Hydrophobic Core Of Membrane Research Articles

Improving the antimicrobial potential of the peptide CIDEM-501 through acylation: A computational approach.

Acylation is a common method used to modify antimicrobial peptides to enhance their effectiveness. It increases the interactions between the peptide and the bacterial cell membranes. However, acylation can also reduce the selectivity of the peptides by making them more active on eukaryotic membranes, which can lead to unintended toxicity. This study examines the potential of using in silico tools to evaluate the interaction and selectivity of the antimicrobial peptide CIDEM-501 when acylated with decanoic acid at the N-terminus, compared to the non-acylated counterpart. Circular dichroism, microdilution, and hemolysis assays were used to determine the peptide's secondary structure, antimicrobial activity, and selectivity to validate the theoretical predictions. The acylated peptide showed a more stable interaction with the bacterial membrane by inserting the acyl chain into the membrane's hydrophobic core, which led to tighter adsorption and a greater buried surface area. Additionally, it significantly altered membrane order more than the non-acylated counterpart, suggesting superior antimicrobial potential. Finally, in vitro activity assays confirmed theoretical predictions, showing that the acylated peptide had lower Minimum Inhibitory Concentration (MIC) values than the non-acylated peptide. Neither peptide showed significant hemolytic activity at their MIC. The computational techniques used in this study displayed strong predictive capability and helped to elucidate the interaction between the peptide and the membranes.

Investigating the Bromoform Membrane Interactions Using Atomistic Simulations and Machine Learning: Implications for Climate Change Mitigation.

Methane emissions from livestock contribute to global warming. Seaweeds used as food additive offer a promising emission mitigation strategy because seaweeds are enriched in bromoform─a methanogenesis inhibitor. Therefore, understanding bromoform storage and production in seaweeds and particularly in a cell-like environment is crucial. As a first step toward this aim, we present an atomistic description of bromoform dynamics, diffusion, and aggregation in the presence of lipid membranes. Using all-atom molecular dynamics simulations with customized CHARMM-formatted bromoform force field files, we investigate the interactions of bromoform and lipid bilayer across various concentrations. Bromoform penetrates membranes and at high concentrations forms aggregates outside the membrane without affecting membrane thickness or lipid tail order. Aggregates outside the membrane influence the membrane curvature. Within the membrane, bromoform preferentially localizes in the membrane hydrophobic core and diffuses the slowest along the membrane normal. Employing general local-atomic descriptors and unsupervised machine learning, we demonstrate the similarity of bromoform local structures between the liquid and aggregated forms.

Anti-Bacterial and Anti-Biofilm Activities of Essential Oil from Citrus reticulata Blanco cv. Tankan Peel Against Listeria monocytogenes.

In recent years, plant essential oils have been confirmed as natural inhibitors of foodborne pathogens. Citrus reticulata Blanco cv. Tankan peel essential oil (CPEO) showed anti-Listeria monocytogenes (LM) activities, and this study investigated the associated mechanisms by using high-resolution electron microscope, fluorescence spectrometer, flow cytometer, potentiometer, and transcriptome sequencing. The results showed that CPEO restrained LM growth at a minimum inhibitory concentration of 2% (v/v). The anti-LM abilities of CPEO were achieved by disrupting the permeability of the cell wall, damaging the permeability, fluidity, and integrity of the cell membrane, disturbing the membrane hydrophobic core, and destroying the membrane protein conformation. Moreover, CPEO could significantly inhibit the LM aggregation from forming biofilm by reducing the extracellular polymeric substances' (protein, polysaccharide, and eDNA) production and bacterial surface charge numbers. The RNA sequencing data indicated that LM genes involved in cell wall and membrane biosynthesis, DNA replication and repair, quorum sensing and two-component systems were expressed differently after CPEO treatment. These results suggested that CPEO could be used as a novel anti-LM agent and green preservative in the food sector. Further studies are needed to verify the anti-LM activities of CPEO in real food.

Atherosclerotic Oxidized Lipids Affect Formation and Biophysical Properties of Supported Lipid Bilayers and Simulated Membranes.

Oxidized lipids arising from oxidative stress are associated with many serious health conditions, including cardiovascular diseases. For example, KDdiA-PC and KOdiA-PC are two oxidized phosphatidylcholines (oxPC) directly linked to atherosclerosis, which precipitate heart failure, stroke, aneurysms, and chronic kidney disease. These oxPCs are well-characterized in small particles such as low-density lipoprotein, but how their presence affects the biophysical properties of larger bilayer membranes is unclear. It is also unclear how membrane mediators, such as cholesterol, affect lipid bilayers containing these oxPCs. Here, we characterize supported lipid bilayers (SLBs) containing POPC, KDdiA-PC, or KOdiA-PC, and cholesterol. We used a quartz crystal microbalance with dissipation monitoring (QCM-D), fluorescence microscopy, and all-atom molecular dynamics (MD) to examine the formation process, biophysical properties, and specific lipid conformations in simulated bilayers. Experimentally, we show that liposomes containing either oxPC form SLBs by rupturing on contact with SiO2 substrates, which differs from the typical adsorption-rupture pathway observed with nonoxidized liposomes. We also show that increasing the oxPC concentration in SLBs results in thinner bilayers that contain defects. Simulations reveal that the oxidized sn-2 tails of KDdiA-PC and KOdiA-PC bend out of the hydrophobic membrane core into the hydrophilic headgroup region and beyond. The altered conformations of these oxPC, which are affected by cholesterol content and protonation state of the oxidized functional groups, contribute to trends of decreasing membrane thickness and increasing membrane area with increasing oxPC concentration. This combined approach provides a comprehensive view of the biophysical properties of membranes containing KDdiA-PC and KOdiA-PC at the molecular level, which is crucial to understanding the role of lipid oxidation in cardiovascular disease and related immune responses.

Dynamic Second Harmonic Imaging of Proton Translocation Through Water Needles in Lipid Membranes.

Proton translocation through lipid membranes is a fundamental process in the field of biology. Several theoretical models have been developed and presented over the years to explain the phenomenon, yet the exact mechanism is still not well understood. Here, we show that proton translocation is directly related to membrane potential fluctuations. Using high-throughput wide-field second harmonic (SH) microscopy, we report apparently universal transmembrane potential fluctuations in lipid membrane systems. Molecular simulations and free energy calculations suggest that H+ permeation proceeds predominantly across a thin, membrane-spanning water needle and that the transient transmembrane potential drives H+ ions across the water needle. This mechanism differs from the transport of other cations that require completely open pores for transport and follows naturally from the well-known Grotthuss mechanism for proton transport in bulk water. Furthermore, SH imaging and conductivity measurements reveal that the rate of proton transport depends on the structure of the hydrophobic core of bilayer membranes.

Long-Chain Lipids Facilitate Insertion of Large Nanoparticles into Membranes of Small Unilamellar Vesicles.

Insertion of hydrophobic nanoparticles into phospholipid bilayers is limited to small particles that can incorporate into a hydrophobic membrane core between two lipid leaflets. Incorporation of nanoparticles above this size limit requires the development of challenging surface engineering methodologies. In principle, increasing the long-chain lipid component in the lipid mixture should facilitate incorporation of larger nanoparticles. Here, we explore the effect of incorporating very long phospholipids (C24:1) into small unilamellar vesicles on the membrane insertion efficiency of hydrophobic nanoparticles that are 5-11 nm in diameter. To this end, we improve an existing vesicle preparation protocol and utilized cryogenic electron microscopy imaging to examine the mode of interaction and evaluate the insertion efficiency of membrane-inserted nanoparticles. We also perform classical coarse-grained molecular dynamics simulations to identify changes in lipid membrane structural properties that may increase insertion efficiency. Our results indicate that long-chain lipids increase the insertion efficiency by preferentially accumulating near membrane-inserted nanoparticles to reduce the thermodynamically unfavorable disruption of the membrane.

Ebola Virus Glycoprotein Strongly Binds to Membranes in the Absence of Receptor Engagement.

Ebola virus (EBOV) is an enveloped virus that must fuse with the host cell membrane in order to release its genome and initiate infection. This process requires the action of the EBOV envelope glycoprotein (GP), encoded by the virus, which resides in the viral envelope and consists of a receptor binding subunit, GP1, and a membrane fusion subunit, GP2. Despite extensive research, a mechanistic understanding of the viral fusion process is incomplete. To investigate GP-membrane association, a key step in the fusion process, we used two approaches: high-throughput measurements of single-particle diffusion and single-molecule measurements with optical tweezers. Using these methods, we show that the presence of the endosomal Niemann-Pick C1 (NPC1) receptor is not required for primed GP-membrane binding. In addition, we demonstrate this binding is very strong, likely attributed to the interaction between the GP fusion loop and the membrane's hydrophobic core. Our results also align with previously reported findings, emphasizing the significance of acidic pH in the protein-membrane interaction. Beyond Ebola virus research, our approach provides a powerful toolkit for studying other protein-membrane interactions, opening new avenues for a better understanding of protein-mediated membrane fusion events.

Archaea membranes in response to extreme acidic environments

Bipolar tetraether lipids (BTL), such as glycerol dialkyl calditol tetraether (GDNT) and glycerol dialkyl glycerol tetraether (GDGT), are the dominating lipid species in thermoacidophiles that inhabit at pH ≤ 4 and temperatures ≥65°C. BTL containing archaea membranes respond to environmental pH changes by varying the number of cyclopentane rings in the isoprenoids, the amount of GDNT relative to GDGT, the ratio of tetraethers to diethers, and the level of glycosylation in polar headgroups. These structural and compositional adjustments can alter the hydrogen bond networks in the membrane polar headgroup regions and the packing tightness and rigidity in the membrane hydrophobic core. It is likely that these changes in non-covalent interactions among archaea lipids are made to retain low membrane volume fluctuations and their low sensitivity to temperature, as illustrated in the case of liposomes made of the polar lipid fraction E (PLFE) of Sulfolobus acidocaldarius. As such, a low passive proton permeability and a near neutral intracellular pH can be maintained, and, as a result, optimal activities of soluble and membrane-bound proteins in thermoacidophiles can be retained in acidic growth conditions at elevated growth temperatures.

Evaluation of Dose-dependent Interactions of Antibiotics using RBC Membrane Model Systems

Background: The present study aims to understand the membrane interactions of three different antibiotics widely used for non-specific viral diseases. Methods: The dose-dependent membrane interactions of the selected antibiotics were elucidated using octanol: water partition coefficient and hemolysis assay. Results: Our octanol: water partition coefficient experiments indicated that among the three antibiotics (Azithromycin, Erythromycin, and Amoxicillin), azithromycin and erythromycin were more hydrophobic compared to amoxicillin. This finding also correlated with hypotonic hemolysis assay experiments where the membrane stabilization effects of both azithromycin and erythromycin were reduced in a dose-dependent manner compared to amoxicillin. Conclusion: This may be attributed to the hydrophobic nature of azithromycin, and erythromycin penetrates deeper into the RBC membrane to the extent of the hydrophobic core compared to amoxicillin. The result is also concurrent with the observations from the hemolysis assay in the isotonic conditions. To conclude, among the three antibiotics, azithromycin, erythromycin localized on the hydrophobic core of the lipid membrane, and amoxicillin localized on the outer surface and slight penetration to the outer leaflet of the membrane.

In vivo characterization of the bacterial intramembrane-cleaving protease RseP using the heme binding tag-based assay iCliPSpy

Regulated intramembrane proteolysis (RIP) describes the protease-dependent cleavage of transmembrane proteins within the hydrophobic core of cellular membranes. Intramembrane-cleaving proteases (I-CliPs) that catalyze these reactions are found in all kingdoms of life and are involved in a wide range of cellular processes, including signaling and protein homeostasis. I-CLiPs are multispanning membrane proteins and represent challenging targets in structural and enzyme biology. Here we introduce iCLiPSpy, a simple assay to study I-CLiPs in vivo. To allow easy detection of enzyme activity, we developed a heme-binding reporter based on TNFα that changes color after I-CLiP-mediated proteolysis. Co-expression of the protease and reporter in Escherichia coli (E. coli) results in white or green colonies, depending on the activity of the protease. As a proof of concept, we use this assay to study the bacterial intramembrane-cleaving zinc metalloprotease RseP in vivo. iCLiPSpy expands the methodological repertoire for identifying residues important for substrate binding or activity of I-CLiPs and can in principle be adapted to a screening assay for the identification of inhibitors or activators of I-CLiPs, which is of great interest for proteases being explored as biomedical targets.

How Exactly Do AIEgens Target Bacteria? Leveraging the Targeting Mechanism to Design Sensitive Fluorescent Immunosensors.

Aggregation-induced emission luminogens (AIEgens) are promising candidates for bacterial imaging and detection because they can "Light-Up" pathogenic bacteria without complicated labeling or washing steps. However, there have been few in-depth analyses of the intrinsic mechanism underlying their utility as fluorescence probes for targeting bacteria. Therefore, using large-scale molecular dynamics simulations, we investigated the mechanism of their bacterial "Light-Up" behavior with N,N-diphenyl-4-(7-(pyridin-4-yl)benzo[c][1,2,5]thiadiazol-4-yl) aniline functionalized with 1-bromoethane (TBP-1). We propose that the triphenylamine motif of TBP-1, rather than the positively charged pyridine group, first contacts the cell membrane. After TBP-1 completely inserts into the cell membrane, the hydrophobic triphenylamine motif localizes in the hydrophobic core of the cell membrane, restricting the molecular variation of TBP-1, which induces the fluorescent "turn-on" and bacterial "Light-Up." On this basis, we established a heterogeneous lateral flow immunoassay (LFIA) for the detection of foodborne pathogens. The LFIA system showed improved sensitivity with a limit of detection as low as 103 CFU mL-1 and strong specificity. Our protocol opened an effective shortcut to the design of more efficient AIEgens and novel AIEgens-based immunoassays.

Passive transport of Ca2+ ions through lipid bilayers imaged by widefield second harmonic microscopy

In biology, release of Ca2+ ions in the cytosol is essential to trigger or control many cell functions. Calcium signaling acutely depends on lipid membrane permeability to Ca2+. For proper understanding of membrane permeability to Ca2+, both membrane hydration and the structure of the hydrophobic core must be taken into account. Here, we vary the hydrophobic core of bilayer membranes and observe different types of behavior in high-throughput wide-field second harmonic imaging. Ca2+ translocation is observed through mono-unsaturated (DOPC:DOPA) membranes, reduced upon the addition of cholesterol, and completely inhibited for branched (DPhPC:DPhPA) and poly-unsaturated (SLPC:SLPA) lipid membranes. We propose, using molecular dynamics simulations, that ion transport occurs through ion-induced transient pores, which requires nonequilibrium membrane restructuring. This results in different rates at different locations and suggests that the hydrophobic structure of lipids plays a much more sophisticated regulating role than previously thought.

ペプチドと膜の動的相互作用

Induction of curvature in lipid bilayers may lead to increased exposure of hydrophobic membrane core to cell surfaces and the eventual hydrophobic interaction with amphiphilic peptides. Not only physicochemical driving force, the membrane curvature and exposure of the hydrophobic core can be elicited by physiological cellular actions including movement of the cell membrane driven by cytoskeletal F–actin restructuring. This review highlights our findings during our studies on developing curvature–inducing peptides and intracellular IgG delivery peptides.

Charged Small Molecule Binding to Membranes in MD Simulations Evaluated against NMR Experiments.

Interactions of charged molecules with biomembranes regulate many of their biological activities, but their binding affinities to lipid bilayers are difficult to measure experimentally and model theoretically. Classical molecular dynamics (MD) simulations have the potential to capture the complex interactions determining how charged biomolecules interact with membranes, but systematic overbinding of sodium and calcium cations in standard MD simulations raises the question of how accurately force fields capture the interactions between lipid membranes and charged biomolecules. Here, we evaluate the binding of positively charged small molecules, etidocaine, and tetraphenylphosphonium to a phosphatidylcholine (POPC) lipid bilayer using the changes in lipid head-group order parameters. We observed that these molecules behave oppositely to calcium and sodium ions when binding to membranes: (i) their binding affinities are not overestimated by standard force field parameters, (ii) implicit inclusion of electronic polarizability increases their binding affinity, and (iii) they penetrate into the hydrophobic membrane core. Our results can be explained by distinct binding mechanisms of charged small molecules with hydrophobic moieties and monoatomic ions. The binding of the former is driven by hydrophobic effects, while the latter has direct electrostatic interactions with lipids. In addition to elucidating how different kinds of charged biomolecules bind to membranes, we deliver tools for further development of MD simulation parameters and methodology.

Aligned peptoid-based macrodiscs for structural studies of membrane proteins by oriented-sample NMR

Development of a robust, uniform, and magnetically orientable lipid mimetic will undoubtedly advance solid-state NMR of macroscopically aligned membrane proteins. Here, we report on a novel lipid membrane mimetic based on peptoid belts. The peptoids, composed of 15 residues, were synthesized by alternating N-(2-phenethyl)glycine with N-(2-carboxyethyl)glycine residues at a 2:1 molar ratio. The chemically synthesized peptoids possess a much lower degree of polydispersity versus styrene-maleic acid polymers, thus yielding uniform discs. Moreover, the peptoid oligomers are more flexible and do not require a specific folding, unlike lipoproteins, in order to wrap around the hydrophobic membrane core. The NMR spectra measured for the membrane-bound form of Pf1 coat protein incorporated in this new lipid mimetics demonstrate a higher order parameter and uniform linewidths compared with the conventional bicelles and peptide-based macrodiscs. Importantly, unlike bicelles, the peptoid-based macrodiscs are detergent free.

Membrane Permeability of Modified Butyltriphenylphosphonium Cations.

The alkyltriphenylphosphonium (TPP) group is the most widely used vector targeted to mitochondria. Previously, the length of the alkyl linker was varied as well as structural modifications in the TPP phenyl rings to obtain the optimal therapeutic effect of a pharmacophore conjugated with a lipophilic cation. In the present work, we synthesized butyltriphenylphosphonium cations halogenated and methylated in phenyl rings (C4TPP-X) and measured electrical current through a planar lipid bilayer in the presence of C4TPP-X. The permeability of C4TPP-X varied in the range of 6 orders of magnitude and correlates well with the previously measured translocation rate constant for dodecyltriphenylphosphonium analogues. The partition coefficient of the butyltriphenylphosphonium analogues obtained by calculating the difference in the free energy of cation solvation in water and octane using quantum chemical methods correlates well with the permeability values. Using an ion-selective electrode, a lower degree of accumulation of analogues with halogenated phenyl groups was found on isolated mitochondria of rat liver, which is in agreement with their permeability decrease. Our results indicate the translocation of the butyltriphenylphosphonium cations across the hydrophobic membrane core as rate-limiting stage in the permeability process rather than their binding/release to/from the membrane.

Lessons from a Single Amino Acid Substitution: Anticancer and Antibacterial Properties of Two Phospholipase A2-Derived Peptides.

The membrane-active nature of phospholipase A2-derived peptides makes them potential candidates for antineoplastic and antibacterial therapies. Two short 13-mer C-terminal fragments taken from snake venom Lys49-PLA2 toxins (p-AppK and p-Acl), differing by a leucine/phenylalanine substitution, were synthesized and their bioactivity was evaluated. Their capacity to interfere with the survival of Gram-positive and Gram-negative bacteria as well as with solid and liquid tumors was assessed in vitro. Toxicity to red blood cells was investigated via in silico and in vitro techniques. The mode of action was mainly studied by molecular dynamics simulations and membrane permeabilization assays. Briefly, both peptides have dual activity, i.e., they act against both bacteria, including multidrug-resistant strains and tumor cells. All tested bacteria were susceptible to both peptides, Pseudomonas aeruginosa being the most affected. RAMOS, K562, NB4, and CEM cells were the main leukemic targets of the peptides. In general, p-Acl showed more significant activity, suggesting that phenylalanine confers advantages to the antibacterial and antitumor mechanism, particularly for osteosarcoma lines (HOS and MG63). Peptide-based treatment increased the uptake of a DNA-intercalating dye by bacteria, suggesting membrane damage. Indeed, p-AppK and p-Acl did not disrupt erythrocyte membranes, in agreement with in silico predictions. The latter revealed that the peptides deform the membrane and increase its permeability by facilitating solvent penetration. This phenomenon is expected to catalyze the permeation of solutes that otherwise could not cross the hydrophobic membrane core. In conclusion, the present study highlights the role of a single amino acid substitution present in natural sequences towards the development of dual-action agents. In other words, dissecting and fine-tuning biomembrane remodeling proteins, such as snake venom phospholipase A2 isoforms, is again demonstrated as a valuable source of therapeutic peptides.

Rotational decoupling between the hydrophilic and hydrophobic regions in lipid membranes

Cells use homeostatic mechanisms to ensure an optimal composition of distinct types of lipids in cellular membranes. The hydrophilic region of biological lipid membranes is mainly composed of several types of phospholipid headgroups that interact with incoming molecules, nanoparticles, and viruses, whereas the hydrophobic region consists of a distribution of acyl chains and sterols affecting membrane fluidity/rigidity related properties and forming an environment for membrane-bound molecules such as transmembrane proteins. A fundamental open question is to what extent the motions of these regions are coupled and, consequently, how strongly the interactions of phospholipid headgroups with other molecules depend on the properties and composition of the membrane hydrophobic core. We combine advanced solid-state nuclear magnetic resonance spectroscopy with high-fidelity molecular dynamics simulations to demonstrate how the rotational dynamics of choline headgroups remain nearly unchanged (slightly faster) with incorporation of cholesterol into a phospholipid membrane, contrasting the well-known extreme slowdown of the other phospholipid segments. Notably, our results suggest a new paradigm in which phospholipid dipole headgroups interact as quasi-freely rotating flexible dipoles at the interface, independent of the properties in the hydrophobic region.

The role of alpha-helix on the structure-targeting drug design of amyloidogenic proteins

The most accredited hypothesis links the toxicity of amyloid proteins to their harmful effects on membrane integrity through the formation of prefibrillar-transient oligomers able to disrupt cell membranes. However, damage mechanisms necessarily assume a first step in which the amyloidogenic protein transfers from the aqueous phase to the membrane hydrophobic core. This determinant step is still poorly understood. However, according to our lipid-chaperon hypothesis, free lipids in solution play a crucial role in facilitating this footfall. Free phospholipid concentration in the aqueous phase acts as a switch between ion channel-like pore and fibril formation, so that high free lipid concentration in solution promotes pore and repress fibril formation. Conversely, low free lipids in the solution favor fibril and repress pore formation. This behavior is due to the formation of stable lipid-protein complexes. Here, we hypothesize that the helix propensity is a fundamental requirement to fulfill the lipid-chaperon model. The alpha-helix region seems to be responsible for the binding with amphiphilic molecules fostering the proposed mechanism. Indeed, our results show the dependency of protein-lipid binding from the helical structure presence. When the helix content is substantially lower than the wild type, the contact probability decreases. Instead, if the helix is broadening, the contact probability increases. Our findings open a new perspective for in silico screening of secondary structure-targeting drugs of amyloidogenic proteins.

Effect of Peptide Sequence Mutations on the Translocation of Charged Peptide Loops across Lipid Bilayers with Molecular Dynamics Simulations

The hydrophobic core of the cell membrane is considered largely impermeable to charged molecules because of the large free energy barrier and corresponding long timescale (∼hours) for charge translocation. Contradicting this view, a variety of water-soluble peptides are known to translocate charged groups across the cell membrane on a surprisingly rapid ∼seconds timescale, for at least some peptides. In this work, we study the interconversion of single α-helix peptides with charged flanking loops between a surface-adsorbed and membrane-embedded state, which requires the translocation (or “flipping”) of charged loops across the bilayer.