Membrane Mimetic Model Research Articles

Calcium-Induced Sculpturing of the Plasma Membrane: Lipid Microclusters Cast by Anionic Lipids

The plasma membrane of the cell is a complex, tightly-controlled heterogeneous environment. While anionic lipids constitute a small part of all phospholipids in the plasma membrane, they play key functional roles in regulation of vital cellular processes such as blood clotting, signaling, vesicle fusion, and apoptosis. Lipid composition and distribution in the plasma membrane are highly dynamic, with anionic phosphatidylserine (PS) lipids typically found on the interior leaflet. In turn, the concentration of divalent Ca2+ ions, an important cellular messenger, is three orders of magnitude greater outside of the cell, spatially separating the ions from anionic lipids. PS lipids can become externalized to the outer leaflet of the plasma membrane in the case of cell injury, disease, or apoptotic changes allowing them to interact with Ca2+, resulting in changes in the plasma membrane structure and initiation of signaling cascades. Nonetheless, the molecular details of lipid-Ca2+ interactions are not completely understood. We performed molecular dynamics simulations of binary mixtures of PS and phosphatidylcholine (PC) lipids using highly mobile membrane mimetic model (HMMM) that enabled extended sampling. Simulations revealed that lipid microclusters are formed by higher order (three or more) lipid oligomers coordinated by Ca2+, with two distinct conformations of the PS headgroup serving as building blocks. In parallel, 13C-dephased 31P-observed solid-state nuclear magnetic resonance (SSNMR) rotational-echo double-resonance (REDOR) experiments were performed with spin-diluted Nanodisc samples to report on 31P-13C distances. Experimental data are consistent with both intra- and inter-molecular lipid headgroup characteristic distances of approximately 4.8 +/- 0.5 A that match well with all-atom MD results. To our knowledge, this is one of the first joint MD and high-resolution SSNMR studies to characterize how lipid microclusters are formed at the atomic level and how they reshape the plasma membrane.

The daunorubicin interplay with mimetic model membranes of cancer cells: A biophysical interpretation

The present work aimed to study the interactions between the anticancer drug daunorubicin and lipid membrane mimetic models of cancer cells composed by their most representative classes of phospholipids, with different degrees of complexity.Regarding these anticancer drug-membrane interactions, several biophysical parameters were assessed using liposomes (LUVs) composed of different molar ratios of DMPC, DOPC, DPPS, DOPE and Chol. In this context, daunorubicin's membrane concentration was determined by calculating its partition coefficient (Kp) between liposomes and water using derivative UV/vis spectrophotometry at 37°C and pH6.3, a typical tumoral microenvironment. Characterization of the zeta potential of such model membranes, in both the absence and presence of the compound, was accomplished through Electrophoretic Light Scattering (ELS). Fluorescence quenching studies, which determine the location of the drug within the bilayer, were carried out using liposomes labelled with DPH and TMA-DPH, fluorescent probes with known membrane position. Temperature dependent steady-state anisotropy assays were also performed to measure the daunorubicin effect on the membranes' microviscosity.The overall results support that daunorubicin permeation depends on the phospholipid membrane composition and causes alterations in the biophysical properties of the bilayers, namely in the membrane fluidity. The interaction of daunorubicin with the studied phospholipids is mainly driven by electrostatic and hydrophobic interactions.These insights demonstrated that not only membranes can affect daunorubicin accumulation in cells but the compound can alter the properties of membranes. The changes produced by daunorubicin on the lipid structure may constitute an additional mechanism of action, which might lead to modifications in the location and, consequently, the activity of membrane signaling proteins.

Langmuir monolayers and Differential Scanning Calorimetry for the study of the interactions between camptothecin drugs and biomembrane models

CPT-11 and SN-38 are camptothecins with strong antitumor activity. Nevertheless, their severe side effects and the chemical instability of their lactone ring have questioned the usual forms for its administration and have focused the current research on the development of new suitable pharmaceutical formulations. This work presents a biophysical study of the interfacial interactions of CPT-11 and SN-38 with membrane mimetic models by using monolayer techniques and Differential Scanning Calorimetry. The aim is to get new insights for the understanding of the bilayer mechanics after drug incorporation and to optimize the design of drug delivery systems based on the formation of stable bilayer structures. Moreover, from our knowledge, the molecular interactions between camptothecins and phospholipids have not been investigated in detail, despite their importance in the context of drug action. The results show that neither CPT-11 nor SN-38 disturbs the structure of the complex liposome bilayers, despite their different solubility, that CPT-11, positively charged in its piperidine group, interacts electrostatically with DOPS, making stable the incorporation of a high percentage of CPT-11 into liposomes and that SN-38 establishes weak repulsive interactions with lipid molecules that modify the compressibility of the bilayer without affecting significantly neither the lipid collapse pressure nor the miscibility pattern of drug–lipid mixed monolayers. The suitability of a binary and a ternary lipid mixture for encapsulating SN-38 and CPT-11, respectively, has been demonstrated.

CHARMM-GUI HMMM Builder for Membrane Simulations with the Highly Mobile Membrane-Mimetic Model

Slow diffusion of the lipids in conventional all-atom simulations of membrane systems makes it difficult to sample large rearrangements of lipids and protein-lipid interactions. Recently, Tajkhorshid and co-workers developed the highly mobile membrane-mimetic (HMMM) model with accelerated lipid motion by replacing the lipid tails with small organic molecules. The HMMM model provides accelerated lipid diffusion by one to two orders of magnitude, and is particularly useful in studying membrane-protein associations. However, building an HMMM simulation system is not easy, as it requires sophisticated treatment of the lipid tails. In this study, we have developed CHARMM-GUI HMMM Builder (http://www.charmm-gui.org/input/hmmm) to provide users with ready-to-go input files for simulating HMMM membrane systems with/without proteins. Various lipid-only and protein-lipid systems are simulated to validate the qualities of the systems generated by HMMM Builder with focus on the basic properties and advantages of the HMMM model. HMMM Builder supports all lipid types available in CHARMM-GUI and also provides a module to convert back and forth between an HMMM membrane and a full-length membrane. We expect HMMM Builder to be a useful tool in studying membrane systems with enhanced lipid diffusion.

Membrane Interaction of the Factor VIIIa Discoidin Domains in Atomistic Detail.

A recently developed membrane-mimetic model was applied to study membrane interaction and binding of the two anchoring C2-like discoidin domains of human coagulation factor VIIIa (FVIIIa), the C1 and C2 domains. Both individual domains, FVIII C1 and FVIII C2, were observed to bind the phospholipid membrane by partial or full insertion of their extruding loops (the spikes). However, the two domains adopted different molecular orientations in their membrane-bound states; FVIII C2 roughly was positioned normal to the membrane plane, while FVIII C1 displayed a multitude of tilted orientations. The results indicate that FVIII C1 may be important in modulating the orientation of the FVIIIa molecule to optimize the interaction with FIXa, which is anchored to the membrane via its γ-carboxyglutamic acid-rich (Gla) domain. Additionally, a structural change was observed in FVIII C1 in the coiled main chain leading the first spike. A tight interaction with one lipid per domain, similar to what has been suggested for the homologous FVa C2, is characterized. Finally, we rationalize known FVIII antibody epitopes and the scarcity of documented hemophilic missense mutations related to improper membrane binding of FVIIIa, based on the prevalent nonspecificity of ionic interactions in the simulated membrane-bound states of FVIII C1 and FVIII C2.

Incorporation of charged residues in the CYP2J2 F-G loop disrupts CYP2J2–lipid bilayer interactions

CYP2J2 epoxygenase is an extrahepatic, membrane bound cytochrome P450 (CYP) that is primarily found in the heart and mediates endogenous fatty acid metabolism. CYP2J2 interacts with membranes through an N-terminal anchor and various non-contiguous hydrophobic residues. The molecular details of the motifs that mediate membrane interactions are complex and not fully understood. To gain better insights of these complex protein–lipid interactions, we employed molecular dynamics (MD) simulations using a highly mobile membrane mimetic (HMMM) model that enabled multiple independent spontaneous membrane binding events to be captured. Simulations revealed that CYP2J2 engages with the membrane at the F-G loop through hydrophobic residues Trp-235, Ille-236, and Phe-239. To explore the role of these residues, three F-G loop mutants were modeled from the truncated CYP2J2 construct (Δ34) which included Δ34-I236D, Δ34-F239H and Δ34-I236D/F239H. Using the HMMM coordinates of CYP2J2, the simulations were extended to a full POPC membrane which showed a significant decrease in the depth of insertion for each of the F-G loop mutants. The CYP2J2 F-G loop mutants were expressed in E. coli and were shown to be localized to the cytosolic fraction at a greater percentage relative to construct Δ34. Notably, the functional data demonstrated that the double mutant, Δ34-I236D/F239H, maintained native-like enzymatic activity. The membrane insertion characteristics were examined by monitoring CYP2J2 Trp-quenching fluorescence spectroscopy upon binding nanodiscs containing pyrene phospholipids. Relative to the Δ34 construct, the F-G loop mutants exhibited lower Trp quenching and membrane insertion. Taken together, the results suggest that the mutants exhibit a different membrane topology in agreement with the MD simulations and provide important evidence towards the involvement of key residues in the F-G loop of CYP2J2.

Strength, Not Depth: An Exploration of Differential Membrane Binding Kinetics of Synaptotagmin-1 and Synaptotagmin-7 C2 Domains

Synaptotagmins (Syt) play an essential role in mediating Ca2+-dependent vesicle fusion, and are believed to function by binding its Ca2+-sensing C2 domains to anionic phospholipids and thereby bring the vesicle near the membrane into which the vesicle will fuse. Many Syt isoforms exist, and despite their close structural similarity, the binding kinetics of each isoform to membranes can vary widely. We have performed an extensive set of molecular dynamics simulations on Syt-1 and Syt-7 C2 domains to characterize the factors that determine the rapid unbinding for Syt-1 and the slow unbinding of Syt-7. Through repeated atomistic binding simulations of Syt-1 and Syt-7 C2 domains to anionic membranes, enabled by our enhanced dynamics membrane mimetic model (HMMM), we found that while the penetration depth of the different loop regions are unchanged, Syt-7 C2 domains form many more contacts with anionic head groups. These additional contacts both strengthen and lengthen duration of the interaction between Syt-7 and the membrane, including contacts between loop 2 of Syt. These results provides the first atomistic picture of differential binding of different isoforms of Syt.

CHARMM Gui Membrane Builder Updates

CHARMM-GUI, http://www.charmm-gui.org, is a web-based user interface designed to generate various molecular simulation systems and input files to facilitate and standardize the usage of common and advanced simulation through an automated optimized process. We have made a significant amount of efforts to implement basic and common molecular dynamics simulation techniques into web interface and the web interface has generated a multitude of positive feedback from our users. In this work, we describe our latest efforts to bringing more advanced molecular modeling and simulation techniques to the web interface, including (1) HMMM builder establishing the high mobile membrane-mimetic model, (2) martini maker building coarse-grain models in Martini force fields, (3) NAMD, GROMACS, OpenMM equilibration and production inputs.

Membrane-Induced Structural Rearrangement and Identification of a Novel Membrane Anchor in Talin F2F3

Experimental challenges associated with characterization of the membrane-bound form of talin have prevented us from understanding the molecular mechanism of its membrane-dependent integrin activation. Here, utilizing what we believe to be a novel membrane mimetic model, we present a reproducible model of membrane-bound talin observed across multiple independent simulations. We characterize both local and global membrane-induced structural transitions that successfully reconcile discrepancies between biochemical and structural studies and provide insight into how talin might modulate integrin function. Membrane binding of talin, captured in unbiased simulations, proceeds through three distinct steps: initial electrostatic recruitment of the F2 subdomain to anionic lipids via several basic residues; insertion of an initially buried, conserved hydrophobic anchor into the membrane; and association of the F3 subdomain with the membrane surface through a large, interdomain conformational change. These latter two steps, to our knowledge, have not been observed or described previously. Electrostatic analysis shows talin F2F3 to be highly polarized, with a highly positive underside, which we attribute to the initial electrostatic recruitment, and a negative top face, which can help orient the protein optimally with respect to the membrane, thereby reducing the number of unproductive membrane collision events.

Interactions of isoniazid with membrane models: Implications for drug mechanism of action

This work focuses on the application of biophysical techniques to assess the interaction of isoniazid (INH) with three-dimensional cell membrane mimetic models. Liposomes made of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were used as cell membrane lipids, being the interactions of the drug studied under different pH conditions to mimic the in vivo near neutral physiological pH found in the cytoplasm and the acidic conditions encountered in the protective gastric barrier and in the macrophage intracellular acidic compartments (i.e., phagosomes and phagolysomes). The effect of INH on the biophysical parameters of the cell membrane lipids was assessed by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). The overall results highlighted the importance of the pH for the interactions of INH with biological membranes. A relationship between the effect of INH on the biophysical properties of the membranes and the pH was established. In fact, the experimental results point to a higher affinity of the drug to the acidic environments, which not only explain its sequestration in the infected cells but also some of its side-effects.

Partitioning of Amino Acids into a Model Membrane: Capturing the Interface

Energetics of protein side chain partitioning between aqueous solution and cellular membranes is of fundamental importance for correctly capturing the membrane binding and specific protein–lipid interactions of peripheral membrane proteins. We recently reported a highly mobile membrane mimetic (HMMM) model that accelerates lipid dynamics by modeling the membrane interior partially as a fluid organic solvent while retaining a literal description of the lipid head groups and the beginning of the tails. While the HMMM has been successfully applied to study spontaneous insertion of a number of peripheral proteins into membranes, a quantitative characterization of the energetics of membrane–protein interactions in HMMM membranes has not been performed. We report here the free energy profiles for partitioning of 10 protein side chain analogues into a HMMM membrane. In the interfacial and headgroup regions of the membrane, the side chain free energy profiles show excellent agreement with profiles previously reported for conventional membranes with full-tail lipids. In regions where the organic solvent is prevalent, the increased dipole and fluidity of the solvent generally result in a less accurate description, most notably overstabilization of aromatic and polar amino acids. As an additional measure of the ability of the HMMM model to describe membrane–protein interactions, the water-to-membrane interface transfer energies were analyzed and found to be in agreement with the previously reported experimental and computational hydrophobicity scales. We discuss strengths and weaknesses of HMMM in describing protein–membrane interactions as well as further development of model membranes.

Capturing Spontaneous Binding of Human Islet Amyloid Polypeptide to Anionic Membranes using a Highly Mobile Membrane Mimetic Model

Human islet amyloid polypeptide (hIAPP) aggregates and forms amyloid fibrils in the pancreas of patients with type2 diabetes. Anionic membranes bind hIAPP and accelerate the fibril formation. During this process, hIAPP disrupts the membrane, a toxic process that has been associated with the N-terminal 19 residues and proposed to lead to the destruction of the insulin-producing β-cells in the pancreas. The mechanism by which the membrane is disrupted is therefore key to our understanding of toxicity of hIAPP, and to design of effective disease modifying treatments.Employing molecular dynamics (MD) simulations, we have investigated the molecular events key to hIAPP binding to anionic phospholipid bilayers. The diffusion of lipids in conventional membrane models is too slow to allow for hIAPP-membrane binding on the time-scale feasible with conventional MD simulations. In order to overcome the slow diffusion of lipids in conventional membranes which might not allow for adequate sampling of the binding of hIAPP binding to the membrane, we have employed a highly mobile membrane mimetic (HMMM) model.Multiple independent MD simulations, amounting to 1.2 μs, both for the N-terminal 19-residue peptide, and for the full (37-residue) hIAPP, resulted in spontaneous binding to mixed anionic/zwitterionic HMMM membranes. Initial contacts between the peptide and lipids, which form rapidly (within the first 10 ns of the simulations), are mediated by a group of positively charged residues, Lys1, Arg11, and His18, indicating that the N-terminal part of hIAPP is responsible for its interaction with anionic membranes. These simulations have provided an unprecedented level of statistic on lipid-protein interaction for hIAPP peptides, characterizing residues and lipid chemical groups key for bindig and interaction, as well as the structure and dynamics of the peptide in its membrane-bound form.

Conformational Analysis of the Frog Skin Peptide, Plasticin-L1 and its Effects on the Production of Proinflammatory Cytokines by Macrophages

Plasticin-L1 (GLVNGLLSSVLGGGQGGGGLLGGIL) is a conformationally flexible glycine/leucine-rich peptide originally isolated from norepinephrine-stimulated skin secretions of the South-American Santa Fe frog Leptodactylus laticeps (Leptodactylidae). A nuclear magnetic resonance (NMR)/molecular dynamics (MD) characterization of plasticin-L1 in the presence of dodecylphosphocholine (DPC) and DPC/sodium dodecylsulphate (SDS) micelles as membrane-mimetic models showed that the peptide has affinity for both neutral and anionic membranes. The peptide adopts a stable helical conformation at the N-terminal region and a more disordered helix at the C-terminal region, separated by an unstructured loop wherein the highest number of glycines is localized. In both micelle environments, plasticin-L1 slowly inserts between the detergent head groups, but always remains localized at the micelle/water interface. Plasticin-L1 lacks direct antimicrobial activity but stimulates cytokine production by macrophages. Incubation with plasticin-L1 (20 µg/mL) significantly (P < 0.05) increased the production of the proinflammatory cytokines IL-1β, IL-12 IL-23 and TNF-α from unstimulated peritoneal macrophages from both C57BL/6 and BALB/C mice. The peptide also increased IL-6 production by unstimulated (P < 0.01) and lipopolysaccharide-stimulated (P < 0.01) macrophages, while the effects on production of the anti-inflammatory cytokine IL-10 were not significant. These findings suggest that plasticin-L1 may play an immunomodulatory role in vivo by stimulating cytokine production from frog skin macrophages in response to microbial pathogens. This peptide may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents.

Conformational Analysis of the Frog Skin Peptide, Plasticin-L1, and Its Effects on Production of Proinflammatory Cytokines by Macrophages

Plasticin-L1 (GLVNGLLSSVLGGGQGGGGLLGGIL) is a conformationally flexible glycine/leucine-rich peptide originally isolated from norepinephrine-stimulated skin secretions of the South-American Santa Fe frog Leptodactylus laticeps (Leptodactylidae). A nuclear magnetic resonance/molecular dynamics characterization of plasticin-L1 in the presence of dodecylphosphocholine (DPC) and DPC/sodium dodecylsulphate micelles as membrane-mimetic models showed that the peptide has affinity for both neutral and anionic membranes. The peptide adopts a stable helical conformation at the N-terminal region and a more disordered helix at the C-terminal region, separated by an unstructured loop wherein the highest number of glycines is localized. In both micelle environments, plasticin-L1 slowly inserts between the detergent head groups but always remains localized at the micelle/water interface. Plasticin-L1 lacks direct antimicrobial activity but stimulates cytokine production by macrophages. Incubation with plasticin-L1 (20 μg/mL) significantly (P < 0.05) increased the production of the proinflammatory cytokines IL-1β, IL-12, IL-23, and TNF-α from unstimulated peritoneal macrophages from both C57BL/6 and BALB/C mice. The peptide also increased IL-6 production by unstimulated (P < 0.01) and lipopolysaccharide-stimulated (P < 0.01) macrophages, whereas the effects on production of the anti-inflammatory cytokine IL-10 were not significant. These findings suggest that plasticin-L1 may play an immunomodulatory role in vivo by stimulating cytokine production from frog skin macrophages in response to microbial pathogens. This peptide may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents.

In Vitro Assessment of NSAIDs-Membrane Interactions: Significance for Pharmacological Actions

To study interactions between nonsteroidal anti-inflammatory drugs (NSAIDs) and membrane mimetic models. The interactions of indomethacin and nimesulide with liposomes of dipalmitoylphosphatidylcholine (DPPC) at two physiological pH conditions (pH 7.4 and 5.0) were investigated by time-resolved and steady-state fluorescence techniques and derivative ultraviolet/visible absorption spectrophotometry. Fluorescence quenching studies that assess the location of the drugs interacting with the membrane were carried out using labeled liposomes with trimethylammonium-diphenylhexatriene (TMA-DPH), a fluorescent probe with well-known membrane localization. Partition of the drugs within membranes was determined by calculating their partition coefficients (K p ) between liposomes and water using derivative ultraviolet/visible absorption spectrophotometry in a temperature range of 37-50°C. The Van't Hoff analysis of the temperature dependence of K p values allowed calculating the membrane-water variation of enthalpy (ΔH w→m) and entropy (ΔS w→m) and consequently the Gibbs free energy (ΔG w→m). Results indicate that quenching, partitioning and thermodynamic parameters inherent to the interaction of the studied drugs with the membrane mimetic model are deeply dependent on the initial organization of the membrane, on the pH medium and on the physical properties of the drug. The interactions between NSAIDs and membranes are manifested as changes in the physical and thermodynamic properties of the bilayers. Depending on the composition and physical state of the membrane and the chemical structure of the NSAID, the interaction can support or prevent drug activity or toxicity.

Mechanisms of P-gp inhibition and effects on membrane fluidity of a new rifampicin derivative, 1,8-dibenzoyl-rifampicin

PurposeTo assess P-glycoprotein (P-gp)-modulation ability and the mechanisms of P-gp inhibition mediated by a new synthetic rifampicin derivative, 1,8-dibenzoyl-rifampicin (DiBenzRif), in an in vitro model of the blood–brain barrier (BBB), RBE4 cells, and in membrane mimetic models (liposomes). MethodsP-gp expression (western blot) and activity [rhodamine 123 accumulation studies] were assessed until 72h of exposure to DiBenzRif. The effects on intracellular ATP levels and on P-gp ATPase activity were studied using luciferin–luciferase bioluminescence assay. Membrane fluidity changes were tracked by steady-state anisotropy measurements. Non-P-gp-related rhodamine 123 accumulation was evaluated using liposomes prepared with the main lipids present in RBE4 cell membranes. ResultsA significant increase in intracellular rhodamine 123 content was observed in DiBenzRif-treated cells at all tested time-points. This effect was associated with a significant reduction in ATP intracellular levels, the inhibition of P-gp ATPase activity and a significant increase in membrane fluidity. DiBenzRif also favoured rhodamine 123 accumulation in a liposomal model of RBE4 cells, suggesting that it may be useful in increasing intracellular levels of substances that passively diffuse into the cells. ConclusionDiBenzRif-induced inhibitory effect on P-gp increases xenobiotic accumulation in BBB cells, which may contribute to the development of therapeutic adjuvants to enhance brain penetration of drugs.

Drug–membrane interaction studies applied to N′-acetyl-rifabutin

This work aims the systematic study of the biophysical interactions of a novel antimycobacterial compound (N′-acetyl-rifabutin, RFB2) with membrane models of different lipid composition and surface charge. Membrane mimetic models were used to evaluate the RFB2’s membrane partition, its preferential location across the membrane, and the effect of RFB2 on the biophysical properties of the membrane, which ultimately might be related with the antimycobacterial compound bioavailability and the membrane toxicity. According to the aforementioned, liposomes of dimyristoyl-sn-glycero-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) were, respectively, used as mimetic models of human and bacterial cell membranes.The antimycobacterial compound lipophilicity was evaluated by spectroscopic methods, which enabled the determination of the partition coefficient (Kp). To study the RFB2 membrane’s location, fluorescence quenching studies and lifetime measurements were executed in liposomes labeled with fluorescent probes. In order to evaluate the changes induced by RFB2 on the membrane biophysical properties, dynamic light scattering (DLS) and steady-state anisotropy were performed.The overall results reveal a strong interaction between RFB2 and the membrane models and allowed the evaluation of its lipophilicity, which is a key molecular descriptor in the characterization of novel potential drugs. Moreover, the higher partition of RFB2 and the more pronounced changes in the biophysical parameters of the negatively charged membrane model suggest that RFB2 has more affinity to the bacterial membrane. For the above-mentioned reasons, this work supports that RFB2 has a potential value as a drug in pharmaceutical formulations used to treat mycobacterial infections.

Biophysical characterization of the drug–membrane interactions: The case of propranolol and acebutolol

The interaction of propranolol and acebutolol with biological membranes was assessed in the present work by using a range of biophysical techniques and liposomes, as membrane mimetic models. Liposomes were made of zwitterionic phosphatidylcholines and experiments were performed at physiologic pH and at various membrane physical states (gel, ripple and fluid phases). Fluorescence techniques were used to study the partition coefficient of β-blockers, the influence of drugs on membrane fluidity and the drugs–membrane binding. Moreover, small and wide angle X-ray scattering techniques were used to evaluate the β-blockers effect on long range bilayer order and hydrocarbon chain packing. The gathered results highlighted the importance of electrostatic interactions between propranolol and acebutolol with membranes. Furthermore, both β-blockers exhibited a membrane-fluidizing effect and the capacity to disturb the membrane organization. In general, propranolol unveiled a more pronounced effect on membrane fluidity and structure than acebutolol. In the current study, the obtained results were also correlated with the cardioprotective properties of the β-blockers studied.

Host Defense Peptides: Molecular Details of Attack on Bacterial and Neoplastic Mammalian Model Membranes

Membrane-active host defense peptides constitute a key part of the initial immune response in multicellular organisms. They interact with anionic lipids of bacterial membranes and are also active against neoplastic mammalian membranes. The non-specific nature of these interactions reduces the propensity for developing resistance to antimicrobial and anticancer therapies but makes development of highly effective peptides a challenging task. Various mechanisms of peptide-membrane activity were proposed, but the molecular picture is still incomplete. We employ our novel highly mobile membrane mimetic (HMMM) model with enhanced lipid mobility, combined with all-atom molecular dynamics simulations to investigate structural and dynamic properties determining initial lipid-peptide interactions of magainin 2 (MAG2) and its highly charged variant, paxiganan (MSI-78). Extended multiple simulations were performed of MAG2 and MSI-78 monomers with HMMM binary membranes representing bacterial (PE/PG) and neoplastic (PC/PS) membranes. Spontaneous association of the peptides with the membranes lead to significant clustering of anionic lipids in both membrane models, lending support to the lipid clustering model as a potential first stage of the interaction. Interestingly, membrane surface rupture was observed as a result of lipid rearrangement. PE/PG membranes appear to be more susceptible than PC/PS to rupture by both peptides, which correlates well with recent experimental observations. PE/PG appears to offer easier entry for the charged residues due to hydrogen bonding and lack of positive charges on the PE head groups that may repel the cationic peptides. We propose a “sweep-and-anchor” mechanism that initiates membrane rupture: initial clustering of charged lipids by the charged residues is creating more loose domain boundaries in which hydrophobic residues can penetrate, creating a pore in membrane leaflet. Prior to the development of the HMMM method these inquiries were out of reach of atomistic simulations.

Accelerating Membrane Insertion of Peripheral Proteins with a Novel Membrane Mimetic Model

Characterizing atomic details of membrane binding of peripheral membrane proteins by molecular dynamics (MD) has been significantly hindered by the slow dynamics of membrane reorganization associated with the phenomena. To expedite lateral diffusion of lipid molecules without sacrificing the atomic details of such interactions, we have developed a novel membrane representation, to our knowledge, termed the highly mobile membrane-mimetic (HMMM) model to study binding and insertion of various molecular species into the membrane. The HMMM model takes advantage of an organic solvent layer to represent the hydrophobic core of the membrane and short-tailed phospholipids for the headgroup region. We demonstrate that using these components, bilayer structures are formed spontaneously and rapidly, regardless of the initial position and orientation of the lipids. In the HMMM membrane, lipid molecules exhibit one to two orders of magnitude enhancement in lateral diffusion. At the same time, the membrane atomic density profile of the headgroup region produced by the HMMM model is essentially identical to those obtained for full-membrane models, indicating the faithful representation of the membrane surface by the model. We demonstrate the efficiency of the model in capturing spontaneous binding and insertion of peripheral proteins by using the membrane anchor (γ-carboxyglutamic-acid-rich domain; GLA domain) of human coagulation factor VII as a test model. Achieving full insertion of the GLA domain consistently in 10 independent unbiased simulations within short simulation times clearly indicates the robustness of the HMMM model in capturing membrane association of peripheral proteins very efficiently and reproducibly. The HMMM model will provide significant improvements to the current all-atom models by accelerating lipid dynamics to examine protein-membrane interactions more efficiently.