Controlling the Adsorption of ß-Glucosidase onto Wrinkled SiO2 Nanoparticles To Boost the Yield of Immobilization of an Efficient Biocatalyst.

ß-Glucosidase (BG) catalyzes the hydrolysis of cellobiose to glucose, a substrate for fermentation to produce the carbon-neutral fuel bioethanol. Enzyme thermal stability and reusability can be improved through immobilization onto insoluble supports. Moreover, nanoscaled matrixes allow for preserving high reaction rates. In this work, BG was physically immobilized onto wrinkled SiO2 nanoparticles (WSNs). The adsorption procedure was tuned by varying the BG:WSNs weight ratio to achieve the maximum controllability and maximize the yield of immobilization, while different times of immobilization were monitored. Results show that a BG:WSNs ratio equal to 1:6 wt/wt provides for the highest colloidal stability, whereas an immobilization time of 24 h results in the highest enzyme loading (135 mg/g of support) corresponding to 80% yield of immobilization. An enzyme corona is formed in 2 h, which gradually disappears as the protein diffuses within the pores. The adsorption into the silica structure causes little change in the protein secondary structure. Furthermore, supported enzyme exhibits a remarkable gain in thermal stability, retaining complete folding up to 90 °C. Catalytic tests assessed that immobilized BG achieves 100% cellobiose conversion. The improved adsorption protocol provides simultaneously high glucose production, enhanced yield of immobilization, and good reusability, resulting in considerable reduction of enzyme waste in the immobilization stage.

Triple Negative Breast Cancer Preclinical Therapeutic Management by a Cationic Ruthenium-Based Nucleolipid Nanosystem.

Based on compelling preclinical evidence concerning the progress of our novel ruthenium-based metallotherapeutics, we are focusing research efforts on challenging indications for the treatment of invasive neoplasms such as the triple-negative breast cancer (TNBC). This malignancy mainly afflicts younger women, who are black, or who have a BRCA1 mutation. Because of faster growing and spreading, TNBC differs from other invasive breast cancers having fewer treatment options and worse prognosis, where existing therapies are mostly ineffective, resulting in a large unmet biomedical need. In this context, we benefited from an experimental model of TNBC both in vitro and in vivo to explore the effects of a biocompatible cationic liposomal nanoformulation, named HoThyRu/DOTAP, able to effectively deliver the antiproliferative ruthenium(III) complex AziRu, thus resulting in a prospective candidate drug. As part of the multitargeting mechanisms featuring metal-based therapeutics other than platinum-containing agents, we herein validate the potential of HoThyRu/DOTAP liposomes to act as a multimodal anticancer agent through inhibition of TNBC cell growth and proliferation, as well as migration and invasion. The here-obtained preclinical findings suggest a potential targeting of the complex pathways network controlling invasive and migratory cancer phenotypes. Overall, in the field of alternative chemotherapy to platinum-based drugs, these outcomes suggest prospective brand-new settings for the nanostructured AziRu complex to get promising goals for the treatment of metastatic TNBC.

Investigating the Interaction of an Anticancer Nucleolipidic Ru(III) Complex with Human Serum Proteins: A Spectroscopic Study.

Ruthenium(III) complexes are very promising candidates as metal-based anticancer drugs, and several studies have supported the likely role of human serum proteins in the transport and selective delivery of Ru(III)-based compounds to tumor cells. Herein, the anticancer nanosystem composed of an amphiphilic nucleolipid incorporating a Ru(III) complex, which we named DoHuRu, embedded into the biocompatible cationic lipid DOTAP, was investigated as to its interaction with two human serum proteins thought to be involved in the mechanism of action of Ru(III)-based anticancer drugs, i.e., human serum albumin (HSA) and human transferrin (hTf). This nanosystem was studied in comparison with the simple Ru(III) complex named AziRu, a low molecular weight metal complex previously designed as an analogue of NAMI-A, decorated with the same ruthenium ligands as DoHuRu but devoid of the nucleolipid scaffold and not inserted in liposomal formulations. For this study, different spectroscopic techniques, i.e., Fluorescence Spectroscopy and Circular Dichroism (CD), were exploited, showing that DoHuRu/DOTAP liposomes can interact with both serum proteins without affecting their secondary structures.

Order vs. Disorder: Cholesterol and Omega-3 Phospholipids Determine Biomembrane Organization.

Lipid structural diversity strongly affects biomembrane chemico-physical and structural properties in addition to membrane-associated events. At high concentrations, cholesterol increases membrane order and rigidity, while polyunsaturated lipids are reported to increase disorder and flexibility. How these different tendencies balance in composite bilayers is still controversial. In this study, electron paramagnetic resonance spectroscopy, small angle neutron scattering, and neutron reflectivity were used to investigate the structural properties of cholesterol-containing lipid bilayers in the fluid state with increasing amounts of polyunsaturated omega-3 lipids. Either the hybrid 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine or the symmetric 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine were added to the mixture of the naturally abundant 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and cholesterol. Our results indicate that the hybrid and the symmetric omega-3 phospholipids affect the microscopic organization of lipid bilayers differently. Cholesterol does not segregate from polyunsaturated phospholipids and, through interactions with them, is able to suppress the formation of non-lamellar structures induced by the symmetric polyunsaturated lipid. However, this order/disorder balance leads to a bilayer whose structural organization cannot be ascribed to either a liquid ordered or to a canonical liquid disordered phase, in that it displays a very loose packing of the intermediate segments of lipid chains.

Structural Organization of Cardiolipin-Containing Vesicles as Models of the Bacterial Cytoplasmic Membrane.

The bacterial cytoplasmic membrane is the innermost bacterial membrane and is mainly composed of three different phospholipid species, i.e., phosphoethanolamine (PE), phosphoglycerol (PG), and cardiolipin (CL). In particular, PG and CL are responsible for the negative charge of the membrane and are often the targets of cationic antimicrobial agents. The growing resistance of bacteria toward the available antibiotics requires the development of new and more efficient antibacterial drugs. In this context, studying the physicochemical properties of the bacterial cytoplasmic membrane is pivotal for understanding drug-membrane interactions at the molecular level as well as for designing drug-testing platforms. Here, we discuss the preparation and characterization of PE/PG/CL vesicle suspensions, which contain all of the main lipid components of the bacterial cytoplasmic membrane. The vesicle suspensions were characterized by means of small-angle neutron scattering, dynamic light scattering, and electron paramagnetic spectroscopy. By combining solution scattering and spectroscopy techniques, we propose a detailed description of the impact of different CL concentrations on the structure and dynamics of the PE/PG bilayer. CL induces the formation of thicker bilayers, which exhibit higher curvature and are overall more fluid. The experimental results contribute to shed light on the structure and dynamics of relevant model systems of the bacterial cytoplasmic membrane.

Physicochemical Approach to Understanding the Structure, Conformation, and Activity of Mannan Polysaccharides.

Extracellular polysaccharides are widely produced by bacteria, yeasts, and algae. These polymers are involved in several biological functions, such as bacteria adhesion to surface and biofilm formation, ion sequestering, protection from desiccation, and cryoprotection. The chemical characterization of these polymers is the starting point for obtaining relationships between their structures and their various functions. While this fundamental correlation is well reported and studied for the proteins, for the polysaccharides, this relationship is less intuitive. In this paper, we elucidate the chemical structure and conformational studies of a mannan exopolysaccharide from the permafrost isolated bacterium Psychrobacter arcticus strain 273-4. The mannan from the cold-adapted bacterium was compared with its dephosphorylated derivative and the commercial product from Saccharomyces cerevisiae. Starting from the chemical structure, we explored a new approach to deepen the study of the structure/activity relationship. A pool of physicochemical techniques, ranging from small-angle neutron scattering (SANS) and dynamic and static light scattering (DLS and SLS, respectively) to circular dichroism (CD) and cryo-transmission electron microscopy (cryo-TEM), have been used. Finally, the ice recrystallization inhibition activity of the polysaccharides was explored. The experimental evidence suggests that the mannan exopolysaccharide from P. arcticus bacterium has an efficient interaction with the water molecules, and it is structurally characterized by rigid-rod regions assuming a 14-helix-type conformation.

Hybrid humic acid/titanium dioxide nanomaterials as highly effective antimicrobial agents against gram(-) pathogens and antibiotic contaminants in wastewater.

Humic acids (HAs) provide an important bio-source for redox-active materials. Their functional chemical groups are responsible for several properties, such as metal ion chelating activity, adsorption ability towards small molecules and antibacterial activity, through reactive oxygen species (ROS) generation. However, the poor selectivity and instability of HAs in solution hinder their application. A promising strategy for overcoming these disadvantages is conjugation with an inorganic phase, which leads to more stable hybrid nanomaterials with tuneable functionalities. In this study, we demonstrate that hybrid humic acid/titanium dioxide nanostructured materials that are prepared via a versatile in situ hydrothermal strategy display promising antibacterial activity against various pathogens and behave as selective sequestering agents of amoxicillin and tetracycline antibiotics from wastewater. A physicochemical investigation in which a combination of techniques were utilized, which included TEM, BET, 13C-CPMAS-NMR, EPR, DLS and SANS, shed light on the structure-property-function relationships of the nanohybrids. The proposed approach traces a technological path for the exploitation of organic biowaste in the design at the molecular scale of multifunctional nanomaterials, which is useful for addressing environmental and health problems that are related to water contamination by antibiotics and pathogens.

Synthesis and Characterization of Multifunctional Nanovesicles Composed of POPC Lipid Molecules for Nuclear Imaging.

The integration of nuclear imaging analysis with nanomedicine has tremendously grown and represents a valid and powerful tool for the development and clinical translation of drug delivery systems. Among the various types of nanostructures used as drug carriers, nanovesicles represent intriguing platforms due to their capability to entrap both lipophilic and hydrophilic agents, and their well-known biocompatibility and biodegradability. In this respect, here we present the development of a labelling procedure of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)-based liposomes incorporating an ad hoc designed lipophilic NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) analogue, derivatized with an oleic acid residue, able to bind the positron emitter gallium-68(III). Based on POPC features, the optimal conditions for liposome labelling were studied with the aim of optimizing the Ga(III) incorporation and obtaining a significant radiochemical yield. The data presented in this work demonstrate the feasibility of the labelling procedure on POPC liposomes co-formulated with the ad hoc designed NOTA analogue. We thus provided a critical insight into the practical aspects of the development of vesicles for theranostic approaches, which in principle can be extended to other nanosystems exploiting a variety of bioconjugation protocols.

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

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-sn-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.

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

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.

Fine-tuning the properties of the thrombin binding aptamer through cyclization: Effect of the 5'-3' connecting linker on the aptamer stability and anticoagulant activity.

A small library of cyclic TBA analogues (named cycTBA I-IV), obtained by covalently connecting its 5'- and 3'-ends with flexible linkers, has been synthesized with the aim of improving its chemical and enzymatic stability, as well as its anticoagulant properties. Two chemical procedures have been exploited to achieve the desired cyclization, based on the oxime ligation method (providing cycTBA I and II) or on Cu(I)-assisted azide-alkyne cycloaddition (CuAAC) protocols (for cycTBA III and IV), leading to analogues containing circularizing linkers with different chemical nature and length, overall spanning from 22 to 48 atoms. The resulting cyclic TBAs have been characterized using a variety of biophysical methods (UV, CD, gel electrophoresis, SE-HPLC analyses) and then tested for their serum resistance and anticoagulant activity under in vitro experiments. A fine-tuning of the length and flexibility of the linker allowed identifying a cyclic analogue, cycTBA II, with improved anticoagulant activity, associated with a dramatically stabilized G-quadruplex structure (ΔTm = +17 °C) and a 6.6-fold higher enzymatic resistance in serum compared to unmodified TBA.

Design, Synthesis and Characterization of Cyclic NU172 Analogues: A Biophysical and Biological Insight.

NU172-a 26-mer oligonucleotide able to bind exosite I of human thrombin and inhibit its activity-was the first aptamer to reach Phase II clinical studies as an anticoagulant in heart disease treatments. With the aim of favoring its functional duplex-quadruplex conformation and thus improving its enzymatic stability, as well as its thrombin inhibitory activity, herein a focused set of cyclic NU172 analogues-obtained by connecting its 5'- and 3'-extremities with flexible linkers-was synthesized. Two different chemical approaches were exploited in the cyclization procedure, one based on the oxime ligation method and the other on Cu(I)-assisted azide-alkyne cycloaddition (CuAAC), affording NU172 analogues including circularizing linkers with different length and chemical nature. The resulting cyclic NU172 derivatives were characterized using several biophysical techniques (ultraviolet (UV) and circular dichroism (CD) spectroscopies, gel electrophoresis) and then investigated for their serum resistance and anticoagulant activity in vitro. All the cyclic NU172 analogues showed higher thermal stability and nuclease resistance compared to unmodified NU172. These favorable properties were, however, associated with reduced-even though still significant-anticoagulant activity, suggesting that the conformational constraints introduced upon cyclization were somehow detrimental for protein recognition. These results provide useful information for the design of improved analogues of NU172 and related duplex-quadruplex structures.

Stability Is Not Everything: The Case of the Cyclisation of a Thrombin-Binding Aptamer.

With the aim of developing a new approach to obtain improved aptamers, a cyclic thrombin-binding aptamer (TBA) analogue (cycTBA) has been prepared by exploiting a copper(I)-assisted azide-alkyne cycloaddition. The markedly increased serum resistance and exceptional thermal stability of the G-quadruplex versus TBA were associated with halved thrombin inhibition, which suggested that some flexibility in the TBA structure was necessary for protein recognition.

On the pH-Modulated Ru-Based Prodrug Activation Mechanism.

The RuIII-based prodrug AziRu efficiently binds to proteins, but the mechanism of its release is still disputed. Herein, in order to test the hypothesis of a reduction-mediated Ru release from proteins, a Raman-assisted crystallographic study on AziRu binding to a model protein (hen egg white lysozyme), in two different oxidation states, RuII and RuIII, was carried out. Our results indicate Ru reduction, but the Ru release upon reduction is dependent on the reducing agent. To better understand this process, a pH-dependent, spectroelectrochemical surface-enhanced Raman scattering (SERS) study was performed also on AziRu-functionalized Au electrodes as a surrogate and simplest model system of RuII- and RuIII-based drugs. This SERS study provided a p Ka of 6.0 ± 0.4 for aquated AziRu in the RuIII state, which falls in the watershed range of pH values separating most cancer environments from their physiological counterparts. These experiments also indicate a dramatic shift of the redox potential E0 by >600 mV of aquated AziRu toward more positive potentials upon acidification, suggesting a selective AziRu reduction in cancer lumen but not in healthy ones. It is expected that the nature of the ligands (e.g., pyridine vs imidazole, present in well-known RuIII complex NAMI-A) will modulate the p Ka and E0, without affecting the underlying reaction mechanism.

Microplastic-induced damage in early embryonal development of sea urchin Sphaerechinus granularis.

Two microplastic sets, polystyrene (PS) and polymethyl methacrylate (PMMA), were tested for adverse effects on early life stages of Sphaerechinus granularis sea urchins. Microparticulate PS (10, 80 and 230 µm diameter) and PMMA (10 and 50 µm diameter) were tested on developing S. granularis embryos from 10 min post-fertilisation (p-f) to the pluteus larval stage (72 h p-f), at concentrations ranging from 0.1 to 5 mg L-1. Both PS and PMMA exposures resulted in significant concentration-related increase of developmental defects and of microplastic uptake in plutei. Moreover, embryo exposures to PS and PMMA (5 and 50 mg L-1) from 10 min to 5 h p-f resulted in a significant increase of cytogenetic abnormalities, expressed as significantly increased mitotic aberrations, while mitotoxicity (as % embryos lacking active mitoses) was observed in embryos exposed to PS, though not to PMMA. When S. granularis sperm suspensions were exposed for 10 min to PS or to PMMA (0.1-5 mg L-1), a significant decrease of fertilisation success was observed following sperm exposure to 0.1 mg L-1 PS, though not to higher PS concentrations nor to PMMA. Sperm pretreatment, however, resulted in significant offspring damage, as excess developmental defects in plutei, both following sperm exposure to PS and PMMA, thus suggesting transmissible damage from sperm pronuclei to the offspring. The overall results point to relevant developmental, cytogenetic and genotoxic effects of PS and PMMA microplastics to S. granularis early life stages, warranting further investigations of other microplastics and other target biota.

A predictive model for the diffusion of a highly non-ideal ternary system.

Diffusion plays a central part in many unit operations. The Maxwell-Stefan model is the dominant model for both gaseous and liquid diffusion. However, it was developed from the kinetic theory of gases, raising the question of whether it can be extended to non-ideal liquid systems. The dynamic fluctuation model is an alternative model based on the Cussler theory and predicts a smaller thermodynamic influence relative to the linear influence of the Maxwell-Stefan model due to dynamic concentration fluctuations. Since the dynamic fluctuation model, which uses the scaling factor α, had improved performance relative to the Maxwell-Stefan model for a wide range of binary systems, it is postulated that this improved performance should also be observed for a ternary system. In this work, the dynamic molecular fluctuation model was extended to a highly non-ideal ternary system, using the same scaling factor α, through matrix manipulation. Using self-diffusion data measured by NMR, mutual diffusion predictions of the developed model and the Maxwell-Stefan model were compared to experimental mutual diffusion data of the partially miscible system ethanol/toluene/n-decane. It is demonstrated that the dynamic fluctuation model gives improved predictions relative to the Maxwell-Stefan approach, consistent with previous observations on binary systems, showing that the reduced thermodynamic influence of the dynamic fluctuation model is an improvement. In addition, we show that the use of local mole fractions, to account for molecular association, in both the dynamic fluctuation and Maxwell-Stefan models, results in improved diffusion predictions for the ternary system. The results confirm that the dynamic fluctuation model improves predictions of mutual diffusion in liquid mixtures, suggesting a non-linear correction to the thermodynamic correction factor. The results also suggest that that the key assumptions in the Maxwell-Stefan model and its derivation, rooted in the kinetic theory of gases, are not entirely accurate for highly non-ideal liquid systems. The optimum α for the ternary system studied here is approximately 0.45, similarly to the optimum α of 0.40 to 0.80 for a range of binary systems previously studied, suggesting that the use of the α scaling factor, which is grounded in scaling laws theory, is of general validity.

Ionophores at work: Exploring the interaction of guanosine-based amphiphiles with phospholipid membranes.

An amphiphilic derivative of guanosine, carrying a myristoyl group at the 5'-position and two methoxy(triethylene glycol) appendages at the 2' and 3'-positions (1), endowed with high ionophoric activity, has been here studied in its interaction mode with a model lipid membrane along with its 5'-spin-labelled analogue 2, bearing the 5-doxyl-stearic in lieu of the myristic residue. Electron spin resonance spectra, carried out on the spin-labelled nucleolipid 2 in mixture with a DOPC/DOPG phospholipid bilayer, on one side, and on spin-labelled lipids mixed with 1, on the other, integrated with dynamic light scattering and neutron reflectivity measurements, allowed getting an in-depth picture of the effect of the ionophores on membrane structure, relevant to clarify the ion transport mechanism through lipid bilayers. Particularly, dehydration of lipid headgroups and lowering of both the local polarity and acyl chains order across the bilayer, due to the insertion of the oligo(ethylene glycol) chains in the bilayer hydrophobic core, have been found to be the main effects of the amphiphilic guanosines interaction with the membrane. These results furnish directions to rationally implement future ionophores design.

Decoration of Chondroitin Polysaccharide with Threonine: Synthesis, Conformational Study, and Ice-Recrystallization Inhibition Activity.

Several threonine (Thr)- and alanine (Ala)-rich antifreeze glycoproteins (AFGPs) and polysaccharides act in nature as ice recrystallization inhibitors. Among them, the Thr-decorated capsular polysaccharide (CPS) from the cold-adapted Colwellia psychrerythraea 34H bacterium was recently investigated for its cryoprotectant activity. A semisynthetic mimic thereof was here prepared from microbial sourced chondroitin through a four-step strategy, involving a partial protection of the chondroitin polysaccharide as a key step for gaining an unprecedented quantitative amidation of its glucuronic acid units. In-depth NMR and computational analysis suggested a fairly linear conformation for the semisynthetic polysaccharide, for which the antifreeze activity by a quantitative ice recrystallization inhibition assay was measured. We compared the structure-activity relationships for the Thr-derivatized chondroitin and the natural Thr-decorated CPS from C. psychrerythraea.

Flow-induced nanostructuring of gelled emulsions.

Although the phase behavior of emulsions has been thoroughly investigated, the effect of flow on emulsion morphology, which is relevant for many applications, is far from being fully elucidated. Here, we investigate an emulsion based on two common nonionic surfactants in a range of water concentration where complex and diverse microstructures are found at rest, such as multilamellar and bicontinuous phases. In spite of such complexity, once subjected to shear flow, all the emulsions investigated are characterized by thinning filaments which eventually break up into a concentrated suspension of micro-sized water-based droplets dispersed in a continuous oil phase. The so-formed droplets tend to align in string-like structures. The emulsions exhibit a yield stress, whose value can be estimated by the plug-core velocity profiles in pressure-driven capillary flow, thus providing evidence of weakly attractive interdroplet interactions. The latter are consistent with droplet clustering and percolation observed at rest. These results can also be relevant to the flow behavior of other liquid-liquid systems, such as polymer blends, where the flow-induced microstructure is under debate as well.

A thermodynamic signature of lipid segregation in biomembranes induced by a short peptide derived from glycoprotein gp36 of feline immunodeficiency virus.

The interactions between proteins/peptides and lipid bilayers are fundamental in a variety of key biological processes, and among these, the membrane fusion process operated by viral glycoproteins is one of the most important, being a fundamental step of the infectious event. In the case of the feline immunodeficiency virus (FIV), a small region of the membrane proximal external region (MPER) of the glycoprotein gp36 has been demonstrated to be necessary for the infection to occur, being able to destabilize the membranes to be fused. In this study, we report a physicochemical characterization of the interaction process between an eight-residue peptide, named C8, modeled on that gp36 region and some biological membrane models (liposomes) by using calorimetric and spectroscopic measurements. CD studies have shown that the peptide conformation changes upon binding to the liposomes. Interestingly, the peptide folds from a disordered structure (in the absence of liposomes) to a more ordered structure with a low but significant helix content. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) results show that C8 binds with high affinity the lipid bilayers and induces a significant perturbation/reorganization of the lipid membrane structure. The type and the extent of such membrane reorganization depend on the membrane composition. These findings provide interesting insights into the role of this short peptide fragment in the mechanism of virus-cell fusion, demonstrating its ability to induce lipid segregation in biomembranes.