Publications by authors named "Giovanna Fragneto"

77 Publications

Lipid bilayer degradation induced by SARS-CoV-2 spike protein as revealed by neutron reflectometry.

Sci Rep 2021 07 21;11(1):14867. Epub 2021 Jul 21.

Institut Laue-Langevin, 71 Avenue des Martyrs, BP 156, 38042, Grenoble, France.

SARS-CoV-2 spike proteins are responsible for the membrane fusion event, which allows the virus to enter the host cell and cause infection. This process starts with the binding of the spike extramembrane domain to the angiotensin-converting enzyme 2 (ACE2), a membrane receptor highly abundant in the lungs. In this study, the extramembrane domain of SARS-CoV-2 Spike (sSpike) was injected on model membranes formed by supported lipid bilayers in presence and absence of the soluble part of receptor ACE2 (sACE2), and the structural features were studied at sub-nanometer level by neutron reflection. In all cases the presence of the protein produced a remarkable degradation of the lipid bilayer. Indeed, both for membranes from synthetic and natural lipids, a significant reduction of the surface coverage was observed. Quartz crystal microbalance measurements showed that lipid extraction starts immediately after sSpike protein injection. All measurements indicate that the presence of proteins induces the removal of membrane lipids, both in the presence and in the absence of ACE2, suggesting that sSpike molecules strongly associate with lipids, and strip them away from the bilayer, via a non-specific interaction. A cooperative effect of sACE2 and sSpike on lipid extraction was also observed.
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http://dx.doi.org/10.1038/s41598-021-93996-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8295359PMC
July 2021

Nanostructural Characterization of Cardiolipin-Containing Tethered Lipid Bilayers Adsorbed on Gold and Silicon Substrates for Protein Incorporation.

Langmuir 2021 Jul 21. Epub 2021 Jul 21.

Univ. Grenoble Alpes, CNRS, UMR 5525, VetAgro Sup, Grenoble INP, TIMC/SyNaBi, 38000 Grenoble, France.

A key to the development of lipid membrane-based devices is a fundamental understanding of how the molecular structure of the lipid bilayer membrane is influenced by the type of lipids used to build the membrane. This is particularly important when membrane proteins are included in these devices since the precise lipid environment affects the ability to incorporate membrane proteins and their functionality. Here, we used neutron reflectometry to investigate the structure of tethered bilayer lipid membranes and to characterize the incorporation of the NhaA sodium proton exchanger in the bilayer. The lipid membranes were composed of two lipids, dioleoyl phosphatidylcholine and cardiolipin, and were adsorbed on gold and silicon substrates using two different tethering architectures based on functionalized oligoethylene glycol molecules of different lengths. In all of the investigated samples, the addition of cardiolipin caused distinct structural rearrangement including crowding of ethylene glycol groups of the tethering molecules in the inner head region and a thinning of the lipid tail region. The incorporation of NhaA in the tethered bilayers following two different protocols is quantified, and the way protein incorporation modulates the structural properties of these membranes is detailed.
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http://dx.doi.org/10.1021/acs.langmuir.1c00119DOI Listing
July 2021

Structural Characterization of Natural Yeast Phosphatidylcholine and Bacterial Phosphatidylglycerol Lipid Multilayers by Neutron Diffraction.

Front Chem 2021 18;9:628186. Epub 2021 Mar 18.

Institut Laue Langevin, Grenoble, France.

Eukaryotic and prokaryotic cell membranes are difficult to characterize directly with biophysical methods. Membrane model systems, that include fewer molecular species, are therefore often used to reproduce their fundamental chemical and physical properties. In this context, natural lipid mixtures directly extracted from cells are a valuable resource to produce advanced models of biological membranes for biophysical investigations and for the development of drug testing platforms. In this study we focused on single phospholipid classes, i.e. phosphatidylcholine (PC) and phosphatidylglycerol (PG) lipids. These lipids were characterized by a different distribution of their respective acyl chain lengths and number of unsaturations. We produced both hydrogenous and deuterated lipid mixtures. Neutron diffraction experiments at different relative humidities were performed to characterize multilayers from these lipids and investigate the impact of the acyl chain composition on the structural organization. The novelty of this work resides in the use of natural extracts with a single class head-group and a mixture of chain compositions coming from yeast or bacterial cells. The characterization of the PC and PG multilayers showed that, as a consequence of the heterogeneity of their acyl chain composition, different lamellar phases are formed.
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http://dx.doi.org/10.3389/fchem.2021.628186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104085PMC
March 2021

Insertion and activation of functional Bacteriorhodopsin in a floating bilayer.

J Colloid Interface Sci 2021 Sep 31;597:370-382. Epub 2021 Mar 31.

Institut Laue-Langevin, 71 av.des Martyrs, BP 156, 38042 Grenoble Cedex, France.

The proton pump transmembrane protein bacteriorhodopsin was successfully incorporated into planar floating lipid bilayers in gel and fluid phases, by applying a detergent-mediated incorporation method. The method was optimized on single supported bilayers by using quartz crystal microbalance, atomic force and fluorescence microscopy techniques. Neutron and X-ray reflectometry were used on both single and floating bilayers with the aim of determining the structure and composition of this membrane-protein system before and after protein reconstitution at sub-nanometer resolution. Lipid bilayer integrity and protein activity were preserved upon the reconstitution process. Reversible structural modifications of the membrane, induced by the bacteriorhodopsin functional activity triggered by visible light, were observed and characterized at the nanoscale.
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http://dx.doi.org/10.1016/j.jcis.2021.03.155DOI Listing
September 2021

Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation.

J Biol Chem 2021 Jan-Jun;296:100602. Epub 2021 Mar 27.

Laboratoire de Biogènese Membranaire, UMR 5200, CNRS, Université de Bordeaux, Villenave d'Ornon Cedex, France. Electronic address:

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph-mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.
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http://dx.doi.org/10.1016/j.jbc.2021.100602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099651PMC
March 2021

Peptide discs as precursors of biologically relevant supported lipid bilayers.

J Colloid Interface Sci 2021 Mar 26;585:376-385. Epub 2020 Nov 26.

Niels Bohr Institute, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark.

Supported lipid bilayers (SLBs) are commonly used to investigate the structure and dynamics of biological membranes. Vesicle fusion is a widely exploited method to produce SLBs. However, this process becomes less favoured when the vesicles contain complex lipid mixtures, e.g. natural lipid extracts. In these cases, it is often necessary to change experimental parameters, such as temperature, to unphysiological values to trigger the SLB formation. This may induce lipid degradation and is also not compatible with including membrane proteins or other biomolecules into the bilayers. Here, we show that the peptide discs, ~10 nm discoidal lipid bilayers stabilized in solution by a self-assembled 18A peptide belt, can be used as precursors for SLBs. The characterizations by means of neutron reflectometry and attenuated total reflectance-FTIR spectroscopy show that SLBs were successfully formed both from synthetic lipid mixtures (surface coverage 90-95%) and from natural lipid mixtures (surface coverage ~85%). Traces of 18A peptide (below 0.02 M ratio) left at the support surface after the bilayer formation do not affect the SLB structure. Altogether, we demonstrate that peptide disc formation of SLBs is much faster than the SLB formation by vesicle fusion and without the need of altering any experimental variable from physiologically relevant values.
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http://dx.doi.org/10.1016/j.jcis.2020.11.086DOI Listing
March 2021

The Antifungal Mechanism of Amphotericin B Elucidated in Ergosterol and Cholesterol-Containing Membranes Using Neutron Reflectometry.

Nanomaterials (Basel) 2020 Dec 6;10(12). Epub 2020 Dec 6.

European Spallation Source ERIC, P.O. Box 176, 22100 Lund, Sweden.

We have characterized and compared the structures of ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl--glycero-3-phosphocholine (POPC) membranes before and after interaction with the amphiphilic antifungal drug amphotericin B (AmB) using neutron reflection. AmB inserts into both pure POPC and sterol-containing membranes in the lipid chain region and does not significantly perturb the structure of pure POPC membranes. By selective per-deuteration of the lipids/sterols, we show that AmB extracts ergosterol but not cholesterol from the bilayers and inserts to a much higher degree in the cholesterol-containing membranes. Ergosterol extraction by AmB is accompanied by membrane thinning. Our results provide new insights into the mechanism and antifungal effect of AmB in these simple models of fungal and mammalian membranes and help understand the molecular origin of its selectivity and toxic side effects.
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http://dx.doi.org/10.3390/nano10122439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762259PMC
December 2020

Encapsulation of Graphene in the Hydrophobic Core of a Lipid Bilayer.

Langmuir 2020 12 24;36(48):14478-14482. Epub 2020 Nov 24.

Department of Supramolecular & Biomaterials Chemistry, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.

Theoretical simulations have predicted that a lipid bilayer forms a stable superstructure when a sheet of graphene is inserted in its hydrophobic core. We experimentally produced for the first time a lipid-graphene-lipid assembly by combining the Langmuir-Blodgett and the Langmuir-Schaefer methods. Graphene is sandwiched and remains flat within the hydrophobic core of the lipid bilayer. Using infrared spectroscopy, ellipsometry, and neutron reflectometry, we characterized the superstructure at every fabrication step. The hybrid superstructure is mechanically stable and graphene does not disturb the natural lipid bilayer structure.
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http://dx.doi.org/10.1021/acs.langmuir.0c01691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726894PMC
December 2020

Aβ Beyond the AD Pathology: Exploring the Structural Response of Membranes Exposed to Nascent Aβ Peptide.

Int J Mol Sci 2020 Nov 5;21(21). Epub 2020 Nov 5.

Department Medical Biotechnologies and Translational Medicine, Università of Milano, Via F.lli Cervi, 93, 20090 Segrate (MI), Italy.

The physiological and pathological roles of nascent amyloid beta (Aβ) monomers are still debated in the literature. Their involvement in the pathological route of Alzheimer's Disease (AD) is currently considered to be the most relevant, triggered by their aggregation into structured oligomers, a toxic species. Recently, it has been suggested that nascent Aβ, out of the amyloidogenic pathway, plays a physiological and protective role, especially in the brain. In this emerging perspective, the study presented in this paper investigated whether the organization of model membranes is affected by contact with Aβ in the nascent state, as monomers. The outcome is that, notably, the rules of engagement and the resulting structural outcome are dictated by the composition and properties of the membrane, rather than by the Aβ variant. Interestingly, Aβ monomers are observed to favor the tightening of adjacent complex membranes, thereby affecting a basic structural event for cell-cell adhesion and cell motility.
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http://dx.doi.org/10.3390/ijms21218295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663943PMC
November 2020

Not just a fluidifying effect: omega-3 phospholipids induce formation of non-lamellar structures in biomembranes.

Soft Matter 2020 Dec 9;16(46):10425-10438. Epub 2020 Nov 9.

Department of Chemical Sciences, University of Naples Federico II, Naples, Italy.

Polyunsaturated omega-3 fatty acid docosahexaenoic acid (DHA) is found in very high concentrations in a few peculiar tissues, suggesting that it must have a specialized role. DHA was proposed to affect the function of the cell membrane and related proteins through an indirect mechanism of action, based on the DHA-phospholipid effects on the lipid bilayer structure. In this respect, most studies have focused on its influence on lipid-rafts, somehow neglecting the analysis of effects on liquid disordered phases that constitute most of the cell membranes, by reporting in these cases only a general fluidifying effect. In this study, by combining neutron reflectivity, cryo-transmission electron microscopy, small angle neutron scattering, dynamic light scattering and electron paramagnetic resonance spectroscopy, we characterize liquid disordered bilayers formed by the naturally abundant 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and different contents of a di-DHA glycero-phosphocholine, 22:6-22:6PC, from both a molecular/microscopic and supramolecular/mesoscopic viewpoint. We show that, below a threshold concentration of about 40% molar percent, incorporation of 22:6-22:6PC in the membrane increases the lipid dynamics slightly but sufficiently to promote the membrane deformation and increase of multilamellarity. Notably, beyond this threshold, 22:6-22:6PC disfavours the formation of lamellar phases, leading to a phase separation consisting mostly of small spherical particles that coexist with a minority portion of a lipid blob with water-filled cavities. Concurrently, from a molecular viewpoint, the polyunsaturated acyl chains tend to fold and expose the termini to the aqueous medium. We propose that this peculiar tendency is a key feature of the DHA-phospholipids making them able to modulate the local morphology of biomembranes.
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http://dx.doi.org/10.1039/d0sm01549kDOI Listing
December 2020

Aggregated Amphiphilic Antimicrobial Peptides Embedded in Bacterial Membranes.

ACS Appl Mater Interfaces 2020 Oct 23;12(40):44420-44432. Epub 2020 Sep 23.

Biological Physics Laboratory, Department of Physics and Astronomy, Faculty of Science and Engineering, The University of Manchester, Oxford Road, Manchester M13 9PL, U.K.

Molecular dynamics (MD) simulations, stochastic optical reconstruction microscopy (STORM), and neutron reflection (NR) were combined to explore how antimicrobial peptides (AMPs) can be designed to promote the formation of nanoaggregates in bacterial membranes and impose effective bactericidal actions. Changes in the hydrophobicity of the designed AMPs were found to have a strong influence on their bactericidal potency and cytotoxicity. G(IIKK)I-NH (G) achieved low minimum inhibition concentrations (MICs) and effective dynamic kills against both antibiotic-resistant and -susceptible bacteria. However, a G derivative with weaker hydrophobicity, KI(KKII)I-NH (KI), exhibited considerably lower membrane-lytic activity. In contrast, the more hydrophobic G(ILKK)L-NH (GL) peptide achieved MICs similar to those observed for G but with worsened hemolysis. Both the model membranes studied by Brewster angle microscopy, zeta potential measurements, and NR and the real bacterial membranes examined with direct STORM contained membrane-inserted peptide aggregates upon AMP exposure. These structural features were well supported by MD simulations. By revealing how AMPs self-assemble in microbial membranes, this work provides important insights into AMP mechanistic actions and allows further fine-tuning of antimicrobial potency and cytotoxicity.
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http://dx.doi.org/10.1021/acsami.0c09931DOI Listing
October 2020

Anesthetics significantly increase the amount of intramembrane water in lipid membranes.

Soft Matter 2020 Nov;16(42):9674-9682

Department of Physics and Astronomy, McMaster University, ABB-241, 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.

The potency of anesthesia was directly linked to the partitioning of the drug molecules in cell membranes by Meyer and Overton. Many molecules interact with lipid bilayers and lead to structural and functional changes. It remains an open question which change in membrane properties is responsible for a potential anesthetic effect or if anesthetics act by binding to direct targets. We studied the effect of ethanol, diethyl ether and isoflurane on the water distribution in lipid bilayers by combining all-atom molecular dynamics simulations and neutron diffraction experiments. The simulations show strong membrane-drug interactions with partitioning coefficients of 38%, 92% and 100% for ethanol, diethyl ether and isoflurane, respectively, and provide evidence for an increased water partitioning in the membrane core. The amount of intramembrane water molecules was experimentally determined by selectively deuterium labeling lipids, anesthetic drug and water molecules in neutron diffraction experiments. Four additional water molecules per lipid were observed in the presence of ethanol. Diethyl ether and isoflurane were found to significantly increase the amount of intramembrane water by 25% (8 water molecules). This increase in intramembrane water may contribute to the non-specific interactions between anesthetics and lipid membranes.
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http://dx.doi.org/10.1039/d0sm01271hDOI Listing
November 2020

Behavior of Hydrated Lipid Bilayers at Cryogenic Temperatures.

Front Chem 2020 18;8:455. Epub 2020 Jun 18.

Institut Laue-Langevin, Grenoble, France.

Neutron diffraction was used to study the behavior of water present in phospholipid multilamellar stacks from 1,2-dimyristoyl--glycero-3-phosphatidylcholine (DMPC) at cryogenic temperatures. Evidence was found for the existence of a highly viscous phase of water that exists between 180 and 220 K based on the observation that water can leave the intermembrane space at these low temperatures. Similar measurements are described in the literature for purple membrane (PM) samples. From a comparison with results from this natural membrane by using the same flash-cooling protocol, it is found that in the case of pure lipid samples, less water is trapped and the water flows out at lower temperatures. This suggests that the water is less hindered in its movements than in the PM case. It is shown that at least the Lβ'-phase of DMPC can be trapped likely by flash cooling; upon heating to about 260 K, it transforms to another phase that was not fully characterized.
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http://dx.doi.org/10.3389/fchem.2020.00455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314993PMC
June 2020

Interaction with Human Serum Proteins Reveals Biocompatibility of Phosphocholine-Functionalized SPIONs and Formation of Albumin-Decorated Nanoparticles.

Langmuir 2020 08 3;36(30):8777-8791. Epub 2020 Jul 3.

CSGI, Center for Colloid and Surface Science, Sesto Fiorentino (FI), Italy.

Nanoparticles (NPs) are increasingly exploited as diagnostic and therapeutic devices in medicine. Among them, superparamagnetic nanoparticles (SPIONs) represent very promising tools for magnetic resonance imaging, local heaters for hyperthermia, and nanoplatforms for multimodal imaging and theranostics. However, the use of NPs, including SPIONs, in medicine presents several issues: first, the encounter with the biological world and proteins in particular. Indeed, nanoparticles can suffer from protein adsorption, which can affect NP functionality and biocompatibility. In this respect, we have investigated the interaction of small SPIONs covered by an amphiphilic double layer of oleic acid/oleylamine and 1-octadecanoyl--glycero-3-phosphocholine with two abundant human plasma proteins, human serum albumin (HSA) and human transferrin. By means of spectroscopic and scattering techniques, we analyzed the effect of SPIONs on protein structure and the binding affinities, and only found strong binding in the case of HSA. In no case did SPIONs alter the protein structure significantly. We structurally characterized HSA/SPIONs complexes by means of light and neutron scattering, highlighting the formation of a monolayer of protein molecules on the NP surface. Their interaction with lipid bilayers mimicking biological membranes was investigated by means of neutron reflectivity. We show that HSA/SPIONs do not affect lipid bilayer features and could be further exploited as a nanoplatform for future applications. Overall, our findings point toward a high biocompatibility of phosphocholine-decorated SPIONs and support their use in nanomedicine.
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http://dx.doi.org/10.1021/acs.langmuir.0c01083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008447PMC
August 2020

Effect of ergosterol on the interlamellar spacing of deuterated yeast phospholipid multilayers.

Chem Phys Lipids 2020 03 9;227:104873. Epub 2020 Jan 9.

Institut Laue-Langevin, 71 Avenue Des Martyrs, 38000, Grenoble, France. Electronic address:

Sterols regulate several physico-chemical properties of biological membranes that are considered to be linked to function. Ergosterol is the main sterol molecule found in the cell membranes of yeasts and other fungi. Like the cholesterol found in mammalian cells, ergosterol has been proposed to have an ordering and condensing effect on saturated phospholipid membranes. The effects of cholesterol have been investigated extensively and result in an increase in the membrane thickness and the lipid acyl chain order. Less information is available on the effects of ergosterol on phospholipid membranes. Neutron Diffraction (ND) was used to characterize the effect of ergosterol on lipid multilayers prepared with deuterated natural phospholipids extracted from the yeast Pichia pastoris. The data show that the effect of ergosterol on membranes prepared from the natural phospholipid extract rich in unsaturated acyl chains, differs from what has been observed previously in membranes rich in saturated phospholipids. In contrast to cholesterol in synthetic phospholipid membranes, the presence of ergosterol up to 30 mol % in yeast phospholipid membranes only slightly altered the multilayer structure. In particular, only a small decrease in the multilayer d-spacing was observed as function of increasing ergosterol concentrations. This result highlights the need for further investigation to elucidate the effects of ergosterol in biological lipid mixtures.
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http://dx.doi.org/10.1016/j.chemphyslip.2020.104873DOI Listing
March 2020

Reflectometry Reveals Accumulation of Surfactant Impurities at Bare Oil/Water Interfaces.

Molecules 2019 Nov 14;24(22). Epub 2019 Nov 14.

Biomaterials Department, Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany.

Bare interfaces between water and hydrophobic media like air or oil are of fundamental scientific interest and of great relevance for numerous applications. A number of observations involving water/hydrophobic interfaces have, however, eluded a consensus mechanistic interpretation so far. Recent theoretical studies ascribe these phenomena to an interfacial accumulation of charged surfactant impurities in water. In the present work, we show that identifying surfactant accumulation with X-ray reflectometry (XRR) or neutron reflectometry (NR) is challenging under conventional contrast configurations because interfacial surfactant layers are then hardly visible. On the other hand, both XRR and NR become more sensitive to surfactant accumulation when a suitable scattering length contrast is generated by using fluorinated oil. With this approach, significant interfacial accumulation of surfactant impurities at the bare oil/water interface is observed in experiments involving standard cleaning procedures. These results suggest that surfactant impurities may be a limiting factor for the investigation of fundamental phenomena involving water/hydrophobic interfaces.
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http://dx.doi.org/10.3390/molecules24224113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891303PMC
November 2019

Attractive Interaction between Fully Charged Lipid Bilayers in a Strongly Confined Geometry.

J Phys Chem Lett 2019 Nov 11;10(22):7195-7199. Epub 2019 Nov 11.

UPR 22/CNRS, Institut Charles Sadron , Université de Strasbourg , 23 rue du Loess, BP 84047 , 67034 Strasbourg Cedex 2, France.

We investigate the interaction between highly charged lipid bilayers in the presence of monovalent counterions. Neutron and X-ray reflectivity experiments show that the water layer between like-charged bilayers is thinner than for zwitterionic lipids, demonstrating the existence of counterintuitive electrostatic attractive interaction between them. Such attraction can be explained by taking into account the correlations between counterions within the Strong Coupling limit, which falls beyond the classical Poisson-Boltzmann theory of electrostatics. Our results show the limit of the Strong Coupling continuous theory in a highly confined geometry and are in agreement with a decrease in the water dielectric constant due to a surface charge-induced orientation of water molecules.
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http://dx.doi.org/10.1021/acs.jpclett.9b02804DOI Listing
November 2019

The Role of Temperature and Lipid Charge on Intake/Uptake of Cationic Gold Nanoparticles into Lipid Bilayers.

Small 2019 06 23;15(23):e1805046. Epub 2019 Apr 23.

Laboratoire TIMC-IMAG, Université Grenoble Alpes, Domaine de la Merci, 38706, La Tronche Cedex, France.

Understanding the molecular mechanisms governing nanoparticle-membrane interactions is of prime importance for drug delivery and biomedical applications. Neutron reflectometry (NR) experiments are combined with atomistic and coarse-grained molecular dynamics (MD) simulations to study the interaction between cationic gold nanoparticles (AuNPs) and model lipid membranes composed of a mixture of zwitterionic di-stearoyl-phosphatidylcholine (DSPC) and anionic di-stearoyl-phosphatidylglycerol (DSPG). MD simulations show that the interaction between AuNPs and a pure DSPC lipid bilayer is modulated by a free energy barrier. This can be overcome by increasing temperature, which promotes an irreversible AuNP incorporation into the lipid bilayer. NR experiments confirm the encapsulation of the AuNPs within the lipid bilayer at temperatures around 55 °C. In contrast, the AuNP adsorption is weak and impaired by heating for a DSPC-DSPG (3:1) lipid bilayer. These results demonstrate that both the lipid charge and the temperature play pivotal roles in AuNP-membrane interactions. Furthermore, NR experiments indicate that the (negative) DSPG lipids are associated with lipid extraction upon AuNP adsorption, which is confirmed by coarse-grained MD simulations as a lipid-crawling effect driving further AuNP aggregation. Overall, the obtained detailed molecular view of the interaction mechanisms sheds light on AuNP incorporation and membrane destabilization.
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http://dx.doi.org/10.1002/smll.201805046DOI Listing
June 2019

End Point Versus Backbone Specificity Governs Characteristics of Antibody Binding to Poly(ethylene glycol) Brushes.

Langmuir 2018 11 6;34(46):13946-13955. Epub 2018 Nov 6.

Max Planck Institute of Colloids and Interfaces , Am Mühlenberg 1 , 14476 Potsdam , Germany.

End-grafted poly(ethylene glycol) (PEG) brushes are widely used in order to suppress undesired protein adsorption to surfaces exposed to blood or other biological fluids. The specific adsorption of antibodies (Abs) to PEG brushes associated with PEG's antigenicity is drawing increasing attention because it can affect clinical applications. Here, the adsorption to PEG brushes of two Ab types, specifically binding the polymer backbone and the polymer endpoints, is structurally characterized by neutron reflectometry. The measurements yield volume fraction profiles of PEG and of the adsorbed Abs with sub-nanometer resolution perpendicular to the surface. For all brush parameters in terms of grafting density and polymerization degree, the Ab profiles clearly differ between backbone binders and endpoint binders. The adsorbed Ab amount per unit area is substantial for both Ab types and for all brush parameters investigated, even for dense brushes, which impose a considerable osmotic barrier to Ab insertion. The results therefore indicate that variation of brush parameters alone is insufficient to prevent undesired Ab adsorption. Instead, our work motivates further efforts in the search for nonantigenic brush chemistry.
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http://dx.doi.org/10.1021/acs.langmuir.8b02774DOI Listing
November 2018

Influence of Acyl Chain Saturation on the Membrane-Binding Activity of a Short Antimicrobial Peptide.

ACS Omega 2017 Nov 1;2(11):7482-7492. Epub 2017 Nov 1.

Biological Physics Laboratory, School of Physics and Astronomy, University of Manchester, Oxford Road, Schuster Building, Manchester M13 9PL, U.K.

Different bacterial types and their living environments can lead to different saturations in the chains of their membrane lipids. Such structural differences may influence the efficacy of antibiotics that target bacterial membranes. In this work, the effects of acyl chain saturation on the binding of an antimicrobial peptide G have been examined as a function of the packing density of lipid monolayers by combining external reflection Fourier transform infrared (ER-FTIR) spectroscopy and neutron reflection (NR) measurements. Langmuir monolayers were formed from 1,2-dipalmitoyl--glycero-3-phospho-(1'--glycerol) (DPPG) and 1-palmitoyl-2-oleoyl--glycero-3-phospho-(1'--glycerol) (POPG), respectively, with the initial surface pressures controlled at 8 and 28 mN/m. A reduction in the order of the acyl chains associated with the increase in the layer thickness upon G binding was revealed from ER-FTIR spectroscopy, with peptide binding reaching equilibration faster in POPG than in DPPG monolayers. Whereas the dynamic DPPG-binding process displayed a steady increase in the amide I band area, the POPG-binding process showed little change in the amide area after the initial period. The peptide amide I area from ER-FTIR spectroscopy could be linearly correlated with the adsorbed G amount from NR, irrespective of time, initial pressure, or chain saturation, with clearly more peptide incorporated into the DPPG monolayer. Furthermore, NR revealed that although the peptide was associated with both POPG and DPPG lipid monolayers, it was more extensively distributed in the latter, showing that acyl chain saturation clearly promoted peptide binding and structural disruption.
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http://dx.doi.org/10.1021/acsomega.7b01270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044940PMC
November 2017

Conformation of Single and Interacting Lipopolysaccharide Surfaces Bearing O-Side Chains.

Biophys J 2018 04;114(7):1624-1635

Biomaterials Department, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany. Electronic address:

The outer surfaces of Gram-negative bacteria are composed of lipopolysaccharide (LPS) molecules exposing oligo- and polysaccharides to the aqueous environment. This unique, structurally complex biological interface is of great scientific interest as it mediates the interaction of bacteria with antimicrobial agents as well as with neighboring bacteria in colonies and biofilms. Structural studies on LPS surfaces, however, have so far dealt almost exclusively with rough mutant LPS of reduced molecular complexity and limited biological relevance. Here, by using neutron reflectometry, we structurally characterize planar monolayers of wild-type LPS from Escherichia coli O55:B5 featuring strain-specific O-side chains in the presence and absence of divalent cations and under controlled interaction conditions. The model used for the reflectivity analysis is self-consistent and based on the volume fraction profiles of all chemical components. The saccharide profiles are found to be bimodal, with dense inner oligosaccharides and more dilute, extended O-side chains. For interacting LPS monolayers, we establish the pressure-distance curve and determine the distance-dependent saccharide conformation.
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http://dx.doi.org/10.1016/j.bpj.2018.02.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5954297PMC
April 2018

The impact of deuteration on natural and synthetic lipids: A neutron diffraction study.

Colloids Surf B Biointerfaces 2018 Aug 5;168:126-133. Epub 2018 Feb 5.

Institut Laue-Langevin, 71 Avenue des Martyrs, 38000, Grenoble, France. Electronic address:

The structural investigation of cellular membranes requires access to model systems where the molecular complexity is representative of the cellular environment and that allow for the exploitation of structural techniques. Neutron scattering, and in particular neutron diffraction can provide unique and detailed information on the structure of lipid membranes. However, deuterated samples are desirable to fully exploit this powerful method. Recently, the extraction of lipids from microorganisms grown in deuterated media was demonstrated to be both an attracting route to obtain complex lipid mixtures resembling the composition of natural membranes, and to producing deuterated molecules in a very convenient way. A full characterization of these deuterated extracts is hence pivotal for their use in building up model membrane systems. Here we report the structural characterization of lipid extracts obtained from Pichia pastoris by means of neutron diffraction measurements. In particular, we compare the structure of membranes extracted from yeast cells grown in a standard culture medium and in a corresponding deuterated culture medium. The results show that the different molecular composition of the deuterated and protiated lipid extracts induce different structural organization of the lipid membranes. In addition, we compare these membranes composed of extracted yeast lipids with stacked bilayers prepared from synthetic lipid mixtures.
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http://dx.doi.org/10.1016/j.colsurfb.2018.02.009DOI Listing
August 2018

Neutron Reflectometry Elucidates Protein Adsorption from Human Blood Serum onto PEG Brushes.

Langmuir 2017 11 26;33(44):12708-12718. Epub 2017 Oct 26.

Max Planck Institute of Colloids and Interfaces , Am Mühlenberg 1, 14476 Potsdam, Germany.

Poly(ethylene glycol) (PEG) brushes are reputed for their ability to prevent undesired protein adsorption to material surfaces exposed to biological fluids. Here, protein adsorption out of human blood serum onto PEG brushes anchored to solid-supported lipid monolayers was characterized by neutron reflectometry, yielding volume fraction profiles of lipid headgroups, PEG, and adsorbed proteins at subnanometer resolution. For both PEGylated and non-PEGylated lipid surfaces, serum proteins adsorb as a thin layer of approximately 10 Å, overlapping with the headgroup region. This layer corresponds to primary adsorption at the grafting surface and resists rinsing. A second diffuse protein layer overlaps with the periphery of the PEG brush and is attributed to ternary adsorption due to protein-PEG attraction. This second layer disappears upon rinsing, thus providing a first observation of the structural effect of rinsing on protein adsorption to PEG brushes.
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http://dx.doi.org/10.1021/acs.langmuir.7b03048DOI Listing
November 2017

Nano-in-Nano Approach for Enzyme Immobilization Based on Block Copolymers.

ACS Appl Mater Interfaces 2017 Aug 21;9(34):29318-29327. Epub 2017 Aug 21.

Dipartimento di Scienze Chimiche, Università di Napoli Federico II , Complesso Monte S. Angelo, Via Cintia, 80126 Napoli, Italy.

We set up a facile approach for fabrication of supports with tailored nanoporosity for immobilization of enzymes. To this aim block copolymers (BCPs) self-assembly has been used to prepare nanostructured thin films with well-defined architecture containing pores of tailorable size delimited by walls with tailorable degree of hydrophilicity. In particular, we employed a mixture of polystyrene-block-poly(l-lactide) (PS-PLLA) and polystyrene-block-poly(ethylene oxide) (PS-PEO) diblock copolymers to generate thin films with a lamellar morphology consisting of PS lamellar domains alternating with mixed PEO/PLLA blocks lamellar domains. Selective basic hydrolysis of the PLLA blocks generates thin films, patterned with nanometric channels containing hydrophilic PEO chains pending from PS walls. The shape and size of the channels and the degree of hydrophilicity of the pores depend on the relative length of the blocks, the molecular mass of the BCPs, and the composition of the mixture. The strength of our approach is demonstrated in the case of physical adsorption of the hemoprotein peroxidase from horseradish (HRP) using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with HO as substrate. The large surface area, the tailored pore sizes, and the functionalization with hydrophilic PEO blocks make the designed nanostructured materials suitable supports for the nanoconfinement of HRP biomolecules endowed with high catalytic performance, no mass-transfer limitations, and long-term stability.
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http://dx.doi.org/10.1021/acsami.7b08959DOI Listing
August 2017

Neutron reflectometry yields distance-dependent structures of nanometric polymer brushes interacting across water.

Soft Matter 2017 Aug;13(34):5767-5777

Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany.

The interaction between surfaces displaying end-grafted hydrophilic polymer brushes plays important roles in biology and in many wet-technological applications. In this context, the conformation of the brushes upon their mutual approach is crucial, because it affects interaction forces and the brushes' shear-tribological properties. While this aspect has been addressed by theory, experimental data on polymer conformations under confinement are difficult to obtain. Here, we study interacting planar brushes of hydrophilic polymers with defined length and grafting density. Via ellipsometry and neutron reflectometry we obtain pressure-distance curves and determine distance-dependent polymer conformations in terms of brush compression and reciprocative interpenetration. While the pressure-distance curves are satisfactorily described by the Alexander-de-Gennes model, the pronounced brush interpenetration as seen by neutron reflectometry motivates detailed simulation-based studies capable of treating brush interpenetration on a quantitative level.
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http://dx.doi.org/10.1039/c7sm01066dDOI Listing
August 2017

The influence of mild acidity on lysyl-phosphatidylglycerol biosynthesis and lipid membrane physico-chemical properties in methicillin-resistant Staphylococcus aureus.

Chem Phys Lipids 2017 08 23;206:60-70. Epub 2017 Jun 23.

Institute of Pharmaceutical Science, King's College London, London, UK; Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany. Electronic address:

The increased biosynthesis of lysyl-phosphatidylglycerol in Staphylococcus aureus when cultured under conditions of mild acidity and the resultant increased proportion of this lipid in the plasma membrane of the bacterium, alters the physico-chemical properties of lipid bilayers in a manner which is itself dependent upon environmental pH. Clinically relevant strains of S. aureus, both methicillin susceptible and resistant, all exhibited increased lysyl-phosphatidylglycerol biosynthesis in response to mild environmental acidity, albeit to differing degrees, from ∼30% to ∼55% total phospholipid. Polar lipid extracts from these bacteria were analysed by P NMR and reconstituted into vesicles and monolayers, which were characterised by zeta potential measurements and Langmuir isotherms respectively. A combination of increased lysyl-phosphatidylglycerol content and mild environmental acidity were found to synergistically neutralise the charge of the membranes, in one instance altering the zeta potential from -56mV to +21mV, and induce closer packing between the lipids. Challenge of reconstituted S. aureus lipid model membranes by the antimicrobial peptide magainin 2 F5W was examined using monolayer subphase injection and neutron diffraction, and revealed that ionisation of the headgroup α-amine of lysyl-phosphatidylglycerol at pH 5.5, which reduced the magnitude of the peptide-lipid interaction by 80%, was more important for resisting peptide partitioning than increased lipid content alone. The significance of these results is discussed in relation to how colonising mildly acidic environments such as human mucosa may be facilitated by increased lysyl-phosphatidylglycerol biosynthesis and the implications of this for further biophysical analysis of the role of this lipid in bacterial membranes.
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http://dx.doi.org/10.1016/j.chemphyslip.2017.06.007DOI Listing
August 2017

The effect of the protein corona on the interaction between nanoparticles and lipid bilayers.

J Colloid Interface Sci 2017 Oct 29;504:741-750. Epub 2017 May 29.

Department of Chemistry, Materials and Chemical Engineering "G. Natta", Politecnico di Milano, Via Mancinelli 7, 20131 Milan, Italy(3). Electronic address:

Hypothesis: It is known that nanoparticles (NPs) in a biological fluid are immediately coated by a protein corona (PC), composed of a hard (strongly bounded) and a soft (loosely associated) layers, which represents the real nano-interface interacting with the cellular membrane in vivo. In this regard, supported lipid bilayers (SLB) have extensively been used as relevant model systems for elucidating the interaction between biomembranes and NPs. Herein we show how the presence of a PC on the NP surface changes the interaction between NPs and lipid bilayers with particular care on the effects induced by the NPs on the bilayer structure.

Experiments: In the present work we combined Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) and Neutron Reflectometry (NR) experimental techniques to elucidate how the NP-membrane interaction is modulated by the presence of proteins in the environment and their effect on the lipid bilayer.

Findings: Our study showed that the NP-membrane interaction is significantly affected by the presence of proteins and in particular we observed an important role of the soft corona in this phenomenon.
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http://dx.doi.org/10.1016/j.jcis.2017.05.086DOI Listing
October 2017

Controlling adsorption of albumin with hyaluronan on silica surfaces and sulfonated latex particles.

J Colloid Interface Sci 2017 Oct 17;504:315-324. Epub 2017 May 17.

Department of Physics and Astronomy & Centre for Neutron Scattering, Uppsala University, Box 516, 75120 Uppsala, Sweden. Electronic address:

Polysaccharides are known to modify binding of proteins at interfaces and this paper describes studies of these interactions and how they are modified by pH. Specifically, the adsorption of human serum albumin on to polystyrene latex and to silica is described, focusing on how this is affected by hyaluronan. Experiments were designed to test how such binding might be modified under relevant physiological conditions. Changes in adsorption of albumin alone and the co-adsorption of albumin and hyaluronan are driven by electrostatic interactions. Multilayer binding is found to be regulated by the pH of the solution and the molecular mass and concentration of hyaluronan. Highest adsorption was observed at pH below 4.8 and for low molecular mass hyaluronan (≤150kDa) at concentrations above 2mgml. On silica with grafted hyaluronan, albumin absorption is reversed by changes in solvent pH due to their strong electrostatic attraction. Albumin physisorbed on silica surfaces is also rinsed away with dilute hyaluronan solution at pH 4.8. The results demonstrate that the protein adsorption can be controlled both by changes of pH and by interaction with other biological macromolecules.
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http://dx.doi.org/10.1016/j.jcis.2017.05.037DOI Listing
October 2017

Building a biomimetic membrane for neutron reflectivity investigation: Complexity, asymmetry and contrast.

Biophys Chem 2017 10 29;229:135-141. Epub 2017 Apr 29.

Università degli Studi di Milano, BIOMETRA, Via F.lli Cervi, 93, 20090 Segrate, Italy.

The preparation and investigation of model membranes is deserving growing interest both for the physics of complex systems, and for biology. The need of simplified models should preserve mimicking the qualifying characteristics of biological membranes, and keep non-invasive and detailed description. As a main feature, biological membranes are non-homogeneous in the disposition of components, both in the lateral and in the transverse direction. We prepared asymmetric supported membranes containing GM1 ganglioside in biomimetic proportion according to different protocols. Then, we studied their internal structure by neutron reflectometry, providing few-Angstrom sensitivity in the cross direction meanwhile avoiding radiation damage. This technique can also be profitably applied to study interactions at the membrane surface. The best protocol has proven to be the Langmuir-Blodgett/Langmuir-Schaefer depositions. Notably, also the simpler and most accessible protocol of vesicle fusion was found to be suitable for straightforward and good quality deposition of compositionally asymmetric membranes.
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http://dx.doi.org/10.1016/j.bpc.2017.04.011DOI Listing
October 2017

Nanoroughness Strongly Impacts Lipid Mobility in Supported Membranes.

Langmuir 2017 03 24;33(9):2444-2453. Epub 2017 Feb 24.

Université Lyon, Université Claude Bernard Lyon 1, CNRS, Institut Lumière Matière, F-69622 Villeurbanne, France.

In vivo lipid membranes interact with rough supramolecular structures such as protein clusters and fibrils. How these features whose size ranges from a few nanometers to a few tens of nanometers impact lipid and protein mobility is still being investigated. Here, we study supported phospholipid bilayers, a unique biomimetic model, deposited on etched surfaces bearing nanometric corrugations. The surface roughness and mean curvature are carefully characterized by AFM imaging using ultrasharp tips. Neutron specular reflectivity supplements this surface characterization and indicates that the bilayers follow the large-scale corrugations of the substrate. We measure the lateral mobility of lipids in both the fluid and gel phases by fluorescence recovery after patterned photobleaching. Although the mobility is independent of the roughness in the gel phase, it exhibits a 5-fold decrease in the fluid phase when the roughness increases from 0.2 to 10 nm. These results are interpreted with a two-phase model allowing for a strong decrease in the lipid mobility in highly curved or defect-induced gel-like nanoscale regions. This suggests a strong link between membrane curvature and fluidity, which is a key property for various cell functions such as signaling and adhesion.
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http://dx.doi.org/10.1021/acs.langmuir.6b03276DOI Listing
March 2017
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