DODAB vesicles containing lysophosphatidylcholines: The relevance of acyl chain saturation on the membrane structure and thermal properties.

The saturated LPC18:0 and unsaturated LPC18:1 lysophosphatidylcholines have important roles in inflammation and immunity and are interesting targets for immunotherapy. The synthetic cationic lipid DODAB has been successfully employed in delivery systems, and would be a suitable carrier for those lysophosphatidylcholines. Here, assemblies of DODAB and LPC18:0 or LPC18:1 were characterized by Differential Scanning Calorimetry (DSC) and Electron Paramagnetic Resonance (EPR) spectroscopy. LPC18:0 increased the DODAB gel-fluid transition enthalpy and rigidified both phases. In contrast, LPC18:1 caused a decrease in the DODAB gel-fluid transition temperature and cooperativity, associated with two populations with distinct rigidities in the gel phase. In the fluid phase, LPC18:1 increased the surface order but, differently from LPC18:0, did not affect viscosity at the membrane core. The impact of the different acyl chains of LPC18:0 and 18:1 on structure and thermotropic behavior should be considered when developing applications using mixed DODAB membranes.

Thermotropic Behavior and Structural Organization of C24:1 Sulfatide Dispersions and Its Mixtures with Cationic Bilayers.

C24:1 sulfatide (SF) is an endogenous activator of type II NKT cells. The thermotropic behavior and structure of SF dispersions and its mixtures (4.8-16.6 mol %) with cationic dioctadecyldimethylammonium bromide (DODAB) bilayers were investigated by differential scanning calorimetry and electron paramagnetic resonance spectroscopy. The non-interdigitated lamellar structures formed by pure SF display broad thermal events around 27.5 °C when heated and cooled. These events disappear upon mixing with DODAB, showing complete lipid miscibility. SF decreases the DODAB gel-phase packing, with a consequent decrease in phase-transition temperatures and cooperativity upon heating. In contrast, SF increases the rigidity of the DODAB fluid phase, resulting in a smaller decrease in transition temperatures upon cooling. The hysteresis between heating and cooling decreased as the SF molar fraction increased. These effects on DODAB are similar to the ones described for other glycolipids, such as αGalCer and ßGlcCer. This might be due to the orientation of the rigid and planar amide bond that connects their sphingoid bases and acyl chains, which result in a V-shaped conformation of the glycolipid molecules. The current results may be important to plan and develop new immunotherapeutic tools based on SF.

Correlating biological activity to thermo-structural analysis of the interaction of CTX with synthetic models of macrophage membranes.

The important pharmacological actions of Crotoxin (CTX) on macrophages, the main toxin in the venom of Crotalus durissus terrificus, and its important participation in the control of different pathophysiological processes, have been demonstrated. The biological activities performed by macrophages are related to signaling mediated by receptors expressed on the membrane surface of these cells or opening and closing of ion channels, generation of membrane curvature and pore formation. In the present work, the interaction of the CTX complex with the cell membrane of macrophages is studied, both using biological cells and synthetic lipid membranes to monitor structural alterations induced by the protein. Here we show that CTX can penetrate THP-1 cells and induce pores only in anionic lipid model membranes, suggesting that a possible access pathway for CTX to the cell is via lipids with anionic polar heads. Considering that the selectivity of the lipid composition varies in different tissues and organs of the human body, the thermostructural studies presented here are extremely important to open new investigations on the biological activities of CTX in different biological systems.

The citrus plant pathogen Xanthomonas citri has a dual polyamine-binding protein.

ATP-Binding Cassette transporters (ABC transporters) are protein complexes involved in the import and export of different molecules, including ions, sugars, peptides, drugs, and others. Due to the diversity of substrates, they have large relevance in physiological processes such as virulence, pathogenesis, and antimicrobial resistance. In Xanthomonas citri subsp. citri, the phytopathogen responsible for the citrus canker disease, 20% of ABC transporters components are expressed under infection conditions, including the putative putrescine/polyamine ABC transporter, PotFGHI. Polyamines are ubiquitous molecules that mediate cell growth and proliferation and play important role in bacterial infections. In this work, we characterized the X. citri periplasmic-binding protein PotF (XAC2476) using bioinformatics, biophysical and structural methods. PotF is highly conserved in Xanthomonas sp. genus, and we showed it is part of a set of proteins related to the import and assimilation of polyamines in X. citri. The interaction of PotF with putrescine and spermidine was direct and indirectly shown through fluorescence spectroscopy analyses, and experiments of circular dichroism (CD) and small-angle X-ray scattering (SAXS), respectively. The protein showed higher affinity for spermidine than putrescine, but both ligands induced structural changes that coincided with the closing of the domains and increasing of thermal stability.

Structure of the Mycobacterium tuberculosis cPknF and conformational changes induced in forkhead-associated regulatory domains.

Mycobacterium tuberculosis (Mtb) has 11 Serine-Threonine Protein Kinases (STPK) that control numerous physiological processes, including cell growth, cell division, metabolic flow, and transcription. PknF is one of the 11 Mtb STPKs that has, among other substrates, two FHA domains (FHA-1 and FHA-2) of the ATP-Binding Cassette (ABC) transporter Rv1747. Phosphorylation in T152 and T210 located in a non-structured linker that connects Rv1747 FHA domains is considerate to be the regulatory mechanism of the transporter. In this work, we resolved the three-dimensional structure of the PknF catalytic domain (cPknF) in complex with the human kinase inhibitor IKK16. cPknF is conserved when compared to other STPKs but shows specific residues in the binding site where the inhibitor is positioned. In addition, using Small Angle X-Ray Scattering analysis we monitored the behavior of the wild type and three FHA-phosphomimetic mutants in solution, and measured the cPknF affinity for these domains. The kinase showed higher affinity for the non-phosphorylated wild type domain and preference for phosphorylation of T152 inducing the rapprochement of the domains and significant structural changes. The results shed some light on the process of regulating the transporter's activity by phosphorylation and arises important questions about evolution and importance of this mechanism for the bacillus.

Correlating biological activity to thermo-structural analysis of the interaction of CTX with synthetic models of macrophage membranes

The important pharmacological actions of Crotoxin (CTX) on macrophages, the main toxin in the venom of Crotalus durissus terrificus, and its important participation in the control of different pathophysiological processes, have been demonstrated. The biological activities performed by macrophages are related to signaling mediated by receptors expressed on the membrane surface of these cells or opening and closing of ion channels, generation of membrane curvature and pore formation. In the present work, the interaction of the CTX complex with the cell membrane of macrophages is studied, both using biological cells and synthetic lipid membranes to monitor structural alterations induced by the protein. Here we show that CTX can penetrate THP-1 cells and induce pores only in anionic lipid model membranes, suggesting that a possible access pathway for CTX to the cell is via lipids with anionic polar heads. Considering that the selectivity of the lipid composition varies in different tissues and organs of the human body, the thermostructural studies presented here are extremely important to open new investigations on the biological activities of CTX in different biological systems.

A new interpretation of the absorption and the dual fluorescence of Prodan in solution.

Remarkable interest is associated with the interpretation of the Prodan fluorescent spectrum. A sequential hybrid Quantum Mechanics/Molecular Mechanics method was used to establish that the fluorescent emission occurs from two different excited states, resulting in a broad asymmetric emission spectrum. The absorption spectra in several solvents were measured and calculated using different theoretical models presenting excellent agreement. All theoretical models [semiempirical, time dependent density functional theory and and second-order multiconfigurational perturbation theory] agree that the first observed band at the absorption spectrum in solution is composed of three electronic excitations very close in energy. Then, the electronic excitation around 340 nm-360 nm may populate the first three excited states (π-π*Lb, n-π*, and π-π*La). The ground state S0 and the first three excited states were analyzed using multi-configurational calculations. The corresponding equilibrium geometries are all planar in vacuum. Considering the solvent effects in the electronic structure of the solute and in the solvent relaxation around the solute, it was identified that these three excited states can change the relative order depending on the solvent polarity, and following the minimum path energy, internal conversions may occur. A consistent explanation of the experimental data is obtained with the conclusive interpretation that the two bands observed in the fluorescent spectrum of Prodan, in several solvents, are due to the emission from two independent states. Our results indicate that these are the n-π* S2 state with a small dipole moment at a lower emission energy and the π-π*Lb S1 state with large dipole moment at a higher emission energy.

Interaction of synthetic antimicrobial peptides of the Hylin a1 family with models of eukaryotic structures: Zwitterionic membranes and DNA.

Antimicrobial peptides (AMPs) have been appointed as a possible alternative to traditional antibiotics in face of pathogens increasing resistance to conventional drugs. Hylin a1 (IFGAILPLALGALKNLIK), an AMP extracted from the skin secretion of a South American frog, Hypsiboas albopunctatus, was found to show a strong cytotoxicity against bacteria and fungus, but also a considerable hemolytic action. Considering the toxicity of the peptide in eukaryotic cells, this work focuses on investigating the effects of the interaction of the Hylin a1 analogues W6Hya1, D0W6Hya1 and K0W6Hya1 with models of eukaryotic structures, namely zwitterionic liposomes of dipalmitoyl phosphatidylcholine (DPPC) and calf-thymus DNA (CT DNA). Through intrinsic Trp fluorescence we determined that the peptide affinity for fluid DPPC bilayers follows the decreasing order: D0W6Hya1 (+2) > W6Hya1 (+3) ¼ K0W6Hya1 (+4). Fluorescence data also indicate that the Trp residue in the more positively charged peptide, K0W6Hya1, is less deep in the bilayer than the residue in the other two peptides. This finding is supported by differential scanning calorimetry (DSC) data, which shows that both D0W6Hya1 and W6Hya1 disturb DPPC gel-fluid transition slightly more effectively than K0W6Hya1. DPPC DSC profiles are homogeneously disturbed by the three peptides, probably related to peptide-membrane diffusion. Surprisingly, the peptide that displays the lowest affinity for PC membranes and is located at the more superficial position in the bilayer, K0W6Hya1, is the most efficient in causing formation of pores on the membrane, as attested by carboxyfluorescein leakage assays. The three peptides were found to interact with CT DNA, with a deep penetration of the Trp residue into hydrophobic pockets of the double helix, as indicated by the significant blue shift on the Trp fluorescence, and the displacement of DNA-bound ethidium bromide by the peptides. The experiments of DNA electrophoresis confirm that Hylin peptides bind DNA in a concentration-dependent manner, inducing complete DNA retardation at the relative AMP/plasmid DNA weight ratio of ~17. These findings could help to better understand the AMPs toxic effects on eukaryotic cells, thus contributing to the design of healthier therapeutic agents.

Supramolecular organization of α-galactosylceramide in pure dispersions and in cationic DODAB bilayers.

α-galactosylceramide (α-GalCer; KRN7000) strongly stimulates NKT cells. The structures of α-GalCer assemblies and of cationic DODAB bilayers containing α-GalCer were investigated by differential scanning calorimetry (DSC) and electron spin resonance (ESR) spectroscopy. Assemblies of α-GalCer have a very tightly packed gel phase, causing spin labels to cluster and display spin exchange interactions. An endothermic phase transition is observed by DSC, leading to a fluid phase. This phase transition peak disappears upon mixing with DODAB, showing that up to 9 mol% α-GalCer is miscible with the cationic lipid. ESR spectra show that α-GalCer decreases DODAB gel phase packing, resulting in a decrease of gel-fluid transition temperature and cooperativity in DSC thermograms of mixed bilayers. In contrast, α-GalCer increases the rigidity of the fluid phase. These effects are probably due to the conformation of the rigid amide bond that connects the phytosphingosine base of α-GalCer to its long and saturated acyl chain. Possibly, α-GalCer adopts a V-shaped conformation because of the perpendicular orientation of the amide bond towards the axes of the hydrocarbon chains. Apparently, the effect of the amide bond configuration is a key structural feature for the interaction between ceramide-based glycolipids and DODAB molecules, since we have previously reported a similar decrease of gel phase packing and increase in fluid phase rigidity for DODAB bilayers containing C24:1ß-glucosylceramide. Since the structure of delivery systems is critical to the biological activity of α-GalCer, this work certainly contributes to the planning and development of novel immunotherapeutic tools.

Structural characterization of cationic DODAB bilayers containing C24:1 ß-glucosylceramide.

The effect of 5 mol%, 9 mol%, and 16 mol% of C24:1 ß-glucosylceramide (ßGlcCer) on the structure of cationic DODAB bilayers was investigated by means of differential scanning calorimetry (DSC), electron spin resonance (ESR) spectroscopy and fluorescence microscopy. ßGlcCer is completely miscible with DODAB at all fractions tested, since no domains were observed in fluorescence microscopy or ESR spectra. The latter showed that ßGlcCer destabilized the gel phase of DODAB bilayers by decreasing the gel phase packing. As a consequence, ßGlcCer induced a decrease in the phase transition temperature and cooperativity of DODAB bilayers, as seen in DSC thermograms. ESR spectra also showed that ßGlcCer induced an increase in DODAB fluid phase order and/or rigidity. Despite their different structures, a similar effect of loosening the gel phase packing and turning the fluid phase more rigid/organized has also been observed when low molar fractions of cholesterol were incorporated in DODAB bilayers. The structural characterization of mixed membranes made of cationic lipids and glucosylceramides may be important for developing novel immunotherapeutic tools such as vaccine adjuvants.

Antimicrobial Peptide K0-W6-Hya1 Induces Stable Structurally Modified Lipid Domains in Anionic Membranes.

Considering the known different mode of action of antimicrobial peptides in zwitterionic and anionic cell membranes, the present work compares the action of the antimicrobial peptide K0-W6-Hya1 (KIFGAIWPLALGALKNLIK-NH2) with zwitterionic and negatively charged model membranes, namely, liposomes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, and a mixture of the two. Differential scanning calorimetry (DSC), steady state fluorescence of the Trp residue, dynamic light scattering (DLS), and measurement of the leakage of an entrapped fluorescent dye (carboxyfluorescein, CF) were performed with large unilamellar vesicles (LUVs). All techniques evidenced the different action of the peptide in zwitterionic and anionic vesicles. Trp fluorescence spectroscopy shows that the differences are related not only to the partition of the cationic peptide in zwitterionic and anionic membranes, but also to the different penetration depth of the peptide into the lipid bilayers: Trp goes deeper into negatively charged membranes, both in the gel and fluid phases, than into zwitterionic ones. DSC shows that the peptide is strongly attached to anionic bilayers, giving rise to the coexistence of two different lipid regions, one depleted of peptide and another one peptide-disturbed, possibly a stable or transient polar pore, considering the leakage of CF. This contrasts with the homogeneous effect produced by the peptide in zwitterionic membranes, probably related to peptide-membrane diffusion. Moreover, in mixed bilayers (PC:PG), the peptide sequesters negatively charged lipids, creating peptide-rich anionic lipid regions, strongly disturbing the membrane. The distinct structural interaction displayed by the peptide in PC and PG membranes could be related to the different mechanisms of action of the peptide in anionic prokaryotic and zwitterionic eukaryotic cell membranes.

Experimental and theoretical studies of emodin interacting with a lipid bilayer of DMPC.

Emodin is one of the most abundant anthraquinone derivatives found in nature. It is the active principle of some traditional herbal medicines with known biological activities. In this work, we combined experimental and theoretical studies to reveal information about location, orientation, interaction and perturbing effects of Emodin on lipid bilayers, where we have taken into account the neutral form of the Emodin (EMH) and its anionic/deprotonated form (EM-). Using both UV/Visible spectrophotometric techniques and molecular dynamics (MD) simulations, we showed that both EMH and EM- are located in a lipid membrane. Additionally, using MD simulations, we revealed that both forms of Emodin are very close to glycerol groups of the lipid molecules, with the EMH inserted more deeply into the bilayer and more disoriented relative to the normal of the membrane when compared with the EM-, which is more exposed to interfacial water. Analysis of several structural properties of acyl chains of the lipids in a hydrated pure DMPC bilayer and in the presence of Emodin revealed that both EMH and EM- affect the lipid bilayer, resulting in a remarkable disorder of the bilayer in the vicinity of the Emodin. However, the disorder caused by EMH is weaker than that caused by EM-. Our results suggest that these disorders caused by Emodin might lead to distinct effects on lipid bilayers including its disruption which are reported in the literature.

Structural insights on biologically relevant cationic membranes by ESR spectroscopy.

Cationic bilayers have been used as models to study membrane fusion, templates for polymerization and deposition of materials, carriers of nucleic acids and hydrophobic drugs, microbicidal agents and vaccine adjuvants. The versatility of these membranes depends on their structure. Electron spin resonance (ESR) spectroscopy is a powerful technique that employs hydrophobic spin labels to probe membrane structure and packing. The focus of this review is the extensive structural characterization of cationic membranes prepared with dioctadecyldimethylammonium bromide or diC14-amidine to illustrate how ESR spectroscopy can provide important structural information on bilayer thermotropic behavior, gel and fluid phases, phase coexistence, presence of bilayer interdigitation, membrane fusion and interactions with other biologically relevant molecules.

Synthesis, High-Resolution Infrared Spectroscopy, and Vibrational Structure of Cubane, C8H8.

Carbon-cage molecules have generated a considerable interest from both experimental and theoretical points of view. We recently performed a high-resolution study of adamantane (C10H16), the smallest hydrocarbon cage belonging to the diamandoid family ( Pirali , O. ; et al. J. Chem. Phys. 2012 , 136 , 024310 ). There exist another family of hydrocarbon cages with additional interesting chemical properties: the so-called platonic hydrocarbons that comprise dodecahedrane (C20H20) and cubane (C8H8). Both possess C-C bond angles that deviate from the tetrahedral angle (109.8°) of the sp(3) hybridized form of carbon. This generates a considerable strain in the molecule. We report a new wide-range high-resolution study of the infrared spectrum of cubane. The sample was synthesized in Bari upon decarboxylation of 1,4-cubanedicarboxylic acid thanks to the improved synthesis of literature. Several spectra have been recorded at the AILES beamline of the SOLEIL synchrotron facility. They cover the 600-3200 cm(-1) region. Besides the three infrared-active fundamentals (ν10, ν11, and ν12), we could record many combination bands, all of them displaying a well-resolved octahedral rotational structure. We present here a preliminary analysis of some of the recorded bands, performed thanks the SPVIEW and XTDS software, based on the tensorial formalism developed in the Dijon group. A comparison with ab initio calculations, allowing to identify some combination bands, is also presented.

Bacterial spores as particulate carriers for gene gun delivery of plasmid DNA.

Bacillus subtilis spores represent a suitable platform for the adsorption of proteins, enzymes and viral particles at physiological conditions. In the present work, we demonstrate that purified spores can also adsorb DNA on their surface after treatment with cationic molecules. In addition, we demonstrate that DNA-coated B. subtilis spores can be used as particulate carriers and act as an alternative to gold microparticles for the biolistic (gene gun) administration of plasmid DNA in mice. Gene gun delivery of spores pre-treated with DODAB (dioctadecyldimethylammonium bromide) allowed efficient plasmid DNA absorption and induced protein expression levels similar to those obtained with gold microparticles. More importantly, we demonstrated that a DNA vaccine adsorbed on spores can be loaded into biolistic cartridges and efficiently delivered into mice, which induced specific cellular and antibody responses. Altogether, these data indicate that B. subtilis spores represent a simple and low cost alternative for the in vivo delivery of DNA vaccines by the gene gun technology.

The effect of an oligonucleotide on the structure of cationic DODAB vesicles.

The effect of a small single-stranded oligonucleotide (ODN) on the structure of cationic DODAB vesicles was investigated by means of differential scanning calorimetry (DSC), small angle X-ray scattering (SAXS) and electron spin resonance (ESR) spectroscopy. ODN adsorption induced coalescence of vesicles and formation of multilamellar structures with close contact between lamellae. It also increased the phase transition temperature by 10 °C but decreased transition cooperativity. The ODN rigidified and stabilized the gel phase. In the fluid phase, a simultaneous decrease of ordering close to the bilayer surface and increase in bilayer core rigidity was observed in the presence of the ODN. These effects may be due not only to electrostatic shielding of DODAB head groups but also to superficial dehydration of the bilayers. The data suggest that oligonucleotides may induce the formation of a multilamellar poorly hydrated coagel-like phase below phase transition. These effects should be taken into account when planning ODN delivery employing cationic bilayer carriers.

Electric dipole moments of the fluorescent probes Prodan and Laurdan: experimental and theoretical evaluations.

Several experimental and theoretical approaches can be used for a comprehensive understanding of solvent effects on the electronic structure of solutes. In this review, we revisit the influence of solvents on the electronic structure of the fluorescent probes Prodan and Laurdan, focusing on their electric dipole moments. These biologically used probes were synthesized to be sensitive to the environment polarity. However, their solvent-dependent electronic structures are still a matter of discussion in the literature. The absorption and emission spectra of Prodan and Laurdan in different solvents indicate that the two probes have very similar electronic structures in both the ground and excited states. Theoretical calculations confirm that their electronic ground states are very much alike. In this review, we discuss the electric dipole moments of the ground and excited states calculated using the widely applied Lippert-Mataga equation, using both spherical and spheroid prolate cavities for the solute. The dimensions of the cavity were found to be crucial for the calculated dipole moments. These values are compared to those obtained by quantum mechanics calculations, considering Prodan in vacuum, in a polarizable continuum solvent, and using a hybrid quantum mechanics-molecular mechanics methodology. Based on the theoretical approaches it is evident that the Prodan dipole moment can change even in the absence of solute-solvent-specific interactions, which is not taken into consideration with the experimental Lippert-Mataga method. Moreover, in water, for electric dipole moment calculations, it is fundamental to consider hydrogen-bonded molecules.

Oligonucleotide adsorption affects phase transition but not interdigitation of diC14-amidine bilayers.

In this work, we investigate the effect of a small single-stranded oligonucleotide (ODN) on the colloid stability and structure of cationic diC14-amidine liposomes. Dynamic light scattering (DLS) shows that small, stable, anionic assemblies are formed in presence of excess ODN negative charge. This charge overcompensation condition was further characterized. A less cooperative bilayer phase transition is observed by differential scanning calorimetry (DSC). Electron spin resonance (ESR) spectra of probes at different bilayer depths show that ODN electrostatic adsorption increases the rigidity of both interdigitated gel and lamellar fluid phases. The increase in gel phase rigidity could be explained by the transformation of an adjacent to an interpenetrated interdigitation. Interdigitated fusogenic bilayers may find interesting applications in delivery of therapeutic oligonucleotides.

Optical characterization of Prodan aggregates in water medium.

The fluorescent probe Prodan (2-dimethylamino-6-propionylnaphthalene) has been widely used in biological systems, mainly due to the high sensitivity of its emission spectrum to the medium polarity. Though mostly used as a membrane probe, in lipid dispersions Prodan partitions in water, mainly in the presence of gel-phase bilayers. Here, optical properties of Prodan in aqueous medium are experimentally studied using absorption and emission spectroscopies, and compared with those of the probe in cyclohexane, where it is supposed to be very soluble. In parallel, theoretical calculations of the absorption spectrum of a monomer and aggregated Prodan in water were performed. Moreover, to understand Prodan-water and Prodan-Prodan interactions, solvation free energies of Prodan in water and in liquid Prodan were calculated. A light scattering profile underneath the optical absorption spectrum of Prodan in water clearly indicates the presence of aggregates at very low Prodan concentrations (0.9 µM). Experimental evidence of Prodan aggregation is theoretically supported by solvation free energy calculations, which demonstrate that Prodan molecules interact preferentially with other Prodan molecules than with water molecules. Theoretical calculations for electronic transition energies of monomers and aggregated Prodan in water show that a Prodan optical absorption band at 358 nm is related to the monomeric form of Prodan. This band saturates as Prodan concentration increases, indicating that aggregated Prodan prevails at higher concentrations. The relative increase in Prodan aggregated population is monitored by the increase in an absorption band at higher energies, at around 250 nm, and by the disappearance of a band at around 280 nm. Surprisingly, it was observed that the fluorescent emission spectrum of Prodan is not sensitive to probe aggregation up to around 15 µM. Hence, Prodan aggregation in water medium, even at very low concentrations, must be considered when using this fluorescent probe in biological systems, having in mind that its fluorescence spectrum is rather insensitive to aggregation.