α-Synuclein colocalizes with AP180 and affects the size of clathrin lattices.

α-Synuclein and family members ß- and γ-synuclein are presynaptic proteins that sense and generate membrane curvature, properties important for synaptic vesicle (SV) cycling. αßγ-synuclein triple knockout neurons exhibit SV endocytosis deficits. Here, we investigated if α-synuclein affects clathrin assembly in vitro. Visualizing clathrin assembly on membranes using a lipid monolayer system revealed that α-synuclein increases clathrin lattices size and curvature. On cell membranes, we observe that α-synuclein is colocalized with clathrin and its adapter AP180 in a concentric ring pattern. Clathrin puncta that contain both α-synuclein and AP180 were significantly larger than clathrin puncta containing either protein alone. We determined that this effect occurs in part through colocalization of α-synuclein with the phospholipid PI(4,5)P2 in the membrane. Immuno-electron microscopy (EM) of synaptosomes uncovered that α-synuclein relocalizes from SVs to the presynaptic membrane upon stimulation, positioning α-synuclein to function on presynaptic membranes during or after stimulation. Additionally, we show that deletion of synucleins impacts brain-derived clathrin-coated vesicle size. Thus, α-synuclein affects the size and curvature of clathrin structures on membranes and functions as an endocytic accessory protein.

Impact of chlorogenic acid on surface and phase properties of cholesterol-enriched phosphatidylcholine membranes.

This study analyses the insertion of Chlorogenic acid (CGA) in phosphatidylcholine (PC) membranes enriched with cholesterol (Chol). While cholesterol decreases the area per lipid and increases the dipole potential, CGA increases and decreases these values, respectively. When CGA is inserted into cholesterol-containing DMPC membranes, these effects cancel out, resulting in values that overlap with those of DMPC monolayers without Chol and CGA. The presence of CGA also compensates the increase of dipole potential produced by Chol which can be explain as a consequence of the orientation of CGA molecule at the interphase opposing the cholesterol dipole moieties and water dipoles. This compensatory effect is less effective when lipids lack carbonyl groups (CO). When monolayers are composed by unsaturated PCs the Chol compensation is found at higher concentrations of CGA due to the direct interaction between CGA and Chol. These results suggest that cholesterol modulates the interaction and distribution of CGA in the lipid membrane, which may have implications for its biological activity.

Cell-free translation system with artificial lipid-monolayer particles as a unique tool for characterizing lipid-monolayer binding proteins.

Methods for functional analysis of proteins specifically localizing to lipid monolayers such as rubber particles and lipid droplets are limited. We have succeeded in establishing a system in which artificially prepared lipid monolayer particles are added to a cell-free translation system to confirm the properties of proteins that specifically bind to lipid monolayers in a translation-coupled manner.

Double-headed binding of myosin II to F-actin shows the effect of strain on head structure.

Force production in muscle is achieved through the interaction of myosin and actin. Strong binding states in active muscle are associated with Mg·ADP bound to the active site; release of Mg·ADP allows rebinding of ATP and dissociation from actin. Thus, Mg·ADP binding is positioned for adaptation as a force sensor. Mechanical loads on the lever arm can affect the ability of myosin to release Mg·ADP but exactly how this is done is poorly defined. Here we use F-actin decorated with double-headed smooth muscle myosin fragments in the presence of Mg·ADP to visualize the effect of internally supplied tension on the paired lever arms using cryoEM. The interaction of the paired heads with two adjacent actin subunits is predicted to place one lever arm under positive and the other under negative strain. The converter domain is believed to be the most flexible domain within myosin head. Our results, instead, point to the segment of heavy chain between the essential and regulatory light chains as the location of the largest structural change. Moreover, our results suggest no large changes in the myosin coiled coil tail as the locus of strain relief when both heads bind F-actin. The method would be adaptable to double-headed members of the myosin family. We anticipate that the study of actin-myosin interaction using double-headed fragments enables visualization of domains that are typically noisy in decoration with single-headed fragments.

Effect of Fluoxetine on the Surface Behavior of the Lipid Monolayers at Different Surface Pressures.

Fluoxetine (FLX), used in the clinic to treat depression, is a well-known cationic amphiphilic antidepressant. However, there is a lack of research on the effect of FLX on the surface behavior of lipid monolayers under different surface pressures. In this study, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/CHOL (DPPC/POPC/CHOL) monolayers were prepared via the Langmuir method, and FLX was added to these monolayers under various surface pressures. The effect of FLX on the surface behavior of DPPC/POPC/CHOL monolayers under various surface pressures was studied using a combination of surface pressure-area isotherms, compressibility modulus-surface pressure curves, and atomic force microscope (AFM). The results showed that the effect of FLX on the lipid monolayers was different under different surface pressures. The interaction between FLX and lipid molecules was weak under low surface pressures, and FLX could easily intercalate between the lipid molecules to inhibit monolayer phase transition. The interaction between FLX and lipid molecules was enhanced and FLX tended to self-aggregate to reduce the monolayer stability when the surface pressure was high. This study lays the foundation for further studies on the interaction between FLX and lipid monolayers.

Proteomic analysis of monolayer-integrated proteins on lipid droplets identifies amphipathic interfacial α-helical membrane anchors.

Despite not spanning phospholipid bilayers, monotopic integral proteins (MIPs) play critical roles in organizing biochemical reactions on membrane surfaces. Defining the structural basis by which these proteins are anchored to membranes has been hampered by the paucity of unambiguously identified MIPs and a lack of computational tools that accurately distinguish monolayer-integrating motifs from bilayer-spanning transmembrane domains (TMDs). We used quantitative proteomics and statistical modeling to identify 87 high-confidence candidate MIPs in lipid droplets, including 21 proteins with predicted TMDs that cannot be accommodated in these monolayer-enveloped organelles. Systematic cysteine-scanning mutagenesis showed the predicted TMD of one candidate MIP, DHRS3, to be a partially buried amphipathic α-helix in both lipid droplet monolayers and the cytoplasmic leaflet of endoplasmic reticulum membrane bilayers. Coarse-grained molecular dynamics simulations support these observations, suggesting that this helix is most stable at the solvent-membrane interface. The simulations also predicted similar interfacial amphipathic helices when applied to seven additional MIPs from our dataset. Our findings suggest that interfacial helices may be a common motif by which MIPs are integrated into membranes, and provide high-throughput methods to identify and study MIPs.

The acid-base and redox properties of menaquinone MK-4, MK-7, and MK-9 (vitamin K2) in DMPC monolayers on mercury.

The acid-base and redox properties of the menaquinones MK-4, MK-7, and MK-9 (vitamin K2) have been studied in DMPC monolayers on mercury electrodes. The monolayers were prepared by adhesion-spreading of menaquinone-spiked DMPC liposomes on a stationary mercury drop electrode. All three menaquinones possess [Formula: see text] constants outside the experimentally accessible range, i.e., they are higher than about 12. The standard potentials of MK-4, MK-7, and MK-9 in the DMPC monolayers are very similar, i.e., 0.351, 0.326, and 0.330 V (corresponding to the biochemical standard potentials - 0.063, - 0.088, and - 0.085 V).

Effect of N-terminal acetylation on lytic activity and lipid-packing perturbation induced in model membranes by a mastoparan-like peptide.

L1A (IDGLKAIWKKVADLLKNT-NH2) is a peptide that displays a selective antibacterial activity to Gram-negative bacteria without being hemolytic. Its lytic activity in anionic lipid vesicles was strongly enhanced when its N-terminus was acetylated (ac-L1A). This modification seems to favor the perturbation of the lipid core of the bilayer by the peptide, resulting in higher membrane lysis. In the present study, we used lipid monolayers and bilayers as membrane model systems to explore the impact of acetylation on the L1A lytic activity and its correlation with lipid-packing perturbation. The lytic activity investigated in giant unilamellar vesicles (GUVs) revealed that the acetylated peptide permeated the membrane at higher rates compared with L1A, and modified the membrane's mechanical properties, promoting shape changes. The peptide secondary structure and the changes in the environment of the tryptophan upon adsorption to large unilamellar vesicles (LUVs) were monitored by circular dichroism (CD) and red-edge excitation shift experiments (REES), respectively. These experiments showed that the N-terminus acetylation has an important effect on both, peptide secondary structure and peptide insertion into the bilayer. This was also confirmed by experiments of insertion into lipid monolayers. Compression isotherms for peptide/lipid mixed films revealed that ac-L1A dragged lipid molecules to the more disordered phase, generating a more favorable environment and preventing the lipid molecules from forming stiff films. Enthalpy changes in the main phase transition of the lipid membrane upon peptide insertion suggested that the acetylated peptide induced higher impact than the non-acetylated one on the thermotropic behavior of anionic vesicles.

Membrane affinity and fluorescent labelling: comparative study of monolayer interaction, cellular uptake and cytotoxicity profile of carboxyfluorescein-conjugated cationic peptides.

Fluorescent labelling is a common approach to reveal the molecular details of cellular uptake, internalisation, transport, distribution processes in biological systems. The conjugation with a fluorescent moiety might affect relevant physico-chemical and in vitro transport properties of the bioactive component. A representative set of seven cationic peptides-including cell-penetrating peptides as well as antimicrobial peptides and synthetic derivatives-was selected for our comparative study. Membrane affinity of the peptides and their 5(6)-carboxyfluorescein (Cf) derivatives was determined quantitatively and compared applying Langmuir monolayer of zwitterionic (DPPC) and negatively charged (DPPC + DPPG) lipids as cell membrane models. The interaction with neutral lipid layer is mainly governed by the overall hydrophobicity of the molecule which is remarkably increased by Cf-conjugation for the most hydrophobic Magainin, Melittin and Transportan. A significantly enhanced membrane affinity was detected in negatively charged lipid model monolayer for all of the peptides since the combination of electrostatic and hydrophobic interaction is active in that case. The Cf-conjugation improved the penetration ability of Penetratin and Dhvar4 suggesting that both the highly charged character (Z/n) and the increased hydrophobicity by Cf-conjugation present important contribution to membrane interaction. This effect might also responsible for the observed high in vitro internalisation rate of Penetratin and Dhvar4, while according to in vitro studies they did not cause damage of cell membrane. From the experiments with the given seven cationic peptides, it can be concluded that the Cf-conjugation alters the degree of membrane interaction of such peptides which are moderately hydrophobic and highly charged.

Artificial plasma membrane models based on lipidomic profiling.

Phospholipid monolayers are often described as membrane models for analyzing drug-lipid interactions. In many works, a single phosphatidylcholine is chosen, sometimes with one or two additional components. Drug penetration is studied at 30mN/m, a surface pressure considered as corresponding to the pressure in bilayers, independently of the density of lipid molecular packing. In this work, we have extracted, identified, and quantified the major lipids constituting the lipidome of plasma and mitochondrial membranes of retinoblastoma (Y79) and retinal pigment epithelium cells (ARPE-19), using liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The results obtained from this lipidomic analysis were used in an attempt to build an artificial lipid monolayer with a composition mimicking that of the plasma membrane of Y79 cells, better than a single phospholipid. The variety and number of lipid classes and species in cell extracts monolayers exceeding by far those of the phospholipids chosen to mimic them, the π-A isotherms of model monolayers differed from those of lipid extracts in shape and apparent packing density. We propose a model monolayer based on the most abundant species identified in the extracts, with a surface compressional modulus at 30mN/m close to the one of the lipid extracts.

Computer simulations of lung surfactant.

Lung surfactant lines the gas-exchange interface in the lungs and reduces the surface tension, which is necessary for breathing. Lung surfactant consists mainly of lipids with a small amount of proteins and forms a monolayer at the air-water interface connected to bilayer reservoirs. Lung surfactant function involves transfer of material between the monolayer and bilayers during the breathing cycle. Lipids and proteins are organized laterally in the monolayer; selected species are possibly preferentially transferred to bilayers. The complex 3D structure of lung surfactant and the exact roles of lipid organization and proteins remain important goals for research. We review recent simulation studies on the properties of lipid monolayers, monolayers with phase coexistence, monolayer-bilayer transformations, lipid-protein interactions, and effects of nanoparticles on lung surfactant. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Antimicrobial activity of lysozyme isoforms: Key molecular features.

Increasing bacterial resistance towards antibiotics has stimulated research for novel antimicrobials. Proteins acting on bacterial membranes could be a solution. Lysozyme has been proven active against E. coli by disruption of both outer and cytoplasmic membranes, with dry-heating increasing lysozyme activity. Dry-heated lysozyme (DH-L) is a mixture of isoforms (isoaspartyl, native-like and succinimide lysozymes), giving rise to two questions: what effects does each form have, and which physicochemical properties are critical as regards the antibacterial activity? These issues were investigated by fractionating DH-L, analyzing structural properties of each fraction, and testing each fraction in vivo on bacteria and in vitro on membrane models. Positive net charge, hydrophobicity and molecular flexibility of the isoforms seem key parameters for their interaction with E. coli membranes. The succinimide lysozyme fraction, the most positive, flexible and hydrophobic, shows the highest antimicrobial activity, induces the strongest bacterial membrane disruption and is the most surface active on model lipid monolayers. Moreover, each fraction appears less efficient than DH-L against E. coli, indicating a synergetic cooperation between lysozyme isoforms. The bacterial membrane modifications induced by one isoform could facilitate the subsequent action of the other isoforms.

The role of C-terminal amidation in the membrane interactions of the anionic antimicrobial peptide, maximin H5.

Maximin H5 is an anionic antimicrobial peptide from amphibians, which carries a C-terminal amide moiety, and was found to be moderately haemolytic (20%). The α-helicity of the peptide was 42% in the presence of lipid mimics of erythrocyte membranes and was found able to penetrate (10.8 mN m(-1)) and lyse these model membranes (64 %). In contrast, the deaminated peptide exhibited lower levels of haemolysis (12%) and α-helicity (16%) along with a reduced ability to penetrate (7.8 m Nm(-1)) and lyse (55%) lipid mimics of erythrocyte membranes. Taken with molecular dynamic simulations and theoretical analysis, these data suggest that native maximin H5 primarily exerts its haemolytic action via the formation of an oblique orientated α-helical structure and tilted membrane insertion. However, the C-terminal deamination of maximin H5 induces a loss of tilted α-helical structure, which abolishes the ability of the peptide's N-terminal and C-terminal regions to H-bond and leads to a loss in haemolytic ability. Taken in combination, these observations strongly suggest that the C-terminal amide moiety carried by maximin H5 is required to stabilise the adoption of membrane interactive tilted structure by the peptide. Consistent with previous reports, these data show that the efficacy of interaction and specificity of maximin H5 for membranes can be attenuated by sequence modification and may assist in the development of variants of the peptide with the potential to serve as anti-infectives.

A constant area monolayer method to assess optimal lipid packing for lipolysis tested with several secreted phospholipase A2.

We present an analysis of lipid monolayer hydrolysis at a constant area to assess the optimal lateral surface pressure value (Πopt) and thus, the surface packing density of the lipid, at which the activity of a given lipolytic enzyme is maximal. This isochoric method consists of a measurement of the decrease down to zero of the Πopt of phospholipid substrate monolayer due to continuous hydrolysis using only one reaction compartment. We performed the comparison of both approaches using several commercially available and literature-evaluated sPLA2s. Also, we characterized for the first time the profile of hydrolysis of DLPC monolayers catalyzed by a sPLA2 from Streptomyces violaceoruber and isoenzymes purified from Bothrops diporus venom. One of these viper venom enzymes is a new isoenzyme, partially sequenced by a mass spectrometry approach. We also included the basic myotoxin sPLA2-III from Bothrops asper. Results obtained with the isochoric method and the standard isobaric one produced quite similar values of Πopt, validating the proposal. In addition, we propose a new classification parameter, a lipolytic ratio of hydrolysis at two lateral pressures, 20 mN·m(-1) and 10 mN·m(-1), termed here as LR20/10 index. This index differentiates quite well "high surface pressure" from "low surface pressure" sPLA2s and, by extension; it can be used as a functional criterion for the quality of a certain enzyme. Also, this index could be added to the grouping systematic criteria for the superfamily proposed for phospholipase A2.

Sticholysin I-membrane interaction: an interplay between the presence of sphingomyelin and membrane fluidity.

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT exclusively found in sea anemones. As for actinoporins, it has been proposed that the presence of sphingomyelin (SM) and the coexistence of lipid phases increase binding to the target membrane. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, presence of lipid domains) on actinoporins' activity or which regions of the membrane are the most favorable platforms for protein insertion. To gain insight into the role of SM on the interaction of St I to lipid membranes we studied their binding to monolayers of phosphatidylcholine (PC) and SM in different proportions. Additionally, the effect of acyl chain length and unsaturation, two features related to membrane fluidity, was evaluated on St I binding to monolayers. This study revealed that St I binds and penetrates preferentially and with a faster kinetic to liquid-expanded films with high lateral mobility and moderately enriched in SM. A high content of SM induces a lower lateral diffusion and/or liquid-condensed phases, which hinder St I binding and penetration to the lipid monolayer. Furthermore, the presence of lipid domain borders does not appear as an important factor for St I binding to the lipid monolayer.

Effect of cholesterol and ergosterol on the antibiotic amphotericin B interactions with dipalmitoylphosphatidylcholine monolayers: X-ray reflectivity study.

Amphotericin B is a Streptomyces nodosus metabolite and one of the oldest polyene antibiotics used in the treatment of invasive systemic fungal infections. Despite its over 50-year existence in clinical practice and the recognition of amphotericin B as the gold standard in the treatment of serious systemic mycosis, it still remains one of the most toxic pharmaceuticals. Understanding of the processes at the molecular levels and the interactions between amphotericin B with lipid membranes containing sterols should elucidate the mechanisms of the action and toxicity of this widely used antibiotic. In this work, we use X-ray reflectivity to study the structural changes on a molecular scale after amphotericin B incorporation. These changes are accompanied by an increase in monolayer surface pressure which is more pronounced for ergosterol - rather than cholesterol-rich membranes. The data indicate that this difference is not due to the higher affinity of amphotericin B towards ergosterol-containing membranes but is rather due to a ~3Angstrom corrugation of the monolayer. Furthermore, the total quantity of amphotericin B incorporated into lipid monolayers containing cholesterol and ergosterol is the same.

Peptide and protein binding to lipid monolayers studied by FT-IRRA spectroscopy.

Lipid monolayers at the air-water interface represent half of a lipid bilayer and are therefore suitable model systems for studying the binding of peripheral proteins and polypeptides as well as proteins containing hydrophobic membrane anchors to membrane interfaces. Infrared reflection-absorption spectroscopy (IRRAS) of these monolayer films at the air-water interface provides information on the state of the lipid monolayers as well as on the conformational and orientational order of the film constituents. We will review shortly the experimental set-up and the possibilities for obtaining structural information before several applications of the method to lipid-protein monolayers will be described. We will focus on examples where the analysis of the protein and peptide bands for pure monolayers of these compounds are combined with experiments where the same compounds are bound to lipid monolayers. Combination of these experiments leads to detailed information about the conformational properties and the orientation of the molecules at the air-water interface in contrast to being bound to the lipid-water interface. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.

Surface mixing of products and substrate of PLA2 in enzyme-free mixed monolayers reproduces enzyme-driven structural topography.

It was proposed that topographic changes in lipid monolayers hydrolyzed by lipolytic enzymes such as Phospholipase A2 (PLA2) are a consequence of enzyme activity at the surface. Lateral packing defects that arise from lipid phase coexistence were suggested as places at which PLA2 activity is preferably localized. Our work employs a method for mixing two lipid monolayers in order to simulate lipid mixing of products and substrate at the surface in the absence of enzyme. In such enzyme-free mixed films, a topographic pattern similar to that actively generated by PLA2 is observed. The main conclusion from our experiments is that mixing-demixing properties of substrate and products generated by PLA2 can determine the evolution of the surface topography.

Fabrication of plasmonic probes for reproducible nanospectroscopic investigation of lipid monolayers - The electrochemical etching with DC-pulsed voltage.

Tip-enhanced Raman spectroscopy (TERS) is a label-free analytical technique that characterizes molecular systems, potentially even with a nanometric resolution. In principle, the metallic plasmonic probe is illuminated with a laser beam generating the localized surface plasmons, which induce a strong local electric field enhancement in close proximity to the probe. Such field enhancement improves the Raman scattering cross-section from the sample volume localized near the probe apex. TERS provides a high spatial resolution and a great sensitivity, however, it is rather rarely used due to technical limitations causing unstable enhancement and the relative lack of data reproducibility. Despite many scientific efforts for the fabrication of effective TER probes providing robust TER enhancement still requires further investigations. In this work, we explore new possibilities based on preparation of scanning tunnelling microscopy (STM) plasmonic probes, since by nature of the tunnelling effect, they potentially could offer a very high spatial resolution in STM guided TERS experiments. Here we compare two methods of STM-TERS probe preparation for effective spectra acquisition. Our results strongly indicate that an application of square pulse voltage upon the etching procedure significantly improves the quality of the TER data over those obtained with a constant voltage one. To demonstrate the efficiency of our probes we present the results of hyperspectral TER mapping of the 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) monolayer deposited on an ultra-pure and atomically flat gold substrate.

Assessing the negative impact of chlorantraniliprole, isoxaflutole, and simazine pesticides on phospholipid membrane models and tilapia gill tissues.

The indiscriminate and, very often, incorrect use of pesticides in Brazil, as well as in other countries, results in severe levels of environmental pollution and intoxication of human life. Herein, we studied plasma membrane models (monolayer and bilayer) of the phospholipid Dioleoyl-sn-glycerol-3-phosphocholine (DOPC) using Langmuir films, and large (LUVs) and giant (GUVs) unilamellar vesicles, to determine the effect of the pesticides chlorantraniliprole (CLTP), isoxaflutole (ISF), and simazine (SMZ), used in sugarcane. CLTP affects the lipid organization of the bioinspired models of DOPC π-A isotherms, while ISF and SMZ pesticides significantly affect the LUVs and GUVs. Furthermore, the in vivo study of the gill tissue in fish in the presence of pesticides (2.0 × 10-10 mol/L for CLTP, 8.3 × 10-9 mol/L for ISF, and SMZ at 9.9 × 10-9 mol/L) was performed using optical and fluorescence images. This investigation was motivated by the gill lipid membranes, which are vital for regulating transporter activity through transmembrane proteins, crucial for maintaining ionic balance in fish gills. In this way, the presence of phospholipids in gills offers a model for understanding their effects on fish health. Histological results show that exposure to CLTP, ISF, and SMZ may interfere with vital gill functions, leading to respiratory disorders and osmoregulation dysfunction. The results indicate that exposure to pesticides caused severe morphological alterations in fish, which could be correlated with their impact on the bioinspired membrane models. Moreover, the effect does not depend on the exposure period (24h and 96h), showing that animals exposed to pesticides for a short period suffer irreparable damage to gill tissue. In summary, we can conclude that the harm caused by pesticides, both in membrane models and in fish gills, occurs due to contamination of the aquatic system with pesticides. Therefore, water quality is vital for the preservation of ecosystems.