cells and a review of literature we predict parameter ranges in which a transition of the membrane is located (15-35°C below growth temperature; 1-3pH units below pH7; at ∼800atm) and propose the corresponding phase diagrams. The latter explain: (i) changes of AP velocity with T,p and pH.(ii) The existence and origin of two qualitatively different forms of loss of nonlinear excitability ("nerve block", anesthesia). (iii) The type and quantity of parameter changes that trigger APs. Finally, a quantitative comparison between the TD behavior of 2D-lipid model membranes with living systems is attempted. The typical shifts in transition temperature with pH and p of model membranes agree with values obtained from cell physiological measurements. Taken together, these results suggest that it is not specific molecules that control the excitability of living systems but rather the TD properties of the membrane interface. The approach as proposed herein can be extended to other quantities (membrane potential, calcium concentration, etc.) and makes falsifiable predictions, for example, that a transition exists within the specified parameter ranges in excitable cells., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The investigation of the intermolecular interactions between platinum-based anticancer drugs and lipid bilayers is of special relevance to unveil the mechanisms involved in different steps of the anticancer mode of action of these drugs. We have simulated the permeation of cisplatin through a model membrane composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine lipids by means of umbrella sampling classical molecular dynamics simulations. The initial physisorption of cisplatin into the polar region of the lipid membrane is controlled by long-range electrostatic interactions with the choline groups in a first step and, in a second step, by long-range electrostatic and hydrogen bond interactions with the phosphate groups. The second half of the permeation pathway, in which cisplatin diffuses through the nonpolar region of the bilayer, is characterized by the drop of the interactions with the polar heads and the rise of attractive interactions with the non-polar tails, which are dominated by van der Waals contributions. The permeation free-energy profile is explained by a complex balance between the drug/lipid interactions and the energy and entropy contributions associated with the dehydration of the drug along the permeation pathway and with the decrease and increase of the membrane ordering along the first and second half of the mechanism, respectively., (© 2021 Wiley-VCH GmbH.)

Membranes surrounding cells or organelles represent barriers to proteins and other molecules. However, specific proteins can cross membranes by different translocation systems, the best studied being the Sec61/SecY channel. This channel forms a hydrophilic, hourglass-shaped membrane channel, with a lateral gate towards the surrounding lipid. However, recent studies show that an aqueous pore is not required in other cases of protein translocation. The Hrd1 complex, mediating the retrotranslocation of misfolded proteins from the endoplasmic reticulum (ER) lumen into the cytosol, contains multispanning proteins with aqueous luminal and cytosolic cavities, and lateral gates juxtaposed in a thinned membrane region. A locally thinned, distorted lipid bilayer also allows protein translocation in other systems, suggesting a new paradigm to overcome the membrane barrier., Competing Interests: Declaration of Interests The authors have no interests to declare., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Biological membranes composed of a lipid bilayer and associated proteins work as a platform for highly selective and sensitive detection in nature. Substrate-supported lipid bilayers (SLBs) are a model system of the biological membrane that are mechanically stable, accessible to highly sensitive analytical techniques, and amenable to micro-fabrication, such as patterning. The surface of SLBs can effectively suppress the non-specific binding of proteins, and enhance selective detection by specific interactions. These features render SLBs highly attractive for the development of devices that utilize artificially mimicked cellular functions. Furthermore, SLBs can be combined with nanoscopic spaces, such as nano-channels and nano-pores, that can reduce the detection volume and suppress the non-specific background noise, enhancing the signal-to-background noise (S/B) ratio. SLBs therefore provide promising platforms for a wide range of biomedical and environmental analyses.

As a class of drugs prescribed to heart disease patients, statins are among the most popular prescription drugs in the world. Over the years, statins have been shown to have beneficial effects on patients via pathways independent of their effect on cholesterol. These pleiotropic effects vary across the different statins, and a growing hypothesis is that they are related to the localization of the statins in and their effect on the membrane. In this study, we use molecular dynamics (MD) simulations with the CHARMM36 all-atom force field to investigate the localization of statins (atorvastatin, cerivastatin, lovastatin, and pravastatin) in a POPC bilayer and how they affect the acyl chain order parameters (S CD ), surface area per lipid (APL), and thicknesses of the bilayer. The data obtained from 500 ns simulations suggests that lovastatin is localized deepest in the membrane, mostly interacting with the hydrophobic core, cerivastatin is slightly closer to the bilayer/solvent interface than lovastatin and interacts with the headgroups via its dihydroxy acid group, and pravastatin is found closest to the bilayer/solvent interface, its hydrophobic rings interacting mostly with the region around the acyl's carbonyl and its dihydroxy acid interacting with the solvent and the headgroups. Consistent binding of atorvastatin to the bilayer is not observed during our simulation due to self-aggregation. The statins differentially alter the S CD and APL and most of the bilayer thicknesses, but these effects are modest. Overall, as expected, the localization of statins seems to follow their hydrophilicity, and given previous data showing the relationship between statins' hydrophobicity and pleiotropic effects, one would expect statins that localize and interact with different regions of the membrane to have different effects. This research provides some important insight into statin localization in a simplified model of a cellular membrane., (Copyright © 2021 Elsevier B.V. All rights reserved.)

CLC-ec1 is a Cl - /H + antiporter that forms stable homodimers in lipid bilayers, with a free energy of -10.9 kcal/mol in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4-5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Membrane-scaffolding proteins (MSPs) derived from apolipoprotein A-1 have become a versatile tool in generating nano-sized discoidal membrane mimetics (nanodiscs) for membrane protein research. Recent efforts have aimed at exploiting their controlled lipid protein ratio and size distribution to arrange membrane proteins in regular supramolecular structures for diffraction studies. Thereby, direct membrane protein crystallization, which has remained the limiting factor in structure determination of membrane proteins, would be circumvented. We describe here the formation of multimers of membrane-scaffolding protein MSP1D1-bounded nanodiscs using the thiol reactivity of engineered cysteines. The mutated positions N42 and K163 in MSP1D1 were chosen to support chemical modification as evidenced by fluorescent labeling with pyrene. Minimal interference with the nanodisc formation and structure was demonstrated by circular dichroism spectroscopy, differential light scattering and size exclusion chromatography. The direct disulphide bond formation of nanodiscs formed by the MSP1D1_N42C variant led to dimers and trimers with low yield. In contrast, transmission electron microscopy revealed that the attachment of oligonucleotides to the engineered cysteines of MSP1D1 allowed the growth of submicron-sized tracts of stacked nanodiscs through the hybridization of nanodisc populations carrying complementary strands and a flexible spacer.

Free standing artificial lipid bilayers are widely used in the study of biological pores. In these types of studies, the free standing planar lipid bilayer is formed over a micron-sized aperture consisting of either polymer such as Polytetrafluoroethylene (PTFE, Teflon) or glass. Teflon is chemically inert, has a low dielectric constant, and has a high electrical resistance which combined allow for obtaining low noise recordings. This study investigates the reproducible generation of micropores in the range of 50-100 microns in diameter in a Teflon film using a high energy discharge set-up. The discharger set-up consists of a microprocessor, a transformer, a voltage regulator, and is controlled by a computer. We compared two approaches for pore creation: single and multi-pulse methods. The results showed that the multi-pulse method produced narrower aperture size distributions and is more convenient for lipid bilayer formation, and thus would have a higher success rate than the single-pulse method. The bilayer stability experiments showed that the lipid bilayer lasts for more than 33 h. Finally, as a proof-of-concept, we show that the single and multi-channel electrophysiology experiments were successfully performed with the apertures created by using the mentioned discharger. In conclusion, the described discharger provides reproducible Teflon-pores in a cheap and easy-to-operate manner.

Background: Organic fluorophores embedded in lipid bilayers can nowadays be described by a multiscale computational approach. Combining different length and time scales, a full characterization of the probe localization and optical properties led to novel insight into the effect of the environments., Scope of Review: Following an introduction on computational advancements, three relevant probes are reviewed that delineate how a multiscale approach can lead to novel insight into the probes' (non) linear optical properties. Attention is paid to the quality of the theoretical description of the optical techniques., Major Conclusions: Computation can assess a priori novel probes' optical properties and guide the analysis and interpretation of experimental data in novel studies. The properties can be used to gain information on the phase and condition of the surrounding biological environment., General Significance: Computation showed that a canonical view on some of the probes should be revisited and adapted., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Membrane proteins are involved in numerous vital biological processes, including transport, signal transduction and the enzymes in a variety of metabolic pathways. Integral membrane proteins account for up to 30% of the human proteome and they make up more than half of all currently marketed therapeutic targets. Unfortunately, membrane proteins are inherently recalcitrant to study using the normal toolkit available to scientists, and one is most often left with the challenge of finding inhibitors, activators and specific antibodies using a denatured or detergent solubilized aggregate. The Nanodisc platform circumvents these challenges by providing a self-assembled system that renders typically insoluble, yet biologically and pharmacologically significant, targets such as receptors, transporters, enzymes, and viral antigens soluble in aqueous media in a native-like bilayer environment that maintain a target's functional activity. By providing a bilayer surface of defined composition and structure, Nanodiscs have found great utility in the study of cellular signaling complexes that assemble on a membrane surface. Nanodiscs provide a nanometer scale vehicle for the in vivo delivery of amphipathic drugs, therapeutic lipids, tethered nucleic acids, imaging agents and active protein complexes. This means for generating nanoscale lipid bilayers has spawned the successful use of numerous other polymer and peptide amphipathic systems. This review, in celebration of the Anfinsen Award, summarizes some recent results and provides an inroad into the current and historical literature., (© 2020 The Protein Society.)

FtsH is a membrane-bound protease that plays a crucial role in proteolytic regulation of many cellular functions. It is universally conserved in bacteria and responsible for the degradation of misfolded or misassembled proteins. A recent study has determined the structure of bacterial FtsH in detergent micelles. To properly study the function of FtsH in a native-like environment, we reconstituted the FtsH complex into lipid nanodiscs. We found that FtsH in membrane scaffold protein (MSP) nanodiscs maintains its native hexameric conformation and is functionally active. We further investigated the effect of the lipid bilayer composition (acyl chain length, saturation, head group charge and size) on FtsH proteolytic activity. We found that the lipid acyl chain length influences AaFtsH activity in nanodiscs, with the greatest activity in a bilayer of di-C18:1 PC. We conclude that MSP nanodiscs are suitable model membranes for further in vitro studies of the FtsH protease complex., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Because of the high sensitivity of lipid bilayers to external pressure fluctuations, a major challenge in functional studies of biological pores or ion channels is the difficulty in exchanging solutions rapidly while maintaining the stability of the lipid bilayer in a model membrane. Here we describe a droplet-interface bilayer-based perfusion system that has been routinely used in our research and is currently the most robust and stable perfusion system that provides prompt solution exchange surrounding a lipid bilayer. In this model membrane system, solutions can be completely exchanged within 1-2 s to obtain prompt responses of a lipid bilayer or membrane pores to the membrane environments. Also, our system is stable enough to sustain continuous perfusions up to at least dozens of minutes. To demonstrate, we show that acidification-induced protein channel insertion, substrate binding to protein channels, and pH gradient-driven protein translocation of anthrax toxin can be sequentially initiated by continuous perfusions in our system. Moreover, by rapidly switching the solutions, the protein translocation based on ratchet mechanisms can be paused and reinitiated iteratively in our system. Overall, this perfusion system provides a controllable and reliable solution exchange platform for investigations of pores and translocations on lipid bilayers.

Pore forming toxins (PFTs) are virulent proteins released by several species, including many strains of bacteria, to attack and kill host cells. In this article, we focus on the utility of molecular dynamics (MD) simulations and the molecular insights gleaned from these techniques on the pore forming pathways of PFTs. In addition to all-atom simulations which are widely used, coarse-grained MARTINI models and structure-based models have also been used to study PFTs. Here, the emphasis is on methods and techniques involved while setting up, monitoring, and evaluating properties from MD simulations of PFTs in a membrane environment. We draw from several case studies to illustrate how MD simulations have provided molecular insights into protein-protein and protein-lipid interactions, lipid dynamics, conformational transitions and structures of both the oligomeric intermediates and assembled pore structures., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In this chapter, we provide a practical guide on how to plan, execute, and interpret atomistic and coarse-grained molecular dynamics (MD) simulations of lipid-modified proteins in model membranes. After outlining some key practical considerations when planning such simulations, we survey resources and techniques to obtain force field parameters for nonconventional amino acids, such as posttranslationally lipid-modified amino acids that are unique to this class of proteins. We then describe the protocols to build, setup, and run the simulations, followed by a brief comment on the analysis and interpretation of the simulations. Finally, examples of insights that could be gained from atomistic and coarse-grained MD simulations of lipidated proteins will be provided, using RAS proteins as illustrative examples. Throughout the chapter, we highlight the main advantages and limitations of simulating RAS and related lipid-modified G-proteins in biomimetic membranes., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Molecular dynamics simulations of membrane proteins have grown dramatically in the last 20 years. Running these simulations first requires embedding the protein's three-dimensional structure in a lipid bilayer of a suitable composition, one that resembles its native environment. This step is far from trivial, especially for modeling heterogeneous mixtures of lipids. CHARMM-GUI, a webserver for simulation system preparation greatly simplifies this step, allowing for the construction of complex heterogeneous and/or asymmetric membranes. Here, we demonstrate how to use CHARMM-GUI to build the membrane for the outer-membrane protein BamA.