Multidimensional Spectral Phasors of LAURDAN's Excitation-Emission Matrices: The Ultimate Sensor for Lipid Phases?

The impact of lipid diversity on the lateral organization of biological membranes remains a topic of debate. While the existence of domains in lamellar membranes is well-established, the nonlamellar phases occurring in biological systems are less explored due to technical constraints. Here, we present the measurement of the excitation-emission matrices (EEM) of LAURDAN in several lipid structures. LAURDAN is a fluorescence probe widely used for characterizing lipid assemblies. The EEMs were analyzed by multidimensional spectral phasors (MdSP), an approach that seizes information from both the excitation and emission spectra. We developed a computer algorithm to construct EEM data based on a model for LAURDAN's photophysics. The MdSP calculated from the simulated EEMs reveals that all feasible possibilities lie inside a universal triangle in the phasor's plot. We use this triangle to propose a ternary representation for the phasors, allowing a better assessment of LAURDAN's surroundings in terms of hydration, water mobility, and local electronic environment. Building upon this foundation, we constructed a theoretical "phase map" that can assess both lamellar and nonlamellar membranes. We thoroughly validated this theory using well-known lipid mixtures under different phase-state conditions and enzymatically generated systems. Our results confirm that the use of MdSP is a powerful tool for obtaining quantitative information on both lamellar and nonlamellar structures. This study not only advances our understanding of the impact of lipid diversity on membrane organization but also provides a robust and general framework for the assessment of fluorescence properties that can be further extended to other probes.

Dengue and Zika virus capsid proteins bind to membranes and self-assemble into liquid droplets with nucleic acids.

Dengue virus (DENV) and Zika virus (ZIKV) capsid proteins efficiently recruit and surround the viral RNA at the endoplasmic reticulum (ER) membrane to yield nascent viral particles. However, little is known either about the molecular mechanisms by which multiple copies of capsid proteins assemble into nucleocapsids (NCs) or how the NC is recruited and wrapped by the ER membrane during particle morphogenesis. Here, we measured relevant interactions concerning this viral process using purified DENV and ZIKV capsid proteins, membranes mimicking the ER lipid composition, and nucleic acids in in vitro conditions to understand the biophysical properties of the RNA genome encapsidation process. We found that both ZIKV and DENV capsid proteins bound to liposomes at liquid-disordered phase regions, docked exogenous membranes, and RNA molecules. Liquid-liquid phase separation is prone to occur when positively charged proteins interact with nucleic acids, which is indeed the case for the studied capsids. We characterized these liquid condensates by measuring nucleic acid partition constants and the extent of water dipolar relaxation, observing a cooperative process for the formation of the new phase that involves a distinct water organization. Our data support a new model in which capsid-RNA complexes directly bind the ER membrane, seeding the process of RNA recruitment for viral particle assembly. These results contribute to our understanding of the viral NC formation as a stable liquid-liquid phase transition, which could be relevant for dengue and Zika gemmation, opening new avenues for antiviral intervention.

Amyloid oligomerization of the Parkinson's disease related protein α-synuclein impacts on its curvature-membrane sensitivity.

The amyloid aggregation of the presynaptic protein α-synuclein (AS) is pathognomonic of Parkinson's disease and other neurodegenerative disorders. Physiologically, AS contributes to synaptic homeostasis by participating in vesicle maintenance, trafficking, and release. Its avidity for highly curved acidic membranes has been related to the distinct chemistry of the N-terminal amphipathic helix adopted upon binding to appropriated lipid interfaces. Pathologically, AS populate a myriad of toxic aggregates ranging from soluble oligomers to insoluble amyloid fibrils. Different gain-of-toxic function mechanisms are linked to prefibrillar oligomers which are considered as the most neurotoxic species. Here, we investigated if amyloid oligomerization could hamper AS function as a membrane curvature sensor. We used fluorescence correlation spectroscopy to quantitatively evaluate the interaction of oligomeric species, produced using a popular method based on lyophilization and rehydration, to lipid vesicles of different curvatures and compositions. We found that AS oligomerization has a profound impact on protein-lipid interaction, altering binding affinity and/or curvature sensitivity depending on membrane composition. Our work provides novel insights into how the formation of prefibrillar intermediate species could contribute to neurodegeneration due to a loss-of-function mechanism. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Myristoylation and Oligonucleotide Interaction Modulate Peptide and Protein Surface Properties: The Case of the HIV-1 Matrix Domain.

Myristoylated proteins typically develop a tight association with membranes. One example is the matrix domain (MA) of the HIV-1 Gag protein. In addition, MA is able to bind the Sel25 RNA sequence, a ligand that can act as a competitor for the interaction with the membrane. These properties make HIV-1 MA an attractive molecule to understand how protein and peptide surface properties can be controlled by myristoylation and oligonucleotide interaction. In this line, we analyzed the stability, thermodynamics, and the topography of Langmuir monolayers composed of the myristoylated or unmyristoylated versions of MA in the presence or the absence of a single-strand DNA (ssDNASel25) analogue of the Sel25 RNA sequence. With a similar approach, we compared the MA surface properties with those obtained from monolayers of myristoylated and unmyristoylated MA-derived peptides (first 21 residues of the MA sequence). Our results show that the protein or peptide films are destabilized by the presence of ssDNASel25, inducing solubilization of the monolayer components into the bulk phase. In addition, the oligonucleotide affects the protein-protein or peptide-peptide lateral interactions, provoking interfacial topography changes of the monolayers, visualized by Brewster angle microscopy. Furthermore, we also show how the myristoyl group has major effects on the lateral stability and the elasticity of the monolayers. Altogether, here we propose a general model considering the effect of myristoylation and the interaction with oligonucleotides on the interfacial properties of MA and derived peptides. In this model, we introduce a new role of the core region of MA (sequence of MA after the 21st residue) that confers higher lateral interfacial stability to the protein.

Membrane Restructuring Events during the Enzymatic Generation of Ceramides with Very Long-Chain Polyunsaturated Fatty Acids.

In rat sperm heads, sphingomyelin (SM) species that contain very long-chain polyunsaturated fatty acid (V-SM) become ceramides (V-Cer) after inducing in vitro the acrosomal reaction. The reason for such a specific location of this conversion, catalyzed by a sphingomyelinase (SMase), has received little investigation so far. Here, the effects of SMase were compared in unilamellar vesicles (large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs)) containing phosphatidylcholine, and either V-SM or a palmitate-rich SM (P-SM). In uniformly sized LUVs at 37 °C, more V-Cer was generated and more rapidly than P-Cer. Nephelometry and dynamic light scattering showed that LUVs tended to form large lipid particles more intensely, and Förster resonance energy transfer (FRET) increases suggested that lateral lipid mixing was more marked when V-Cer rather than P-Cer was produced. As reported by 6-dodecanoyl-2-dimethyl-aminopnaphthalene (Laurdan) and 1,6-diphenyl-1,3,5,-hexatriene (DPH), the production of V-Cer resulted in higher and faster restriction in lipid mobility than that of P-Cer, implying a stronger increase in membrane dehydration and microviscosity. Moreover, DPH anisotropy suggested a higher solubility of V-Cer than that of P-Cer in the liquid-disordered phase. At room temperature, liquid-condensed lateral domains appeared in P-SM- but not in V-SM-containing GUVs. The former maintained their size while losing their contents gradually during SMase action, whereas the latter became permeable earlier and reduced their size in few minutes until suddenly collapsing. The fast and potent generation of V-Cer may contribute to the membrane restructuring events that occur on the acrosome-reacted sperm head.

Differential Interaction of Antimicrobial Peptides with Lipid Structures Studied by Coarse-Grained Molecular Dynamics Simulations.

In this work; we investigated the differential interaction of amphiphilic antimicrobial peptides with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid structures by means of extensive molecular dynamics simulations. By using a coarse-grained (CG) model within the MARTINI force field; we simulated the peptide-lipid system from three different initial configurations: (a) peptides in water in the presence of a pre-equilibrated lipid bilayer; (b) peptides inside the hydrophobic core of the membrane; and (c) random configurations that allow self-assembled molecular structures. This last approach allowed us to sample the structural space of the systems and consider cooperative effects. The peptides used in our simulations are aurein 1.2 and maculatin 1.1; two well-known antimicrobial peptides from the Australian tree frogs; and molecules that present different membrane-perturbing behaviors. Our results showed differential behaviors for each type of peptide seen in a different organization that could guide a molecular interpretation of the experimental data. While both peptides are capable of forming membrane aggregates; the aurein 1.2 ones have a pore-like structure and exhibit a higher level of organization than those conformed by maculatin 1.1. Furthermore; maculatin 1.1 has a strong tendency to form clusters and induce curvature at low peptide-lipid ratios. The exploration of the possible lipid-peptide structures; as the one carried out here; could be a good tool for recognizing specific configurations that should be further studied with more sophisticated methodologies.

Phosphatidylserine lipids and membrane order precisely regulate the activity of Polybia-MP1 peptide.

Polybia-MP1 (IDWKKLLDAAKQIL-NH2) is a lytic peptide from the Brazilian wasp venom with known anti-cancer properties. Previous evidence indicates that phosphatidylserine (PS) lipids are relevant for the lytic activity of MP1. In agreement with this requirement, phosphatidylserine lipids are translocated to the outer leaflet of cells, and are available for MP1 binding, depending on the presence of liquid-ordered domains. Here, we investigated the effect of PS on MP1 activity when this lipid is reconstituted in membranes of giant or large liposomes with different lipid-phase states. By monitoring the membrane and soluble luminal content of giant unilamellar vesicles (GUVs), using fluorescence confocal microscopy, we were able to determine that MP1 has a pore-forming activity at the membrane level. Liquid-ordered domains, which were phase-separated within the membrane of GUVs, influenced the pore-forming activity of MP1. Experiments evaluating the membrane-binding and lytic activity of MP1 on large unilamellar vesicles (LUVs), with the same lipid composition as GUVs, demonstrated that there was synergy between liquid-ordered domains and PS, which enhanced both activities. Based on our findings, we propose that the physicochemical properties of cancer cell membranes, which possess a much higher concentration of PS than normal cells, renders them susceptible to MP1 binding and lytic pore formation. These results can be correlated with MP1's potent and selective anti-cancer activity and pave the way for future research to develop cancer therapies that harness and exploit the properties of MP1.

The rheological properties of beta amyloid Langmuir monolayers: Comparative studies with melittin peptide.

We determined the rheological properties of ß-amyloid Langmuir films at the air/water interface, a peptide whose interfacial structure is extended ß-sheet, and compared them with those of films composed of Melittin (Mel), which adopts an α-helical conformation at neutral pH. To determine the dilatational and shear moduli we evaluated the response of pure peptide monolayers to an oscillatory anisotropic compressive work. Additionally, a micro-rheological characterization was performed by tracking the diffusion of micrometer sized latex beads onto the interface. This technique allowed us the detection of different rheological behaviour between monolayers presenting a low shear response. Monolayers of the ß-sheet structure-adopting peptides, such as ß-amyloid peptides, exhibited a marked shear (elastic) modulus even at low surface pressures. In contrast, Mel monolayers exhibited negligible shear modulus and the micro-rheological shear response was markedly lower than that observed for either Aß1-40 or Aß1-42 amyloid peptides. When Mel monolayers were formed at the interface of an aqueous solution at pH 11, we observed an increase in both the lateral stability and film viscosity as detected by a slower diffusion of the latex beads, in keeping with an increase in ß-sheet structure at this high pH (verified by ATR and FT-IR measurements). We suggest that the interactions responsible for the marked response upon shear observed for ß-amyloid peptide monolayers are the hydrogen bonds of the ß-sheet structure that can form an infinite planar network at the interface. Conversely, α-helical Mel peptide lack of these inter-molecular interactions and, therefore the shear contribution was negligible. We propose that the secondary structure is important for modulating the rheological behavior of short peptide monolayers regardless of the mass density or surface charge at the surface.

The amphiphilic alkyl ester derivatives of l-ascorbic acid induce reorganization of phospholipid vesicles.

l-ascorbic acid alkyl esters (ASCn) are lipophilic forms of vitamin C, which maintain some of its antioxidant power. Those properties make this drug family attractive to be used in pharmacological preparations protecting other redox-sensible drugs or designed to reduce possible toxic oxidative processes. In this work, we tested the ability of l-ascorbic acid alkyl esters (ASCn) to modulate the structure, permeability, and rheological properties of phospholipid bilayers. The ASCn studied here (ASC16, ASC14, and ASC12) alter the structural integrity as well as the rheological properties of phospholipid membranes without showing any evident detergent activity. ASC14 appeared as the most efficient drug in destabilize the membrane structure of nano- and micro-size phospholipid liposomes inducing vesicle content leakage and shape elongation on giant unilamellar vesicles. It also was the most potent enhancer of membrane microviscosity and surface water structuring. Only ASC16 induced the formation of drug-enriched condensed domains after its incorporation into the lipid bilayer, while ASC12 appeared as the less membrane-disturbing compound, likely because of its poor, and more superficial, partition into the membrane. We also found that incorporation of ASCn into the lipid bilayers enhanced the reduction of membrane components, compared with soluble vitamin C. Our study shows that ASCn compounds, which vary in the length of the acyl chain, show different effects on phospholipid vesicles used as biomembrane models. Those variances may account for subtly differences in the effectiveness on their pharmacological applications.

Reversing the peptide sequence impacts on molecular surface behaviour.

The protein's primary structure has all the information for specific protein/peptide folding and, in many cases, can define specific amphiphilic regions along molecules that are important for interaction with membranes. In order to shed light on how peptide sequence is important for the surface properties of amphiphilic peptides, we designed three pairs of peptides with the following characteristics: (1) all molecules have the same hydrophobic residues; (2) the couples differ from each other in their hydrophilic amino acids: positively, negatively and non-charged; (3) each pair has the same residues (same global molecular hydrophobicity) but the primary structure is reversed in comparison to its partner (retro-isomer), giving a molecule with a hydrophilic N or C-terminus and a hydrophobic C or N-terminus. Using the Langmuir monolayer approach, we observed that sequence reversal has a central role in the lateral stability of peptide monolayers, in the ability of the molecules to partition into the air-water interface and in the rheological properties of peptide films, whereas the peptide's secondary structure, determined by ATR-FTIR, was the same for all peptides. Reversing the sequence also gives a differential way of peptide/lipid interaction when peptides are in the presence of POPC lipid bilayers. Our results show how sequence inversion confers a distinctive peptide surface behaviour and lipid interaction for molecules with a similar structure.

Arf1 and membrane curvature cooperate to recruit Arfaptin2 to liposomes.

Arfaptin2 contains a Bin/Amphiphysin/Rvs (BAR) domain and directly interacts with proteins of the Arf/Arl family in their active GTP-bound state. It has been proposed that BAR domains are able to sense membrane curvature and to induce membrane tubulation. We report here that active Arf1 is required for the recruitment of Arfaptin2 to artificial liposomes mimicking the Golgi apparatus lipid composition. The Arf1-dependent recruitment of Arfaptin2 increases with membrane curvature, while the recruitment of Arf1 itself is not sensitive to curvature. At high protein concentrations, the binding of Arfaptin2 induces membrane tubulation. Finally, membrane-bound Arfaptin2 is released from the liposome when ArfGAP1 catalyzes the hydrolysis of GTP to GDP in Arf1. These results show that both Arf1 activation and high membrane curvature are required for efficient recruitment of Arfaptin2 to membranes.

Lipid-like behavior of signal sequence peptides at air-water interface.

Several protein transport processes in the cell are mediated by signal sequence peptides located at the N-terminal side of the mature protein sequence. To date, the specific interaction and the stability of these peptides at the amphipathic interface of biological membranes and the relevance of the peptide conformation when they interact with lipids is not clear. We report the surface properties and the peptide-lipid interaction of three signal sequence peptides at the air-NaCl 145 mM interface by using the Langmuir monolayer approach. These synthetic peptides have a natural sequence with a non-periodic amphiphilicity, where hydrophobic and hydrophilic residues are located on opposed sides of the peptide primary sequence. We show that signal sequence peptides form insoluble monolayers of high stability against lateral compression. At close packing, peptide molecular area, surface potential and the high stability of the peptide monolayer are indicative that signal sequence peptides are compatible with a ß-sheet conformation at the interface. Structure was confirmed with PM-IRRAS and transmission FT-IR studies. The peptides show lateral miscibility with either POPC (a liquid-expanded lipid) or DPPC (a liquid-condensed lipid) in mixed peptide-lipid monolayers. This indicates that signal sequence peptides studied are laterally miscible with phospholipids independent of the phase state of the lipid.

Biophysical properties of a synthetic transit peptide from wheat chloroplast ribulose 1,5-bisphosphate carboxylase.

The surface properties of pure RuBisCo transit peptide (RTP) and its interaction with zwitterionic, anionic phospholipids and chloroplast lipids were studied by using the Langmuir monolayer technique. Pure RTP is able to form insoluble films and the observed surface parameters are compatible with an alpha-helix perpendicular to the interface. The alpha-helix structure tendency was also observed by using transmission FT-IR spectroscopy in bulk system of a membrane mimicking environment (SDS). On the other hand, RTP adopts an unordered structure in either aqueous free interface or in the presence of vesicles composed of a zwitterionic phospholipid (POPC). Monolayer studies show that in peptide/lipid mixed monolayers, RTP shows no interaction with zwitterionic phospholipids, regardless of their physical state. Also, with the anionic POPG at high peptide ratios RTP retains its individual surface properties and behaves as an immiscible component of the peptide/lipid mixed interface. This behaviour was also observed when the mixed films were composed by RTP and the typical chloroplast lipids MGDG or DGDG (mono- and di-galactosyldiacylglycerol). Conversely, RTP establishes a particular interaction with phosphatidylglycerol and cardiolipin at low peptide to lipid area covered relation. This interaction takes place with an increase in surface stability and a reduction in peptide molecular area (intermolecular interaction). Data suggest a dynamic membrane modulation by which the peptide fine-tunes its membrane orientation and its lateral stability, depending on the quality (lipid composition) of the interface.

Interfacial properties of the M1 segment of the nicotinic acetylcholine receptor.

We have studied the thermodynamic, surface, and structural properties of alphaM1 transmembrane sequence of the nicotinic acetylcholine receptor (nAChR) by using Langmuir monolayer, FT-IR spectroscopy and molecular dynamics simulation techniques in membrane-mimicking environments. M1 spontaneously incorporates into a lipid-free air-water interface, showing a favourable adsorption free energy of -7.2 kcal/mol. A cross-sectional molecular area of 210 A(2)/molecule, a surface potential of 4.2 fV/molecule and a high stability of the film were deducted from pure M1 monolayers. FT-IR experiments and molecular dynamics simulations in membrane-mimicking environments (sodium-dodecyl-sulfate and CCl(4), respectively) indicate coexistence between helical and non-helical structures. Furthermore, mixed peptide-lipid monolayers and monolayer penetration experiments were performed in order to study the peptide-lipid interaction. Mixed with condensed lipids (dipalmitoyl-phosphocholine, and dipalmitoyl-phosphoglycerol), M1 shows immiscible/miscible behaviour at low/high peptide concentration, respectively. Conversely, a complete miscible peptide-lipid interface is observed with liquid-expanded lipids (palmitoyl-oleoyl-phosphocholine, and palmitoyl-oleoyl-phosphoglycerol). Peptide penetration experiments demonstrate that the M1 peptide preferentially interacts with zwitterionic phosphocholine interfaces.

Amyloid-beta peptide disruption of lipid membranes and the effect of metal ions.

Beta-amyloid peptide (Abeta), which is cleaved from the larger trans-membrane amyloid precursor protein, is found deposited in the brain of patients suffering from Alzheimer's disease and is linked with neurotoxicity. We report the results of studies of Abeta1-42 and the effect of metal ions (Cu2+ and Zn2+) on model membranes using 31P and 2H solid-state NMR, fluorescence and Langmuir Blodgett monolayer methods. Both the peptide and metal ions interact with the phospholipid headgroups and the effects on the lipid bilayer and the peptide structure were different for membrane incorporated or associated peptides. Copper ions alone destabilise the lipid bilayer and induced formation of smaller vesicles but when Abeta1-42 was associated with the bilayer membrane copper did not have this effect. Circular dichroism spectroscopy indicated that Abeta1-42 adopted more beta-sheet structure when incorporated in a lipid bilayer in comparison to the associated peptide, which was largely unstructured. Incorporated peptides appear to disrupt the membrane more severely than associated peptides, which may have implications for the role of Abeta in disease states.

Direct visualization of membrane leakage induced by the antibiotic peptides: maculatin, citropin, and aurein.

Membrane lysis caused by antibiotic peptides is often rationalized by means of two different models: the so-called carpet model and the pore-forming model. We report here on the lytic activity of antibiotic peptides from Australian tree frogs, maculatin 1.1, citropin 1.1, and aurein 1.2, on POPC or POPC/POPG model membranes. Leakage experiments using fluorescence spectroscopy indicated that the peptide/lipid mol ratio necessary to induce 50% of probe leakage was smaller for maculatin compared with aurein or citropin, regardless of lipid membrane composition. To gain further insight into the lytic mechanism of these peptides we performed single vesicle experiments using confocal fluorescence microscopy. In these experiments, the time course of leakage for different molecular weight (water soluble) fluorescent markers incorporated inside of single giant unilamellar vesicles is observed after peptide exposure. We conclude that maculatin and its related peptides demonstrate a pore-forming mechanism (differential leakage of small fluorescent probe compared with high molecular weight markers). Conversely, citropin and aurein provoke a total membrane destabilization with vesicle burst without sequential probe leakage, an effect that can be assigned to a carpeting mechanism of lytic action. Additionally, to study the relevance of the proline residue on the membrane-action properties of maculatin, the same experimental approach was used for maculatin-Ala and maculatin-Gly (Pro-15 was replaced by Ala or Gly, respectively). Although a similar peptide/lipid mol ratio was necessary to induce 50% of leakage for POPC membranes, the lytic activity of maculatin-Ala and maculatin-Gly decreased in POPC/POPG (1:1 mol) membranes compared with that observed for the naturally occurring maculatin sequence. As observed for maculatin, the lytic action of Maculatin-Ala and maculatin-Gly is in keeping with the formation of pore-like structures at the membrane independently of lipid composition.

Surface behavior and lipid interaction of Alzheimer beta-amyloid peptide 1-42: a membrane-disrupting peptide.

Amyloid aggregates, found in patients that suffer from Alzheimer's disease, are composed of fibril-forming peptides in a beta-sheet conformation. One of the most abundant components in amyloid aggregates is the beta-amyloid peptide 1-42 (Abeta 1-42). Membrane alterations may proceed to cell death by either an oxidative stress mechanism, caused by the peptide and synergized by transition metal ions, or through formation of ion channels by peptide interfacial self-aggregation. Here we demonstrate that Langmuir films of Abeta 1-42, either in pure form or mixed with lipids, develop stable monomolecular arrays with a high surface stability. By using micropipette aspiration technique and confocal microscopy we show that Abeta 1-42 induces a strong membrane destabilization in giant unilamellar vesicles composed of palmitoyloleoyl-phosphatidylcholine, sphingomyelin, and cholesterol, lowering the critical tension of vesicle rupture. Additionally, Abeta 1-42 triggers the induction of a sequential leakage of low- and high-molecular-weight markers trapped inside the giant unilamellar vesicles, but preserving the vesicle shape. Consequently, the Abeta 1-42 sequence confers particular molecular properties to the peptide that, in turn, influence supramolecular properties associated to membranes that may result in toxicity, including: 1), an ability of the peptide to strongly associate with the membrane; 2), a reduction of lateral membrane cohesive forces; and 3), a capacity to break the transbilayer gradient and puncture sealed vesicles.

Surface behaviour and peptide-lipid interactions of the antibiotic peptides, Maculatin and Citropin.

Surface behaviour of Maculatin 1.1 and Citropin 1.1 antibiotic peptides have been studied using the Langmuir monolayer technique in order to understand the peptide-membrane interaction proposed as critical for cellular lysis. Both peptides have a spontaneous adsorption at the air-water interface, reaching surface potentials similar to those obtained by direct spreading. Collapse pressures (Pi(c), stability to lateral compression), molecular areas at maximal packing and surface potentials (DeltaV) obtained from compression isotherms of both pure peptide monolayers are characteristic of peptides adopting mainly alpha-helical structure at the interface. The stability of Maculatin monolayers depended on the subphase and increased when pH was raised. In an alkaline environment, Maculatin exhibits a molecular reorganization showing a reproducible discontinuity in the Pi-A compression isotherm. Both peptides in lipid films with the zwitterionic palmitoyl-oleoyl-phosphatidylcholine (POPC) showed an immiscible behaviour at all lipid-peptide proportions studied. By contrast, in films with the anionic palmitoyl-oleoyl-phosphatidylglycerol (POPG), the peptides showed miscible behaviour when the peptides represented less than 50% of total surface area. Additional penetration experiments also demonstrated that both peptides better interact with POPG compared with POPC monolayers. This lipid preference is discussed as a possible explanation of their antibiotic properties.

Péptidos sintéticos beta-amilode 1-40 y 1-42, propiedades de superficie e interacción con modelos de membranas biológicas

Dados los resultados obtenidos en nuestros estudios, es posible afirmar que AB 1-40 y AB 1-42 conforman capas monomoleculares insolubles en interfases agua-aire con una alta estabilidad frente al trabajo de compresión. La alta estabilidad de un film peptídico en una interfase agua-aire y los bajos potencíales de superficie pueden deberse a que dichas moléculas estén adoptando una estructura del tipo p en la misma. Sin embargo por las áreas moleculares medidas hace sugerir que parte de la secuencia podría estar adoptando una estructura del tipo helicoidal. Esto también se puede interpretar por los valores de los potenciales de superficie, ya que, si bien son bajos, no son tan pequeños en comparación con péptidos que adoptan totalmente una estructura del tipo p. El estudio de las propiedades de superficie de ambos péptidos en presencia de cationes tales como Cu , Zn y Fe arrojó como resultado que dichos metales no afectan considerablemente la estabilidad de las capas monomoleculares de estas proteínas pero sí alteran levemente el potencial de superficie. Esto podría deberse a la unión del metal por la proteína ya que, como se conoce, AB 1-40 y AB 1-42 forman complejos con dichos cationes reduciéndolos lo que genera la formación de peróxido de hidrógeno, lo que ocasionaría un stress oxídativo en interfases. De los estudios de films mezcla con lípidos hay que recalcar que AB 1-40 y Ab 1-40 presentan misciblidad parcial con los lípidos estudiados y la estabilidad de la interfase a presiones laterales donde la fase rica en péptido no colapso se encuentra dentro del rango de presiones que están descriptas para membranas celulares lo que implica que ambos péptidos se encontrarían en la membrana formando parte de ella. Es importante el análisis del cambio de estabilidad de una membrana cuando en el medio se encuentran dichas moléculas. Por manipulación de vesículas gigantes mediante la técnica de aspiración con micropipeta, se puede afirmar que ambos péptidos disrumpen bicapas lipídicas formadas por POPC o por una mezcla ternaria de lípidos tal como la que se ha estudiado (POPC/SM/Chol 1:1:1). Estos resultados son importantes en el sentido de que era desconocido que AB 1-40 y AB 1-42 afectan la estabilidad y la cohesión de lípidos en bicapas lipídicas. Nuestros resultados muestran que cuando una vesícula gigante unilamelar es expuesta en una solución con ambos péptidos, la membrana es totalmente desestabilizada. En el caso de vesículas de POPC la presión crítica de lisís disminuye de 9 mN/m a 0,3 mN/m, mientras que vesículas formadas por (POPC/SM/Choi 1:1:1) dicha tensión baja de 12 mN/m a 4 mN/m y como se puede observar en la figura 3 B, la membrana se hace más elástica ya que una prolongación de la misma se manifiesta en la micropipeta cuando una presión es aplicada. Dicha prolongación también significa la inserción de la proteína en la bícapa.

Péptidos sintéticos beta-amilode 1-40 y 1-42, propiedades de superficie e interacción con modelos de membranas biológicas

Dados los resultados obtenidos en nuestros estudios, es posible afirmar que AB 1-40 y AB 1-42 conforman capas monomoleculares insolubles en interfases agua-aire con una alta estabilidad frente al trabajo de compresión. La alta estabilidad de un film peptídico en una interfase agua-aire y los bajos potencíales de superficie pueden deberse a que dichas moléculas estén adoptando una estructura del tipo p en la misma. Sin embargo por las áreas moleculares medidas hace sugerir que parte de la secuencia podría estar adoptando una estructura del tipo helicoidal. Esto también se puede interpretar por los valores de los potenciales de superficie, ya que, si bien son bajos, no son tan pequeños en comparación con péptidos que adoptan totalmente una estructura del tipo p. El estudio de las propiedades de superficie de ambos péptidos en presencia de cationes tales como Cu , Zn y Fe arrojó como resultado que dichos metales no afectan considerablemente la estabilidad de las capas monomoleculares de estas proteínas pero sí alteran levemente el potencial de superficie. Esto podría deberse a la unión del metal por la proteína ya que, como se conoce, AB 1-40 y AB 1-42 forman complejos con dichos cationes reduciéndolos lo que genera la formación de peróxido de hidrógeno, lo que ocasionaría un stress oxídativo en interfases. De los estudios de films mezcla con lípidos hay que recalcar que AB 1-40 y Ab 1-40 presentan misciblidad parcial con los lípidos estudiados y la estabilidad de la interfase a presiones laterales donde la fase rica en péptido no colapso se encuentra dentro del rango de presiones que están descriptas para membranas celulares lo que implica que ambos péptidos se encontrarían en la membrana formando parte de ella. Es importante el análisis del cambio de estabilidad de una membrana cuando en el medio se encuentran dichas moléculas. Por manipulación de vesículas gigantes mediante la técnica de aspiración con micropipeta, se puede afirmar que ambos péptidos disrumpen bicapas lipídicas formadas por POPC o por una mezcla ternaria de lípidos tal como la que se ha estudiado (POPC/SM/Chol 1:1:1). Estos resultados son importantes en el sentido de que era desconocido que AB 1-40 y AB 1-42 afectan la estabilidad y la cohesión de lípidos en bicapas lipídicas. Nuestros resultados muestran que cuando una vesícula gigante unilamelar es expuesta en una solución con ambos péptidos, la membrana es totalmente desestabilizada. En el caso de vesículas de POPC la presión crítica de lisís disminuye de 9 mN/m a 0,3 mN/m, mientras que vesículas formadas por (POPC/SM/Choi 1:1:1) dicha tensión baja de 12 mN/m a 4 mN/m y como se puede observar en la figura 3 B, la membrana se hace más elástica ya que una prolongación de la misma se manifiesta en la micropipeta cuando una presión es aplicada. Dicha prolongación también significa la inserción de la proteína en la bícapa.