Lysine Acetylation Changes the Mechanism of Aß25-35 Peptide Binding and Dimerization in the DMPC Bilayer.

The impact of Lys28 acetylation on Alzheimer's Aß peptide binding to the lipid bilayer has not been previously studied, either experimentally or computationally. To probe this common post-translational modification, we performed all-atom replica exchange molecular dynamics simulations targeting binding and aggregation of acetylated acAß25-35 peptide within the DMPC bilayer. Using the unmodified Aß25-35 studied previously as a reference, our results can be summarized as follows. First, Lys28 acetylation strengthens the Aß25-35 hydrophobic moment and consequently promotes the helical structure across the peptide extending it into the N-terminus. Second, because Lys28 acetylation disrupts electrostatic contact between Lys28 and lipid phosphate groups, it reduces the binding affinity of acAß25-35 peptides to the DMPC bilayer. Accordingly, although acetylation preserves the bimodal binding featuring a preferred inserted state and a less probable surface bound state, it decreases the stability of the former. Third, acetylation promotes acAß25-35 aggregation and eliminates monomers as thermodynamically viable species. More importantly, acAß25-35 retains as the most thermodynamically stable the inserted dimer with unique head-to-tail helical aggregation interface. However, due to enhanced helix structure, this dimer state becomes less stable and is less likely to propagate into higher order aggregates. Thus, acetylation is predicted to facilitate the formation of low-molecular-weight oligomers. Other post-translational modifications, including phosphorylation and oxidation, reduce helical propensity and have divergent impact on aggregation. Consequently, acetylation, when considered in its totality, has distinct consequences on Aß25-35 binding and aggregation in the lipid bilayer.

De Novo Transmembrane Aggregation of Aß10-40 Peptides in an Anionic Lipid Bilayer.

Using the all-atom model and 10 µs serial replica-exchange molecular dynamics (SREMD), we investigated the binding of Alzheimer's Aß10-40 peptides to the anionic dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) lipid bilayer. Our objective was to probe de novo transmembrane Aß10-40 aggregation and to test the utility of SREMD. Our results are threefold. First, upon binding, Aß10-40 adopts a helical structure in the C-terminus and deeply inserts into the bilayer. Binding is primarily controlled by electrostatic interactions of the peptides with water, ions, and lipids, particularly, anionic DMPG. Second, Aß-bilayer interactions reorganize lipids in the proximity of the bound peptides, causing an influx of DMPG lipids into the Aß binding footprint. Third and most important, computed free energy landscapes reveal that Aß10-40 peptides partition into monomeric and dimeric species. The dimers result from transmembrane aggregation of the peptides and induce a striking lipid density void throughout both leaflets in the bilayer. There are multiple factors stabilizing transmembrane dimers, including van der Waals and steric interactions, electrostatic interactions, and hydrogen bonding, hydration, and entropic gains originating from dimer conformations and lipid disorder. We argue that helix dipole-dipole interactions underestimated in the all-atom force field must be a contributing factor to stabilizing antiparallel transmembrane dimers. We propose that transmembrane aggregates serve as mechanistic links between the populations of extra- and intracellular Aß peptides. From the computational perspective, SREMD is found to be a viable alternative to traditional replica-exchange simulations.

Reduced DMPC and PMPC in lung surfactant promote SARS-CoV-2 infection in obesity.

OBJECTIVE: Obesity is an established risk factor for higher SARS-CoV-2 viral loads, severe COVID-19 pneumonia requiring hospitalization, and worse outcomes. However, the underlying mechanisms for the increased risk are not well understood. SARS-CoV-2 is a respiratory virus with the primary route of entry through the lungs, where the Spike protein of SARS-CoV-2 binds to the ACE2 receptor on pneumocytes. Lung surfactant produced by type II pneumocytes plays a major role in respiratory defense against infections. Surfactant predominantly contains lipids, especially phosphatidylcholines (PC), and obesity is characterized by aberrant lipid metabolism. We hypothesized that altered lipid composition in lung surfactant in obesity may promote SARS-CoV-2 infection, leading to severe COVID-19 disease. METHODS: Lipidomic analysis of lung tissue and bronchoalveolar lavage fluid (BALF) was performed using LC-MS/MS. The effects of PCs on SARS-CoV-2 pseudovirus infection were studied in HEK293T cells with ACE2 overexpression and in Vero-E6 cells with endogenous ACE2 expression. For the cell-cell fusion assay, HEK293T-ACE2 and HEK293T expressing SARS-CoV-2 Spike/eGFP were used as the target and effector cells, respectively. RESULTS: Lipidomic analysis revealed that myristic acid-containing dimyristoyl-PC (DMPC) and palmitoylmyristoyl-PC (PMPC) were reduced in lung tissue and BALF from high fat diet-induced obese mice. DMPC and PMPC markedly inhibited wild type and D614G mutant SARS-CoV-2 infection in HEK293T-ACE2 and Vero-E6 cells. Feeding obese mice with trimyristin, the triglycerides of myristic acid, increased DMPC and PMPC levels in lung surfactant. Lipid extract from BALF of trimyristin-treated obese mice mitigated the elevated wild type and D614G mutant SARS-CoV-2 infection. The inhibitory effects of DMPC and PMPC on SARS-CoV-2 infection were reversed by cholesterol. CONCLUSIONS: The reduced DMPC and PMPC in lung surfactant may promote SARS-CoV-2 infection. Increasing DMPC and PMPC in lung surfactant could be an innovative strategy for preventing and treating severe COVID-19 disease in obesity.

Mechanisms of Binding of Antimicrobial Peptide PGLa to DMPC/DMPG Membrane.

PGLa belongs to a class of antimicrobial peptides showing strong affinity to anionic bacterial membranes. Using all-atom explicit solvent replica exchange molecular dynamics with solute tempering, we studied binding of PGLa to a model anionic dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bilayer. Due to a strong hydrophobic moment, PGLa upon binding adopts a helical structure and two distinct bound states separated by a significant free energy barrier. In these states, the C-terminus helix is either surface bound or inserted into the bilayer, whereas the N-terminus remains anchored in the bilayer. Analysis of the free energy landscape indicates that the transition between the two states involves a C-terminus helix rotation permitting the peptide to preserve the interactions between cationic Lys amino acids and anionic lipid phosphorus groups. We calculated the free energy of PGLa binding and showed that it is mostly governed by the balance between desolvation of PGLa positive charges and formation of electrostatic PGLa-lipid interactions. PGLa binding induces minor bilayer thinning but causes pronounced lipid redistribution resulting from an influx of DMPG lipids into the binding footprint and efflux of DMPC lipids. Our in silico results rationalize the S-state detected in NMR experiments.

Computational study of DMPC liposomes loaded with the N-(2-Hydroxyphenyl)-2-propylpentanamide (HO-AAVPA) and determination of its antiproliferative activity in vitro in NIH-3T3 cells.

N-(2-Hydroxyphenyl)-2-propylpentanamide (HO-AAVPA) is a valproic acid (VPA) derivative that has shown promising antiproliferative effects in different cancer cell lines, such as A204, HeLa, and MDA-MB-231. However, its low water solubility could reduce its therapeutic effectiveness. To solve this problem, in this work, we incorporated HO-AAVPA into dimyristoyl-phosphatidylcholine (DMPC) liposomes in the presence or absence of cholesterol (CHOL). Using differential scanning calorimetry (DSC), we found that the transition enthalpy (ΔHtr) of DMPC liposomes is reduced in the presence of CHOL and/or HO-AAVPA, indicating the favorable interactions between CHOL and/or HO-AAVPA and DMPC. Further, by molecular dynamics simulations it was possible to observed that HO-AAVPA migrates from the center of the bilayer toward the water and lipid interface of the DPMC bilayer systems exposing the amine group to water and the aliphatic chain toward the interior of the bilayer. As a consequence, we observed an ordering of the lipid bilayer. Moreover, CHOL harbors into the inner bilayer membrane, increasing the order parameter of the system. The liposomal solutions loaded with HO-AAVPA were tested in the NIH3T3 cell line, showing a reduction in cell proliferation compared to those cells presented without liposomes.Communicated by Ramaswamy H. Sarma.

Early-stage culprit in protein misfolding diseases investigated using electrochemical parameters: New insights over peptide-membrane interactions.

The dysfunctioning of ß-cells caused by the unspecific misfolding of the human islet amyloid polypeptide (hIAPP) at the membrane results in type 2 diabetes mellitus. Here, we report for the first time, the early-stage interaction of hIAPP oligomers on the DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) lipid membrane using electrochemical parameters. Electrochemical techniques are better than other techniques to detect hIAPP at significantly lower concentrations. The surface level interactions between the peptide (hIAPP) and lipid membrane (DMPC) were investigated using atomic force microscopy (AFM), confocal microscopy (CM) and electrochemical techniques such as Tafel polarization, cyclic voltammetry (CV), differential pulse voltammetry (DPV), linear sweep voltammetry (LSV) and electrochemical impedance spectroscopy (EIS). Inserting IAPP into the fluid domains results in breaking the lipid-to-lipid interaction, leading to restriction of membrane mobility. The SLateral values of the liposome and IAPP co-solubilized liposome indicates the cooperative insertion of IAPP. Further, a new method of immobilizing a membrane to the gold surface has been employed, resulting in an electrical contact with the buffer, preventing the direct utilization of a steady-state voltage across the bilayer. The electrochemical studies revealed that the charge transfer resistance decreased for 3-mercaptopropanoic acid modified gold (MPA-Au) electrode coated with the liposome and after the addition of IAPP, followed by an increase in the capacitance. The present study has opened up new dimensions to the understanding of peptide-membrane interactions and shows different experimental approaches for the future researchers in this domain.

Microscopic Interactions of Melatonin, Serotonin and Tryptophan with Zwitterionic Phospholipid Membranes.

The interactions at the atomic level between small molecules and the main components of cellular plasma membranes are crucial for elucidating the mechanisms allowing for the entrance of such small species inside the cell. We have performed molecular dynamics and metadynamics simulations of tryptophan, serotonin, and melatonin at the interface of zwitterionic phospholipid bilayers. In this work, we will review recent computer simulation developments and report microscopic properties, such as the area per lipid and thickness of the membranes, atomic radial distribution functions, angular orientations, and free energy landscapes of small molecule binding to the membrane. Cholesterol affects the behaviour of the small molecules, which are mainly buried in the interfacial regions. We have observed a competition between the binding of small molecules to phospholipids and cholesterol through lipidic hydrogen-bonds. Free energy barriers that are associated to translational and orientational changes of melatonin have been found to be between 10-20 kJ/mol for distances of 1 nm between melatonin and the center of the membrane. Corresponding barriers for tryptophan and serotonin that are obtained from reversible work methods are of the order of 10 kJ/mol and reveal strong hydrogen bonding between such species and specific phospholipid sites. The diffusion of tryptophan and melatonin is of the order of 10-7 cm2/s for the cholesterol-free and cholesterol-rich setups.

Membrane insertion mechanism and molecular assembly of the bacteriophage lysis toxin ΦX174-E.

The bacteriophage ΦX174 causes large pore formation in Escherichia coli and related bacteria. Lysis is mediated by the small membrane-bound toxin ΦX174-E, which is composed of a transmembrane domain and a soluble domain. The toxin requires activation by the bacterial chaperone SlyD and inhibits the cell wall precursor forming enzyme MraY. Bacterial cell wall biosynthesis is an important target for antibiotics; therefore, knowledge of molecular details in the ΦX174-E lysis pathway could help to identify new mechanisms and sites of action. In this study, cell-free expression and nanoparticle technology were combined to avoid toxic effects upon ΦX174-E synthesis, resulting in the efficient production of a functional full-length toxin and engineered derivatives. Pre-assembled nanodiscs were used to study ΦX174-E function in defined lipid environments and to analyze its membrane insertion mechanisms. The conformation of the soluble domain of ΦX174-E was identified as a central trigger for membrane insertion, as well as for the oligomeric assembly of the toxin. Stable complex formation of the soluble domain with SlyD is essential to keep nascent ΦX174-E in a conformation competent for membrane insertion. Once inserted into the membrane, ΦX174-E assembles into high-order complexes via its transmembrane domain and oligomerization depends on the presence of an essential proline residue at position 21. The data presented here support a model where an initial contact of the nascent ΦX174-E transmembrane domain with the peptidyl-prolyl isomerase domain of SlyD is essential to allow a subsequent stable interaction of SlyD with the ΦX174-E soluble domain for the generation of a membrane insertion competent toxin.

An in vitro study on the interaction of the anti-Alzheimer drug rivastigmine with human erythrocytes.

The inhibition of the enzyme acetylcholinesterase (AChE) is a frequently used therapeutic option to treat Alzheimer's disease (AD). By decreasing the levels of acetylcholine degradation in the synaptic space, some cognitive functions of patients suffering from this disease are significantly improved. Rivastigmine is one of the most widely used AChE inhibitors. The objective of this work was to determine the effects of this drug on human erythrocytes, which have a type of AChE in the cell membrane. To that end, human erythrocytes and molecular models of its membrane constituted by dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were used. They correspond to classes of phospholipids present in the outer and inner monolayers of the human erythrocyte membrane, respectively. The experimental results obtained by X-ray diffraction and differential scanning calorimetry (DSC) indicated that rivastigmine molecules were able to interact with both phospholipids. Fluorescence spectroscopy results showed that rivastigmine produce a slight change in the acyl chain packing order and a weak displacement of the water molecules of the hydrophobic-hydrophilic membrane interface. On the other hand, observations by scanning electron microscopy (SEM) showed that the drug changed the normal biconcave shape of erythrocytes in stomatocytes (cup-shaped cells) and echinocytes (speculated shaped).

The influence of the stereochemistry and C-end chemical modification of dermorphin derivatives on the peptide-phospholipid interactions.

In this work the conformation of dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, an opioid peptide and its analogues with different stereochemistry of alanine and different C-terminus is studied in aqueous and membrane environments. Using two-dimensional NMR techniques we demonstrate that in D2O/H2O peptides with D-alanine have extended conformation, while for the L-isomers more compact conformation is preferred. The analysis of ROESY HR MAS spectra of the peptides interacting with the DMPC bilayer indicates that both stereoisomers have still more extended conformation compared to aqueous phase, as shown by much weaker intermolecular interactions. The influence of Ala residue stereochemistry is also reflected in the interactions of the studied peptides with model membranes, as shown by the 31P NMR static spectra, in which the shapes of the phosphorus NMR signals originating from D-isomers correspond to spherically shaped vesicles in the presence of external magnetic field, in comparison to a more elongated ones observed for L-isomers, while TEM photographs shows that upon addition of D-isomers larger lipid vesicles are formed, in contrast to smaller ones for L-isomers. The location of aromatic fragments of dermorphins in the membrane is determined based on static 2H NMR and 1H1H RFDR MAS experiments. All aromatic rings were found to be inserted in the hydrophobic part of the bilayer, with the exception of the Tyr5 rings of D-Ala dermorphins. The influence of the C-terminal modification was found to be almost imperceptible.

Binding and dynamics of melatonin at the interface of phosphatidylcholine-cholesterol membranes.

The characterization of interactions between melatonin, one main ingredient of medicines regulating sleeping rhythms, and basic components of cellular plasma membranes (phospholipids, cholesterol, metal ions and water) is very important to elucidate the main mechanisms for the introduction of melatonin into cells and also to identify its local structure and microscopic dynamics. Molecular dynamics simulations of melatonin inside mixtures of dimyristoylphosphatidylcholine and cholesterol in NaCl solution at physiological concentration have been performed at 303.15 K to systematically explore melatonin-cholesterol, melatonin-lipid and melatonin-water interactions. Properties such as the area per lipid and thickness of the membrane as well as selected radial distribution functions, binding free energies, angular distributions, atomic spectral densities and translational diffusion of melatonin are reported. The presence of cholesterol significantly affects the behavior of melatonin, which is mainly buried into the interfaces of membranes. Introducing cholesterol into the system helps melatonin change from folded to extended configurations more easily. Our results suggest that there exists a competition between the binding of melatonin to phospholipids and to cholesterol by means of hydrogen-bonds. Spectral densities of melatonin reported in this work, in overall good agreement with experimental data, revealed the participation of each atom of melatonin to its complete spectrum. Melatonin self-diffusion coefficients are of the order of 10-7 cm2/s and they significantly increase when cholesterol is addeed to the membrane.

Methionine Oxidation Changes the Mechanism of Aß Peptide Binding to the DMPC Bilayer.

Using all-atom explicit solvent replica exchange molecular dynamics simulations with solute tempering, we study the effect of methionine oxidation on Aß10-40 peptide binding to the zwitterionic DMPC bilayer. By comparing oxidized and reduced peptides, we identified changes in the binding mechanism caused by this modification. First, Met35 oxidation unravels C-terminal helix in the bound peptides. Second, oxidation destabilizes intrapeptide interactions and expands bound peptides. We explain these outcomes by the loss of amphiphilic character of the C-terminal helix due to oxidation. Third, oxidation "polarizes" Aß binding to the DMPC bilayer by strengthening the interactions of the C-terminus with lipids while largely releasing the rest of the peptide from bilayer. Fourth, in contrast to the wild-type peptide, oxidized Aß induces significantly smaller bilayer thinning and drop in lipid density within the binding footprint. These observations are the consequence of mixing oxidized peptide amino acids with lipids promoted by enhanced Aß conformational fluctuations. Fifth, methionine oxidation reduces the affinity of Aß binding to the DMPC bilayer by disrupting favorable intrapeptide interactions upon binding, which offset the gains from better hydration. Reduced binding affinity of the oxidized Aß may represent the molecular basis for its reduced cytotoxicity.

Dynamics of heroin molecule inside the lipid membrane: a molecular dynamics study.

Heroin, or diamorphine (C21H23NO5), is an opium product used for various pharmaceutical and euphoric purposes. In this work, the molecular dynamics simulation study of the heroin inside the two lipid bilayers, dipalmitoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) are presented. The whole study was conducted at three different temperatures. The location of the heroin drug, the nature of the diffusion, rotational correlation function and structural variation inside both lipid bilayers is studied. Moreover, the free energy of the solvation of the drug inside both lipid bilayers is calculated. It is found that during the whole molecular dynamics study, the drug locates at the center of both lipid membranes. The effect of the temperature is not seen at the drug location. The nature of the diffusion of the heroin drug is anomalous. The radius of gyration is calculated to study the structural variations of the heroin molecule inside both lipid bilayers. It is found that the heroin molecule does not change its structure at three temperatures. From the rotational correlation function, it is seen that the drug is more hindered for rotation inside the DPPC lipid bilayer as compared to the DMPC lipid bilayer. It is applicable for all three temperatures. The rotational correlation time of the drug is decreased while the temperature of the system is increased. In the case of DMPC, there is an abrupt change in rotational correlation time while the phase is changed.

The acetylcholinesterase (AChE) inhibitor and anti-Alzheimer drug donepezil interacts with human erythrocytes.

Donepezil is used to treat symptomatically the Alzheimer's disease (AD). This drug is a specific inhibitor of the enzyme acetylcholinesterase (AChE), whose main physiological function is to hydrolyze the neurotransmitter acetylcholine. The main objective of this work was to study the effect of donepezil on human erythrocytes as AChE is present in its membrane. For this purpose, human erythrocytes and molecular model of its membrane built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were used. The latter correspond to classes of phospholipids present in the outer and inner monolayers of the human erythrocyte membrane, respectively. Our experimental evidences obtained from X-ray diffraction and differential scanning calorimetry (DSC) analysis indicated that donepezil was capable of interacting with both phospholipids. Fluorescence spectroscopy results showed a moderate increase in the fluidity of the hydrophobic tails of DMPC and isolated unsealed human erythrocyte membranes (IUM). On the other hand, results by scanning electron microscopy (SEM) and optical defocusing microscopy (DM) showed that the drug changed the normal biconcave shape of the erythrocytes inducing the formation of stomatocytes (cup-shaped cells). This effect was explained by the incorporation of donepezil molecules into the erythrocyte membrane and interactions with AChE.

Methanol Accelerates DMPC Flip-Flop and Transfer: A SANS Study on Lipid Dynamics.

Methanol is a common solubilizing agent used to study transmembrane proteins/peptides in biological and synthetic membranes. Using small angle neutron scattering and a strategic contrast-matching scheme, we show that methanol has a major impact on lipid dynamics. Under increasing methanol concentrations, isotopically distinct 1,2-dimyristoyl-sn-glycero-3-phosphocholine large unilamellar vesicle populations exhibit increased mixing. Specifically, 1,2-dimyristoyl-sn-glycero-3-phosphocholine transfer and flip-flop kinetics display linear and exponential rate enhancements, respectively. Ultimately, methanol is capable of influencing the structure-function relationship associated with bilayer composition (e.g., lipid asymmetry). The use of methanol as a carrier solvent, despite better simulating some biological conditions (e.g., antimicrobial attack), can help misconstrue lipid scrambling as the action of proteins or peptides, when in actuality it is a combination of solvent and biological agent. As bilayer compositional stability is crucial to cell survival and protein reconstitution, these results highlight the importance of methanol, and solvents in general, in biomembrane and proteolipid studies.

Biophysical interaction of temozolomide and its active metabolite with biomembrane models: The relevance of drug-membrane interaction for Glioblastoma Multiforme therapy.

Temozolomide (TMZ) is the first-line treatment for Glioblastoma Multiforme (GBM). After administration, TMZ is rapidly converted into its active metabolite (MTIC). However, its pharmacological activity is reduced due MTIC low bioavailability in the brain. Since drugs' permeability through biological barriers and tumor cell membranes affects its bioavailability, the ability of MTIC to interact with the biological membranes presents a major contribution on its pharmacological properties and activity. Biomembrane models mimic the physiological conditions, allowing to predict the drug's behavior at biological membranes and its effects on drug biodistribution profiles. In this work, lipid bilayer models using liposomes were applied for the drug-membrane interaction studies. The zwitterionic phospholipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and cholesterol were chosen for the composition of the model, since they represent the major components of the membranes of GBM cells and brain capillary endothelial cell. Thus, the molecular interactions between MTIC and these models were studied by the evaluation of the partition of the drug into the phospholipid's membrane, its location within the bilayer and its effect on the fluidity of the membrane. The attained results suggest that the composition of membranes affects drugs partition, showing that drug biodistribution depends not only on its physicochemical features, but also depends on the characteristics of the membrane such as the packing of the lipid molecules. Also, MTIC exhibited low affinity to biological membranes, explaining its low bioavailability on the target cells.

An in vitro study of the protective effect of caffeic acid on human erythrocytes.

The interaction and protective effect of caffeic acid (CA) on human erythrocytes (RBC) and molecular models of its membrane were studied. The latter consisted of bilayers built up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. X-ray diffraction and differential scanning calorimetry results indicated that CA induced structural and thermotropic perturbations in multilayers and vesicles of DMPC. Fluorescence spectroscopy analysis showed that CA increased the fluidity of DMPC vesicles and of human erythrocyte ghosts. Scanning electron microscopy observations displayed that CA induced morphological alterations to RBC from their normal discoid form to echinocytes. The assessment of its protective capacity showed that CA inhibits RBC morphological alterations and lysis induced by HClO. These findings imply that CA molecules were located in the outer monolayer of the erythrocyte membrane, and that this preferential location might effectively protect the red cells from damage caused by oxidizing species.

Stability of the two-dimensional lattice of bacteriorhodopsin reconstituted in partially fluorinated phosphatidylcholine bilayers.

This study aims to investigate bacteriorhodopsin (bR) molecules reconstituted in lipid bilayers composed of di(nonafluorotetradecanoyl)-phosphatidylcholine (F4-DMPC), a partially fluorinated analogue of dimyristoyl-phosphatidylcholine (DMPC) to clarify the effects of partially fluorinated hydrophobic chains of lipids on protein's stability. Calorimetry measurements showed that the chain-melting transition of F4-DMPC/bR systems occurs at 3.5 °C, whereas visible circular dichroism (CD) and X-ray diffraction measurements showed that a two-dimensional (2D) hexagonal lattice formed by bR trimers in F4-DMPC bilayers remains intact even above 30 °C, similar to bR in a native purple membrane. Complete dissociation of the trimers into the monomers detected by visible CD almost coincides with the complete melting of 2D lattice observed by X-ray diffraction, in which both take place at around 65 °C (10 °C lower than that for bR in a native purple membrane). However, it is extremely high in comparison with the bR reconstituted in DMPC bilayers in which the dissociation of bR trimer in DMPC bilayers occurs near the chain-melting transition temperature of DMPC bilayers at approximately 18 °C. In order to explore the rationale behind the difference in stability, a further investigation of the detailed structural features of pure F4-DMPC bilayers was performed by analyzing the lamellar diffraction data using simple electron density models. The results suggested that the perfluoroalkyl groups do not exhibit any conformation change even if the chain-melting transition occurs, which is likely to contribute to the stability of the 2D hexagonal lattice formed by the bR trimers.

Membrane localization of the Repeats-in-Toxin (RTX) Leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans.

The oral bacterium, Aggregatibacter actinomycetemcomitans, which is associated with localized aggressive periodontitis, as well as systemic infections including endocarditis, produces numerous virulence factors, including a repeats-in-toxin (RTX) protein called leukotoxin (LtxA), which kills human immune cells. The strains of A. actinomycetemcomitans most closely associated with disease have been shown to produce the most LtxA, suggesting that LtxA plays a significant role in the virulence of this organism. LtxA, like many of the RTX toxins, can be divided into four functional domains: an N-terminal hydrophobic domain, which contains a significant fraction of hydrophobic residues and has been proposed to play a role in the membrane interaction of the toxin; the central domain, which contains two lysine residues that are the sites of post-translational acylation; the repeat domain that is characteristic of the RTX toxins, and a C-terminal domain thought to be involved in secretion. In its initial interaction with the host cell, LtxA must bind to both cholesterol and an integrin receptor, lymphocyte function-associated antigen-1 (LFA-1). While both interactions are essential for toxicity, the domains of LtxA involved remain unknown. We therefore undertook a series of experiments, including tryptophan quenching and trypsin digestion, to characterize the structure of LtxA upon interaction with membranes of various lipid compositions. Our results demonstrate that LtxA adopts a U-shaped conformation in the membrane, with the N- and C-terminal domains residing outside of the membrane.

Topotecan effect on the structure of normal and cancer plasma membrane lipid models: A multi-model approach.

Topotecan is a relatively large, planar, asymmetric and polar molecule with a lactone moiety. In neutral or basic aqueous solutions, this ring opens forming the carboxylate form of Topotecan that is biologically inactive and uncapable of passively cross membranes. Nevertheless, despite this inability to cross membranes at this form, Topotecan may still be able to interact with phospholipid bilayers, disturbing them. In this context, phospholipid models, mimicking normal (DMPC at pH 7.4) and cancer cell lipid membranes (DMPC:DMPS (5:1) at pH 6.5), were used to assess structural modifications upon interaction with Topotecan. Langmuir isotherms of monolayers coupled with Brewster angle microscopy, differential scanning calorimetry of liposomes and X-ray scattering of small and wide angle of stacked multilayers were used as complementary techniques. The overall results show that the interaction of Topotecan with lipid membranes is deeply conditioned by their composition and that Topotecan seems to have a preferential interaction with the glycerol backbone of phosphatidylcholine phospholipids.