Lipid Membrane Systems Research Articles

Binding and Interaction of Human Beta Defensin Type 3 with Mixed PIP2 Lipid Membranes

Defensins are cationic cysteine-rich small molecules with molecular masses ranging from 3 to 5 kDa. Human β defensin type 3 (hBD-3) belongs to the human innate immune system, and is mainly secreted from the epithelial cells. It has a broad-spectrum of antimicrobial activities on virus, fungi, and both Gram-positive and Gram-negative bacteria. hBD-3 can bind to the negatively charged and even zwitterionic (but charge-neutral) lipid membrane, disrupt and then translocate through the lipid membrane. In order to understand its dynamics and functional mechanism in molecular level, all-atom molecular dynamics simulations on hBD-3 binding with and crossing mixed lipid membranes were investigated. Since PIP2 is the key minor component in cell membranes, mixed PIP2 lipid membrane represented by neutrally charged POPC lipids mixed with 10% of PIP2 lipids were studied. Simulations on hBD-3 placed inside the mixed lipid bilayer, and on the surface of the lipid bilayer were conducted. In order to consider the influence of the initial binding orientation on the hBD-3 binding structure, in total five different initial binding orientations of hBD-3 on the lipid bilayer were studied. Since hBD-3 can form a dimer, the binding of hBD-3 in both monomer and dimer forms with lipid membranes were explored. The structure of hBD-3 on lipid membranes and the key residues on the binding interface were predicted and compared. During the micro-second long simulations, the assembly of PIP2 lipids towards hBD-3 was observed in both hBD-3 on the lipid membrane surface systems and inside the lipid membrane systems. hBD-3 staying inside the mixed lipid bilayer can behave as the channel to facilitate water molecules to cross the mixed lipid bilayer. The result emphasized the importance of PIP2 in hBD-3's disruption capability with the mixed cell membrane.

Visualization of Protein-Lipid Interactions in Connexin-46/50 Intercellular Channels by Cryo-EM and MD-Simulation

Cell-to-cell communication by gap junctions is facilitated by a unique macromolecular architecture, where intercellular channels directly couple the plasma membranes of two neighboring cells. In each membrane, a “hemi-channel” is formed by the oligomerization of six individual subunits (called connexins). To gain insight toward how gap junctions interact with their local membrane environment, we used lipid nanodisc technology to incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid membrane system – closely mimicking a native cell-to-cell junction. Structural characterization of Cx46/50 lipid-embedded channels by single particle CryoEM revealed a drastic lipid-induced stabilization to the channel architecture, resulting in a 3D reconstruction at 2.1 Å resolution – providing an unprecedented level of detail for the class of protein. The subunit packing within each hemi-channel is buttressed by a bouquet of highly-stabilized lipid acyl-chains. Remarkably, the stabilizing effects to the lipid-bilayer extends well beyond the annular layer of lipids and effectively immobilizes the extra-cellular leaflets of the two connected membranes. In addition, over 150 water molecules are resolved within the channel architecture. Time-averaged densities of the lipids and water obtained by MD simulation display the same pattern of stabilization seen in the CryoEM reconstruction. MD analysis suggest lipid headgroups remain highly dynamic relative to the lipid acyl-chains, rationalizing the absence of headgroup densities in the 2.1 Å CryoEM density map. 3D heterogeneity analysis of the CryoEM data identified 4 distinct classes of lipid headgroup configurations, which were also detected by network analysis of extracellular lipids in MD-simulations. Lipid and water binding sites identified in Cx46/50 are remarkably conserved across the connexin family, suggesting these stabilizing features are key contributors to the structure and function of gap junction intercellular communication.

Sulfolobus acidocaldarius Microvesicles Exhibit Unusually Tight Packing Properties as Revealed by Optical Spectroscopy.

In this study, we used optical spectroscopy to characterize the physical properties of microvesicles released from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sa-MVs). The most abundant proteins in Sa-MVs are the S-layer proteins, which self-assemble on the vesicle surface forming an array of crystalline structures. Lipids in Sa-MVs are exclusively bipolar tetraethers. We found that when excited at 275 nm, intrinsic protein fluorescence of Sa-MVs at 23 °C has an emission maximum at 303 nm (or 296 nm measured at 75 °C), which is unusually low for protein samples containing multiple tryptophans and tyrosines. In the presence of 10–11 mM of the surfactant n-tetradecyl-β-d-maltoside (TDM), Sa-MVs were disintegrated, the emission maximum of intrinsic protein fluorescence was shifted to 312 nm, and the excitation maximum was changed from 288 nm to 280.5 nm, in conjunction with a significant decrease (>2 times) in excitation band sharpness. These data suggest that most of the fluorescent amino acid residues in native Sa-MVs are in a tightly packed protein matrix and that the S-layer proteins may form J-aggregates. The membranes in Sa-MVs, as well as those of unilamellar vesicles (LUVs) made of the polar lipid fraction E (PLFE) tetraether lipids isolated from S. acidocaldarius (LUVPLFE), LUVs reconstituted from the tetraether lipids extracted from Sa-MVs (LUVMV) and LUVs made of the diester lipids, were investigated using the probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The generalized polarization (GP) values of Laurdan in tightly packed Sa-MVs, LUVMV, and LUVPLFE were found to be much lower than those obtained from less tightly packed DPPC gel state, which echoes the previous finding that the GP values from tetraether lipid membranes cannot be directly compared with the GP values from diester lipid membranes, due to differences in probe disposition. Laurdan’s GP and red-edge excitation shift (REES) values in Sa-MVs and LUVMV decrease with increasing temperature monotonically with no sign for lipid phase transition. Laurdan’s REES values are high (9.3–18.9 nm) in the tetraether lipid membrane systems (i.e., Sa-MVs, LUVMV and LUVPLFE) and low (0.4–5.0 nm) in diester liposomes. The high REES and low GP values suggest that Laurdan in tetraether lipid membranes, especially in the membrane of Sa-MVs, is in a very motionally restricted environment, bound water molecules and the polar moieties in the tetraether lipid headgroups strongly interact with Laurdan’s excited state dipole moment, and “solvent” reorientation around Laurdan’s chromophore in tetraether lipid membranes occurs very slowly compared to Laurdan’s lifetime.

Synthetic organelles

One approach towards the creation of bottom-up synthetic biological systems of higher complexity relies on the subcompartmentalization of synthetic cell structures using artificially generated organelles - roughly mimicking the architecture of eukaryotic cells. Organelles create dedicated chemical environments for specific synthesis tasks - they separate incompatible processes from each other and help to create or maintain chemical gradients that drive other chemical processes. Artificial organelles have been used to compartmentalize enzyme reactions, to generate chemical fuels via photosynthesis and oxidative phosphorylation, and they have been utilized to spatially organize cell-free gene expression reactions. In this short review article, we provide an overview of recent developments in this field, which involve a wide variety of compartmentalization strategies ranging from lipid and polymer membrane systems to membraneless compartmentalization via coacervation.

Quantitative analysis for lipophilic drug transport through a model lipid membrane with membrane retention

Drug absorption after oral administration mostly occurs in the intestine, and is controlled mainly by the aqueous solubility and the intestinal permeability of the drug. In modern drug discovery, the proportion of lipophilic drug candidates with poor water solubility has increased, and these lipophilic drugs are known to undergo membrane retention during transport across the cell membrane, depending on the hydrophobic interaction between the drug and lipid molecules. Hence, a precise and effective permeability assay for lipophilic drug compounds, which can also quantify membrane retention, is required. In this study, we developed a permeability assay for lipophilic drugs with poor water solubility using a freestanding lipid bilayer. The lipid bilayer was created within a UV cuvette, and the number of transported molecules through the bilayer was estimated by measuring the UV absorbance over time. We then measured the permeability of six tested compounds, and there was a significant difference in permeability between the Biopharmaceutics Classification System (BCS) class 2 and class 4 compounds. In addition, the fraction of molecules trapped in the lipid bilayer were estimated for each compound as well. As a result, the drugs with higher lipophilicity were found to undergo more membrane retention, which agrees with the previous reports, supporting that our model lipid membrane system could also be applied to investigate the drug-lipid interaction.

Hybrids of Polymeric Capsules, Lipids, and Nanoparticles: Thermodynamics and Temperature Rise at the Nanoscale and Emerging Applications.

The importance of thermodynamics does not need to be emphasized. Indeed, elevated temperature processes govern not only industrial scale production but also self-assembly, chemical reaction, interaction between molecules, etc. Not surprisingly, biological processes typically take place at a specific temperature. Here, we look at possibilities to raise the localized temperature by a laser around noble-metal nanoparticles incorporated into shells of layer-by-layer polyelectrolyte microcapsules-freely suspended delivery vehicles in an aqueous solution, developed in the Department of Interfaces, Max Planck Institute of Colloids and Interfaces, headed by Helmuth Möhwald. Understanding the mechanisms of localized temperature rise is essential, that is why we analyze the influence of incident intensity, nanoparticle size, their distribution and aggregation state, as well as thermodynamics at the nanoscale. This leads us to scrutinize "global" (used for thermal encapsulation) versus "local" (used for release of encapsulated materials) temperature rise. Similar analysis is extended to planar polymeric coatings, the lipid membrane system of vesicles and cells, on which nanoparticles are adsorbed. Insights are provided into the mechanisms of physicochemical and biological effects, the nature of which has always been profoundly, interactively, and engagingly discussed in the Department of Interfaces. This analysis is combined with recent developments providing outlook and highlighting a broad range of emerging applications.

Inhibition of copper-induced lipid peroxidation by sinapic acid and its derivatives in correlation to their effect on the membrane structural properties

The efficiencies of sinapic acid and its derivatives syringic acid, syringaldehyde, three sinapoyl esters (ethyl, propyl, butyl sinapates), 4-vinylsyringol and sinapine were investigated for prevention of lipid peroxidation in correlation with their interactions with model lipid membrane systems. Significant antioxidant activities of propyl and butyl sinapates were seen by fluorimetric assay in phosphatidylcholine liposomes as model membrane using C11-BODIPY581/591 lipophilic fluorescent probe. The sinapic acid esters also had the highest impact on membrane structural properties, as observed by differential scanning calorimetry and fluorescence polarisation measurements. The greatest protection of phospholipids from peroxidation by these esters correlated well with their polarity and insertion into the lipid bilayer.

Interaction of Oil and Lipids in Freestanding Lipid Bilayer Membranes Studied with Label-Free High-Throughput Wide-Field Second-Harmonic Microscopy

The interaction of oils and lipids is relevant for membrane biochemistry since the cell uses bilayer membranes, lipid droplets, and oily substances in its metabolic cycle. In addition, a variety of model lipid membrane systems, such as freestanding horizontal membranes and droplet interface bilayers, are made using oil to facilitate membrane monolayer apposition. We characterize the behavior of excess oil inside horizontal freestanding lipid bilayers using different oils, focusing on hexadecane and squalene. Using a combination of second-harmonic (SH) and white-light imaging, we measure how oil redistributes within the membrane bilayer after formation. SH imaging shows that squalene forms a wider annulus compared with hexadecane, suggesting that there is a higher quantity of squalene remaining in the bilayer compared with hexadecane. Excess oil droplets that appear right after membrane formation are tracked with white-light microscopy. Hexadecane droplets move directionally to the edge of the membrane with diffusion constants similar to those of single lipids, whereas squalene oil droplets move randomly with lower diffusion speeds similar to lipid condensed domains and remain trapped in the center of the bilayer for ∼1-3 h. We discuss the observed differences in terms of different coupling mechanisms between the oil and lipid molecules induced by the different chemical structures of the oils.

Thermo-Responsive Behavior of Polymer Solutions Coated with Phospholipids

Various kinds of cell model systems have been utilized for fundamental studies on physical properties of cells. Water-in-Oil (W/O) droplet system coated with phospholipids is one of the beneficial simple model systems of living cells. For instance, a biomimetic phase-separated membrane was constructed in the W/O systems with phospholipid mixture (Hamada et al., J. Phys. Chem. Lett., 2010) since phase separation in lipid membrane system corresponds to the separation between lipid raft domains and other soft domains in the living cell membranes. Phase transition of a polymer has been studied for many years, such as coil-globule transition (Nishio et al., Nature, 1979), and helix-coil transition (Tanaka et al., J. Chem. Phys., 1973). In addition to the phase transition of polymers, phase separation in a binary polymer solution was also reported (Yanagisawa et al., J. Mol. Liq., 2014). However, cell-sized confinement effects on polymer solutions have been poorly confirmed. To clarify the cell-sized confinement effects on polymer solutions, we previously observed the dynamic behavior of polymer droplets (Yoshida et al., Polymers, 2017). The previous study revealed that droplet size influences the start time of phase separation in the case of temperature change from 10 to 65 degrees Celsius. In this study, we observed the dynamic behavior of hydroxypropyl cellulose (HPC) solution droplets in response to temperature change from 10 to 65 degrees Celsius using a fluorescence microscope. The droplets were coated with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). As a control, we observed non-coated water droplets. DPPC and DPPE are widely used for studies on model cell membranes. Using time-lapse images, we confirmed the water/HPC distribution using a spatial autocorrelation function. The results revealed that water-rich region appeared in the case of lipid coated droplets with increase in temperature while such phenomenon was not observed in the case of non-coated droplets.

Catalytic Activity and Proton Translocation of Reconstituted Respiratory Complex I Monitored by Surface-Enhanced Infrared Absorption Spectroscopy.

Respiratory complex I (CpI) is a key player in the way organisms obtain energy, being an energy transducer, which couples nicotinamide adenine dinucleotide (NADH)/quinone oxidoreduction with proton translocation by a mechanism that remains elusive so far. In this work, we monitored the function of CpI in a biomimetic, supported lipid membrane system assembled on a 4-aminothiophenol (4-ATP) self-assembled monolayer by surface-enhanced infrared absorption spectroscopy. 4-ATP serves not only as a linker molecule to a nanostructured gold surface but also as pH sensor, as indicated by concomitant density functional theory calculations. In this way, we were able to monitor NADH/quinone oxidoreduction-induced transmembrane proton translocation via the protonation state of 4-ATP, depending on the net orientation of CpI molecules induced by two complementary approaches. An associated change of the amide I/amide II band intensity ratio indicates conformational modifications upon catalysis which may involve movements of transmembrane helices or other secondary structural elements, as suggested in the literature [ Di Luca , Proc. Natl. Acad. Sci. U.S.A. , 2017 , 114 , E6314 - E6321 ].

Cytochrome P450 Prefers to be in Liquid-Ordered Domains in the Endoplasmic Reticulum

High-resolution mapping of protein/lipid interactions is essential to fully understand the biological function of a membrane protein, but poses tremendous challenges to most biophysical and biochemical approaches. These challenges are more severe for membrane proteins like cytochrome P450 that contain a very large soluble domain and exhibit a completely different time scale of dynamics as compared to its transmembrane domain [Durr et al., BBA Biomemb, 1768(12), 2007; Yamamoto et al., Sci Rep, 3, 2013; Yamamoto et al., Sci Rep, 7, 2017]. In this study, we use peptide-based lipid nanodiscs to “trap” the microsomal cytochrome P450 2B4 alone or also in complex with its redox partners [Zhang et al., Angew Chem, 55(14), 2016]. We effectively utilized the unique nature of peptide-based nanodiscs, that allows for the lipid exchange among nanodiscs, in order to characterize the immediate membrane environment preferred/stabilized by cytochrome P450. We report the first evidence that CYP2B4 is able to induce the formation of “raft”-like domains in a biomimetic of the endoplasmic reticulum (ER). NMR experiments were used to identify and quantitatively determine the lipids present in the nanodiscs. In addition, biophysical experiments were supported by molecular dynamics simulations to identify a sphingomyelin binding motif in P450, which is largely conserved among microsomal P450s. The protein-induced raft formation increased the thermal stability of P450 and also dramatically altered the ligand binding kinetics. These results unveil lipid/protein dynamics which can possibly contribute to the delicate mechanism of redox catalysis in lipid membrane systems [Barnaba et al., F1000Research, 6, 2017].

Role of the Lipid Membrane on the Oligomeric Assembly and Function of Proteorhodopsin

Transmembrane proteins are biomolecules in the lipid bilayers of cells that accomplish critical tasks such as sensing, transport, and enzymatic catalysis. Previous studies indicated that transmembrane proteins could tune their function by forming oligomeric complexes. However, it remains unclear whether oligomerization modulates function significantly more than other factors in native-like lipid bilayer environments. In this study, we aim to elucidate how protein oligomerization and other environmental factors tune the function of transmembrane proteins in lipid membrane systems. Proteorhodopsin (PR), a model transmembrane protein, was used to examine this functional modulation because of its assessable light-responsive proton transferring functions. Our results showed that oligomerization remained an important factor in tuning PR's functions in a liposome environment. Analyses of PR photocycle kinetics measured by a time-resolved optical absorbance technique indicated that faster photocycle kinetics can be observed with the monomeric PR E50Q mutant than with the pentameric/hexameric wild type PR in a liposome environment. These results concur with what has been observed for surfactant-solubilized PR. Interestingly, the pKa of the PR D97 residue that determines the pH at which proton transport switches direction was insensitive to oligomeric distribution in the lipid bilayer environment, but was sensitive to changes in the hydration gradient across lipid bilayers. Therefore, our work suggested that besides oligomerization, the lipid hydration gradient could play a role in modulating PR's function. The new understanding developed here is expected to yield guidelines for functional studies of other therapeutically relevant transmembrane proteins.

Teaching an old scaffold new recognition tricks: oligopyrrolamide antagonists of IAPP aggregation.

A library of N-substituted oligopyrrolamides was designed to modulate the aggregation kinetics of islet amyloid polypeptide (IAPP). IAPP is a hormonal peptide, co-secreted with insulin in the pancreatic β-cells. IAPP samples a variety of conformations, starting from a native random coil to membrane-associated α-helical intermediates and eventually terminates in the amyloid plaques rich in β-sheet structures. A growing body of evidence suggests that membrane bound α-helical intermediates are the key cytotoxic species that impair the functionality and viability of β-cells and contribute to the onset of type 2 diabetes mellitus (DM2). The N-substituted oligopyrrolamides were screened against the aggregation of IAPP using amyloid kinetic assays. A tripyrrole, ADH-101, was the most effective antagonist of IAPP fibrillation in a physiologically relevant lipid membrane system as well as under de novo conditions. ADH-101 induces/stabilizes a secondary structure in IAPP which potentially affects its downstream functions. ADH-101 efficiently affects IAPP-mediated liposome leakage and cell toxicity in insulin secreting cells. ADH-101 inhibits the elongation process potentially binding to the monomeric IAPP and attenuating its access to the preformed fibers. More importantly, oligopyrrolamides are better inhibitors of IAPP aggregation than analogous oligopyridylamides and have more desirable biological properties reflected by their partition coefficients. In essence, an oligopyrrolamide scaffold has been designed which modulates the membrane bound helical intermediates of IAPP and affects their downstream functions such as oligomerization, membrane poration, and cytotoxicity.

Erratum to: Phase-Separated Liposomes Enhance the Efficiency of Macromolecular Delivery to the Cellular Cytoplasm.

[This corrects the article DOI: 10.1007/s12195-017-0489-4.].

Interaction of cholesterol with artificial bilayer lipid membrane system and development of an electrochemical sensor

Interaction of Cholesterol with the bilayer arrangement of phospholipid molecules was studied using electrochemical impedance spectroscopy in Sodium Chloride (NaCl) bath solutions. The membrane resistance (Rm) was decreased from 3.35GΩ in 1.0M NaCl bath to 0.756GΩ in 0.01M NaCl bath. The cholesterol molecules were found to penetrate into Bilayer Lipid Membrane (BLM) and fluidized the BLM phase. Due to fluidization, the membrane resistance was decreased. The fluidization effect of cholesterol was dependent on the concentration of bath solutions. In 1.0M NaCl bath solution, the membrane was stable up to 200µM concentration of cholesterol. With the addition of cholesterol in NaCl bath solutions, the membrane capacitance was increased. An impedimetric sensor was developed based on the membrane resistance in the presence of cholesterol at various concentrations. The detection limit of cholesterol by impedimetric sensor was dependent on the concentration of NaCl in the bath.

Phase-Separated Liposomes Enhance the Efficiency of Macromolecular Delivery to the Cellular Cytoplasm.

From viruses to organelles, fusion of biological membranes is used by diverse biological systems to deliver macromolecules across membrane barriers. Membrane fusion is also a potentially efficient mechanism for the delivery of macromolecular therapeutics to the cellular cytoplasm. However, a key shortcoming of existing fusogenic liposomal systems is that they are inefficient, requiring a high concentration of fusion-promoting lipids in order to cross cellular membrane barriers. Toward addressing this limitation, our experiments explore the extent to which membrane fusion can be amplified by using the process of lipid membrane phase separation to concentrate fusion-promoting lipids within distinct regions of the membrane surface. We used confocal fluorescence microscopy to investigate the integration of fusion-promoting lipids into a ternary lipid membrane system that separated into liquid-ordered and liquid-disordered membrane phases. Additionally, we quantified the impact of membrane phase separation on the efficiency with which liposomes transferred lipids and encapsulated macromolecules to cells, using a combination of confocal fluorescence imaging and flow cytometry. Here we report that concentrating fusion-promoting lipids within phase-separated lipid domains on the surfaces of liposomes significantly increases the efficiency of liposome fusion with model membranes and cells. In particular, membrane phase separation enhanced the delivery of lipids and model macromolecules to the cytoplasm of tumor cells by at least 4-fold in comparison to homogenous liposomes. Our findings demonstrate that phase separation can enhance membrane fusion by locally concentrating fusion-promoting lipids on the surface of liposomes. This work represents the first application of lipid membrane phase separation in the design of biomaterials-based delivery systems. Additionally, these results lay the ground work for developing fusogenic liposomes that are triggered by physical and molecular cues associated with target cells.

Multilayered Lipid Membrane Stacks for Biocatalysis Using Membrane Enzymes

Multilayered or stacked lipid membranes are a common principle in biology and have various functional advantages compared to single‐lipid membranes, such as their ability to spatially organize processes, compartmentalize molecules, and greatly increase surface area and hence membrane protein concentration. Here, a supramolecular assembly of a multilayered lipid membrane system is reported in which poly‐l‐lysine electrostatically links negatively charged lipid membranes. When suitable membrane enzymes are incorporated, either an ubiquinol oxidase (cytochrome bo 3 from Escherichia coli) or an oxygen tolerant hydrogenase (the membrane‐bound hydrogenase from Ralstonia eutropha), cyclic voltammetry (CV) reveals a linear increase in biocatalytic activity with each additional membrane layer. Electron transfer between the enzymes and the electrode is mediated by the quinone pool that is present in the lipid phase. Using atomic force microscopy, CV, and fluorescence microscopy it is deduced that quinones are able to diffuse between the stacked lipid membrane layers via defect sites where the lipid membranes are interconnected. This assembly is akin to that of interconnected thylakoid membranes or the folded lamella of mitochondria and has significant potential for mimicry in biotechnology applications such as energy production or biosensing.

Droplet Interface Bilayers as a Platform for a Spatially Segregated Nanoreactor

Compartmentalisation is a defining feature of biological processes. Within eukaryotic cells, organelles serve as segregated microenvironments for specific biochemical processes. These microenvironments allow distinct chemical environments to be maintained within the cell and concentrate enzymes involved in catalytic cycles in the same space. This concentration of enzymes and substrates within a small segregated environment contributes to the great efficiency of biochemical processes. I aim to investigate the potential of a novel artificial lipid membrane system, the droplet interface bilayer (DIB), as a platform for a spatially segregated compartmentalised nanoreactor. DIB technology serves as a simple method for producing small aqueous compartments bounded by lipid membranes. The incorporation of membrane transport proteins between the aqueous compartments will allow distinct chemical environments to be maintained and for the controlled separation of substrates and products. The nanoreactor design will aim to recreate the segregated microenvironments and enhanced catalytic efficiencies found in biological processes in a synthetic setting.

Molecular Dynamics Simulation and Analysis of the Antimicrobial Peptide-Lipid Bilayer Interactions.

A great deal of research has been undertaken in order to discover antimicrobial peptides (AMPs) with unexploited mechanisms of action to counteract the health-threatening issues associated with bacterial resistance. The intrinsic effectiveness of AMPs is strongly influenced by their initial interactions with the bacterial cell membrane. Understanding these interactions in the atomistic details is important for the design of the less prone bacteria-resistant peptides. However, these studies always require labor-intensive and difficult steps. With this regard, modeling studies of the AMPs binding to simple lipid membrane systems, e.g., lipid bilayers, is of great advantage. In this chapter, we present an applicable step-by-step protocol to run the molecular dynamics (MD) simulation of the interaction between cyclo-RRWFWR (c-WFW) (a small cyclic AMP) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer using the Groningen machine for chemical simulations (GROMACS) package. The protocol as described here may simply be optimized for other peptide-lipid systems of interest.

The Association of VDAC with Cell Viability of PC12 Model of Huntington's Disease.

It is becoming increasingly apparent that mitochondria dysfunction plays an important role in the pathogenesis of Huntington’s disease (HD), but the underlying mechanism is still elusive. Thus, there is a still need for further studies concerning the upstream events in the mitochondria dysfunction that could contribute to cell death observed in HD. Taking into account the fundamental role of the voltage-dependent anion-selective channel (VDAC) in mitochondria functioning, it is reasonable to consider the channel as a crucial element in HD etiology. Therefore, we applied inducible PC12 cell model of HD to determine the relationship between the effect of expression of wild type and mutant huntingtin (Htt and mHtt, respectively) on cell survival and mitochondria functioning in intact cells under conditions of undergoing cell divisions. Because after 48 h of Htt and mHtt expression differences in mitochondria functioning co-occurred with differences in the cell viability, we decided to estimate the effect of Htt and mHtt expression lasted for 48 h on VDAC functioning. Therefore, we isolated VDAC from the cells and tested the preparations by black lipid membrane system. We observed that the expression of mHtt, but not Htt, resulted in changes of the open state conductance and voltage-dependence when compared to control cells cultured in the absence of the expression. Importantly, for all the VDAC preparations, we observed a dominant quantitative content of VDAC1, and the quantitative relationships between VDAC isoforms were not changed by Htt and mHtt expression. Thus, Htt and mHtt-mediated functional changes of VDAC, being predominantly VDAC1, which occur shortly after these protein appearances in cells, may result in differences concerning mitochondria functioning and viability of cells expressing Htt and mHtt. The assumption is important for better understanding of cytotoxicity as well as cytoprotection mechanisms of potential clinical application.