Intersite communication in dimeric enzymes highlighted by structural and thermodynamic analysis of didansyltyrosine binding to thymidylate synthases.

Intersite communication in dimeric enzymes, triggered by ligand binding, represents both a challenge and an opportunity in enzyme inhibition strategy. Though often understestimated, it can impact on the in vivo biological mechansim of an inhibitor and on its pharmacokinetics. Thymidylate synthase (TS) is a homodimeric enzyme present in almost all living organisms that plays a crucial role in DNA synthesis and cell replication. While its inhibition is a valid strategy in the therapy of several human cancers, designing specific inhibitors of bacterial TSs poses a challenge to the development of new anti-infective agents. N,O-didansyl-l-tyrosine (DDT) inhibits both Escherichia coli TS (EcTS) and Lactobacillus casei TS (LcTS). The available X-ray structure of the DDT:dUMP:EcTS ternary complex indicated an unexpected binding mode for DDT to EcTS, involving a rearrangement of the protein and addressing the matter of communication between the two active sites of an enzyme dimer. Combining molecular-level information on DDT binding to EcTS and LcTS extracted from structural and FRET-based fluorometric evidence with a thermodynamic characterization of these events obtained by fluorometric and calorimetric titrations, this study unveiled a negative cooperativity between the DDT bindings to the two monomers of each enzyme dimer. This result, complemented by the species-specific thermodynamic signatures of the binding events, implied that communication across the protein dimer was triggered by the first DDT binding. These findings could challenge the conventional understanding of TS inhibition and open the way for the development of novel TS inhibitors with a different mechanism of action and enhanced efficacy and specificity.

Oxidases as Oxygen Scavengers in Hypoxic Conditions: A Kinetic Model.

A simple kinetic model allowed for the description of the observed decay of the oxygen content in hypoxic aqueous samples with and without headspace, in the presence of glucose oxidase (Glucox) or laccase and their substrates (glucose for Glucox and ABTS for Laccase). The experimental tests involved both the direct measurement of the oxygen content with a fluorescence-based probe and the indirect stopped-flow spectroscopic detection of colored compounds generated from suitable chromogenic reagents. The complete depletion of dissolved oxygen occurred in the no-headspace samples, whereas some residual oxygen remained in a steady state in the samples with headspace. Simple pseudo-first-order kinetics was adequate to describe the behavior of the system, as long as oxygen was the rate-limiting compound, i.e., in the presence of excess substrates. The values of the kinetic constants drawn from best-fit routines of the data from both experimental approaches were quite comparable. The oxygen residues in the samples with headspace seemed related to the low solubility of O2 in the aqueous phase, especially if compared with the large amount of oxygen in the headspace. The extent of such residue decreased by increasing the concentration of the enzyme. The kinetic model proposed in this paper can be of help in assembling suitable sensors to be used for food safety and quality control.

Impact of Thermal Treatment on the Starch-Protein Interplay in Red Lentils: Connecting Molecular Features and Rheological Properties.

Thermal treatments are widely applied to gluten-free (GF) flours to change their functionality. Despite the interest in using pulses in GF formulations, the effects of thermal treatment at the molecular level and their relationship with dough rheology have not been fully addressed. Raw and heat-treated red lentils were tested for starch and protein features. Interactions with water were assessed by thermogravimetric analysis and water-holding capacity. Finally, mixing properties were investigated. The thermal treatment of red lentils induced a structural modification of both starch and proteins. In the case of starch, such changes consequently affected the kinetics of gelatinization. Flour treatment increased the temperature required for gelatinization, and led to an increased viscosity during both gelatinization and retrogradation. Regarding proteins, heat treatment promoted the formation of aggregates, mainly stabilized by hydrophobic interactions between (partially) unfolded proteins. Overall, the structural modifications of starch and proteins enhanced the hydration properties of the dough, resulting in increased consistency during mixing.

Thermodynamic Evaluation of the Interactions between Anticancer Pt(II) Complexes and Model Proteins.

In this work, we have analysed the binding of the Pt(II) complexes ([PtCl(4'-phenyl-2,2':6',2″-terpyridine)](CF3SO3) (1), [PtI(4'-phenyl-2,2':6',2″-terpyridine)](CF3SO3) (2) and [PtCl(1,3-di(2-pyridyl)benzene) (3)] with selected model proteins (hen egg-white lysozyme, HEWL, and ribonuclease A, RNase A). Platinum coordination compounds are intensively studied to develop improved anticancer agents. In this regard, a critical issue is the possible role of Pt-protein interactions in their mechanisms of action. Multiple techniques such as differential scanning calorimetry (DSC), electrospray ionization mass spectrometry (ESI-MS) and UV-Vis absorbance titrations were used to enlighten the details of the binding to the different biosubstrates. On the one hand, it may be concluded that the affinity of 3 for the proteins is low. On the other hand, 1 and 2 strongly bind them, but with major binding mode differences when switching from HEWL to RNase A. Both 1 and 2 bind to HEWL with a non-specific (DSC) and non-covalent (ESI-MS) binding mode, dominated by a 1:1 binding stoichiometry (UV-Vis). ESI-MS data indicate a protein-driven chloride loss that does not convert into a covalent bond, likely due to the unfavourable complexes' geometries and steric hindrance. This result, together with the significant changes of the absorbance profiles of the complex upon interaction, suggest an electrostatic binding mode supported by some stacking interaction of the aromatic ligand. Very differently, in the case of RNase A, slow formation of covalent adducts occurs (DSC, ESI-MS). The reactivity is higher for the iodo-compound 2, in agreement with iodine lability higher than chlorine.

Influence of Free Fatty Acids on Lipid Membrane-Nisin Interaction.

The influence of free fatty acids (FFAs) on the nisin-membrane interaction was investigated through micro-DSC and fluorescence spectroscopy. A simple but informative model membrane was prepared (5.7 DMPC:3.8 DPPS:0.5 DOPC molar ratio) by considering the presence of different phospholipid headgroups in charge and size and different phospholipid tails in length and unsaturation level, allowing the discrimination of the combined interaction of nisin and FFAs with the single phospholipid constituents. The effects of six FFAs on membrane stability were evaluated, namely two saturated FFAs (palmitic acid and stearic acid), two monounsaturated FFAs (cis-unsaturated oleic acid and trans-unsaturated elaidic acid) and two cis-polyunsaturated FFAs (ω-6 linoleic acid and ω-3 docosahexaenoic acid). The results permitted assessment of a thermodynamic picture of such interactions which indicates that the peptide-membrane interaction does not overlook the presence of FFAs within the lipid bilayer since both FFAs and nisin are able to selectively promote thermodynamic phase separations as well as a general lipid reorganization within the host membrane. Furthermore, the magnitude of the effects may be different depending on the FFA chemical structure as well as the membrane lipid composition.

Thermogenic flux induced by lignoceric acid in peroxisomes isolated from HepG2 cells and from X-adrenoleukodystrophy and control fibroblasts.

This work analyzes the thermogenic flux induced by the very long-chain fatty acid (VLCFA) lignoceric acid (C24:0) in isolated peroxisomes. Specific metabolic alterations of peroxisomes are related to a variety of disorders, the most frequent one being the neurodegenerative inherited disease X-linked adrenoleukodystrophy (X-ALD). A peroxisomal transport protein is mutated in this disorder. Due to reduced catabolism and enhanced fatty acid (FA) elongation, VLCFA accumulates in plasma and in all tissues, contributing to the clinical manifestations of this disorder. During peroxisomal metabolism, heat is produced but it is considered lost. Instead, it is a form of energy that could play a role in molecular mechanisms of this pathology and other neurodegenerative disorders. The thermogenic flux induced by lignoceric acid (C24:0) was estimated by isothermal titration calorimetry in peroxisomes isolated from HepG2 cells and from fibroblasts obtained from patients with X-ALD and healthy subjects. Heat flux induced by lignoceric acid in HepG2 peroxisomes was exothermic, indicating normal peroxisomal metabolism. In X-ALD peroxisomes the heat flux was endothermic, indicating the requirement of heat/energy, possibly for cellular metabolism. In fibroblasts from healthy subjects, the effect was less pronounced than in HepG2, a kind of cell known to have greater FA metabolism than fibroblasts. Our hypothesis is that heat is not lost but it could act as an activator, for example on the heat-sensitive pathway related to TRVP2 receptors. To investigate this hypothesis we focused on peroxisomal metabolism, considering that impaired heat generation could contribute to the development of peroxisomal neurodegenerative disorders.

Advanced Drug Delivery Nanosystems for Shikonin: A Calorimetric and Electron Paramagnetic Resonance Study.

Drug delivery is considered a mature scientific and technological platform for producing innovative medicines with nanosystems composed of intelligent bio-materials that carry active pharmaceutical ingredients forming advanced drug delivery nanosystems (aDDnSs). Shikonin and its enantiomer alkannin are natural products that have been extensively studied in vitro and in vivo for, among others, their antitumor activity, and various efforts have been made to prepare shikonin-loaded drug delivery systems. This study is focused on chimeric aDDnSs and specifically on liposomal formulations combining three lipids (egg-phosphatidylcholine; dipalmitoyl phosphatidylcholine; and distearoyl phosphatidylcholine) and a hyperbranched polymer (PFH-64-OH). Furthermore, PEGylated liposomal formulations of all samples were also prepared. Calorimetric techniques and electron paramagnetic resonance were used to explore and evaluate the interactions and stability of the liposomal formulations, showing that the presence of hyperbranched polymers promote the overall stability of the chimeric aDDnSs based on the drug release profile enhancement. Furthermore, results showed that polyethylene glycol enhances drug stabilization inside the liposomes, forming a stable and promising carrier for shikonin with improved characteristics.

The crystal structure of the AgamOBP1•Icaridin complex reveals alternative binding modes and stereo-selective repellent recognition.

Anopheles gambiae Odorant Binding Protein 1 in complex with the most widely used insect repellent DEET, was the first reported crystal structure of an olfactory macromolecule with a repellent, and paved the way for OBP1-structure-based approaches for discovery of new host-seeking disruptors. In this work, we performed STD-NMR experiments to directly monitor and verify the formation of a complex between AgamOBP1 and Icaridin, an efficient DEET alternative. Furthermore, Isothermal Titration Calorimetry experiments provided evidence for two Icaridin-binding sites with different affinities (Kd = 0.034 and 0.714 mM) and thermodynamic profiles of ligand binding. To elucidate the binding mode of Icaridin, the crystal structure of AgamOBP1•Icaridin complex was determined at 1.75 Å resolution. We found that Icaridin binds to the DEET-binding site in two distinct orientations and also to a novel binding site located at the C-terminal region. Importantly, only the most active 1R,2S-isomer of Icaridin's equimolar diastereoisomeric mixture binds to the AgamOBP1 crystal, providing structural evidence for the possible contribution of OBP1 to the stereoselectivity of Icaridin perception in mosquitoes. Structural analysis revealed two ensembles of conformations differing mainly in spatial arrangement of their sec-butyl moieties. Moreover, structural comparison with DEET indicates a common recognition mechanism for these structurally related repellents. Ligand interactions with both sites and binding modes were further confirmed by 2D 1H-15N HSQC NMR spectroscopy. The identification of a novel repellent-binding site in AgamOBP1 and the observed structural conservation and stereoselectivity of its DEET/Icaridin-binding sites open new perspectives for the OBP1-structure-based discovery of next-generation insect repellents.

Interaction study between maltose-modified PPI dendrimers and lipidic model membranes.

The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers - OS) or completely (dense shell glycodendrimers - DS) modified with maltose residues. As a model membrane, two types of 100nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D2O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between maltose shell of glycodendrimers and surface membrane of liposome.

Crystal and solution studies of the "Plus-C" odorant-binding protein 48 from Anopheles gambiae: control of binding specificity through three-dimensional domain swapping.

Much physiological and behavioral evidence has been provided suggesting that insect odorant-binding proteins (OBPs) are indispensable for odorant recognition and thus are appealing targets for structure-based discovery and design of novel host-seeking disruptors. Despite the fact that more than 60 putative OBP-encoding genes have been identified in the malaria vector Anopheles gambiae, the crystal structures of only six of them are known. It is therefore clear that OBP structure determination constitutes the bottleneck for structure-based approaches to mosquito repellent/attractant discovery. Here, we describe the three-dimensional structure of an A. gambiae "Plus-C" group OBP (AgamOBP48), which exhibits the second highest expression levels in female antennae. This structure represents the first example of a three-dimensional domain-swapped dimer in dipteran species. A combined binding site is formed at the dimer interface by equal contribution of each monomer. Structural comparisons with the monomeric AgamOBP47 revealed that the major structural difference between the two Plus-C proteins localizes in their N- and C-terminal regions, and their concerted conformational change may account for monomer-swapped dimer conversion and furthermore the formation of novel binding pockets. Using a combination of gel filtration chromatography, differential scanning calorimetry, and analytical ultracentrifugation, we demonstrate the AgamOBP48 dimerization in solution. Eventually, molecular modeling calculations were used to predict the binding mode of the most potent synthetic ligand of AgamOBP48 known so far, discovered by ligand- and structure-based virtual screening. The structure-aided identification of multiple OBP binders represents a powerful tool to be employed in the effort to control transmission of the vector-borne diseases.

Protein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase.

Human thymidylate synthase is a homodimeric enzyme that plays a key role in DNA synthesis and is a target for several clinically important anticancer drugs that bind to its active site. We have designed peptides to specifically target its dimer interface. Here we show through X-ray diffraction, spectroscopic, kinetic, and calorimetric evidence that the peptides do indeed bind at the interface of the dimeric protein and stabilize its di-inactive form. The "LR" peptide binds at a previously unknown binding site and shows a previously undescribed mechanism for the allosteric inhibition of a homodimeric enzyme. It inhibits the intracellular enzyme in ovarian cancer cells and reduces cellular growth at low micromolar concentrations in both cisplatin-sensitive and -resistant cells without causing protein overexpression. This peptide demonstrates the potential of allosteric inhibition of hTS for overcoming platinum drug resistance in ovarian cancer.

Quantitative determination of alkannins and shikonins in endemic Mediterranean Alkanna species.

The optical antipodes alkannin/shikonin (A/S) and their esters are potent pharmaceutical substances found in the roots of 150 Boraginaceous species. This study estimated and compared total and free A/S content and A/S enantiomeric ratio in roots of 11 Alkanna species (A. corcyrensis, A. tinctoria, A. pindicola, A. orientalis, A. methanaea, A. calliensis, A. graeca, A. primuliflora, A. stribrnyi, A. sieberi and A. noneiformis) growing wild in various Greek regions, to compare with cultivated species. It also re-characterized the chirality of A/S commercial samples, since most of them were misnamed by the providers. Several Alkanna species were collected (groups 1 and 3) and botanically identified, whereas some Alkanna species were cultivated from collected seeds (group 2). Free A/S and derivatives were extracted from the dried roots of Alkanna species and analyzed by high performance liquid chromatography-diode array detection (HPLC-DAD). For total A/S content the hexane extracts of Alkanna roots were hydrolyzed and analyzed by HPLC-DAD. Chirality determination and A/S enantiomeric ratio estimation was performed for several commercial samples by polarimetry,chiral LC-DAD and circular dichroism studies. Quantitative analysis revealed that A/S content varied from one region to another even within the same species. Most of the cultivated samples contained greater amounts of free and total A/S compared with the wild ones, wheras no difference was observed in A/S enantiomeric ratio. All the Alkanna samples tested contain mainly alkannin derivatives. Some of the examined Alkanna species of the Greek flora that are endemic to the Mediterranean area could serve as alternative sources for medicinally valuable A/S derivatives. Most of the commercial A/S samples tested were misnamed in terms of chirality and re-characterized.

Valorization of citrus peel industrial wastes for facile extraction of extractives, pectin, and cellulose nanocrystals through ultrasonication: An in-depth investigation.

In this work we developed an eco-friendly valorisation of Citrus wastes (CWs), through a solvent-assisted ultrasonication extraction technique, thus having access to a wide range of bio-active compounds and polysaccharides, extremely useful in different industrial sectors (food, cosmetics, nutraceutical). Water-based low-amplitude ultrasonication was examined as a potential method for pectin extraction as well as polar and non-polar citrus extractives (CEs), among which hesperidin and triglycerides of 18 carbon fatty acids were found to be the most representative ones. In addition, citric acid:glycerol (1:4)-based deep eutectic solvent (DES) in combination with ultrasonic extraction was utilized to extract microcellulose (CMC), from which stable cellulose nanocrystals (CNCs) with glycerol-assisted high amplitude ultrasonication were obtained. The physical and chemical properties of the extracted polysaccharides (pectin, micro and nanocellulose) were analysed through DLS, ζ-potential, XRD, HP-SEC, SEM, AFM, TGA-DSC, FTIR, NMR, and PMP-HPLC analyses. The putative structure of the extracted citrus pectin (CP) was analysed and elucidated through enzyme-assisted hydrolysis in correlation with ESI-MS and monosaccharide composition. The developed extraction methods are expected to influence the industrial process for the valorisation of CWs and implement the circular bio-economy.

Molecular insights into the role of amylose/amylopectin ratio on gluten protein organization.

Waxy (WX) and high-amylose (HA) wheat flours have interesting functional and/or nutritional characteristics, but low technological properties compared to regular wheat. Here a set of three wheat lines, having different amylose content but sharing the same varietal background, were compared to shed light on the role of the amylose/amylopectin ratio on the protein conformational changes that lead to gluten formation. Despite the absence of differences in their protein profile, as also confirmed by thiolomic approaches, both WX and HA lines developed a weaker gluten than the control sample. The altered amylose/amylopectin ratio exerts a matrix effect establishing a competition for water with proteins, leading to a different protein structure and three-dimensional organization of the gluten network. These results add a piece to the understanding of the molecular aspects that oversee matrix effects on gluten formation in wheat, which description can be helpful for a rational optimization of the transformation process.

The structure of AgamOBP5 in complex with the natural insect repellents Carvacrol and Thymol: Crystallographic, fluorescence and thermodynamic binding studies.

Among several proteins participating in the olfactory perception process of insects, Odorant Binding Proteins (OBPs) are today considered valid targets for the discovery of compounds that interfere with their host-detection behavior. The 3D structures of Anopheles gambiae mosquito AgamOBP1 in complex with the known synthetic repellents DEET and Icaridin have provided valuable information on the structural characteristics that govern their selective binding. However, no structure of a plant-derived repellent bound to an OBP has been available until now. Herein, we present the novel three-dimensional crystal structures of AgamOBP5 in complex with two natural phenolic monoterpenoid repellents, Carvacrol and Thymol, and the MPD molecule. Structural analysis revealed that both monoterpenoids occupy a binding site (Site-1) by adopting two alternative conformations. An additional Carvacrol was also bound to a secondary site (Site-2) near the central cavity entrance. A protein-ligand hydrogen-bond network supplemented by van der Waals interactions spans the entire binding cavity, bridging α4, α6, and α3 helices and stabilizing the overall structure. Fluorescence competition and Differential Scanning Calorimetry experiments verified the presence of two binding sites and the stabilization effect on AgamOBP5. While Carvacrol and Thymol bind to Site-1 with equal affinity in the submicromolar range, they exhibit a significantly lower and distinct binding capacity for Site-2 with Kd's of ~7 µΜ and ~18 µΜ, respectively. Finally, a comparison of AgamOBP5 complexes with the AgamOBP4-Indole structure revealed that variations of ligand-interacting aminoacids such as A109T, I72M, A112L, and A105T cause two structurally similar and homologous proteins to display different binding specificities.

Influence of pH on indole-dependent heterodimeric interactions between Anopheles gambiae odorant-binding proteins OBP1 and OBP4.

Insect Odorant Binding Proteins (OBPs) constitute important components of their olfactory apparatus, as they are essential for odor recognition. OBPs undergo conformational changes upon pH change, altering their interactions with odorants. Moreover, they can form heterodimers with novel binding characteristics. Anopheles gambiae OBP1 and OBP4 were found capable of forming heterodimers possibly involved in the specific perception of the attractant indole. In order to understand how these OBPs interact in the presence of indole and to investigate the likelihood of a pH-dependent heterodimerization mechanism, the crystal structures of OBP4 at pH 4.6 and 8.5 were determined. Structural comparison to each other and with the OBP4-indole complex (3Q8I, pH 6.85) revealed a flexible N-terminus and conformational changes in the α4-loop-α5 region at acidic pH. Fluorescence competition assays showed a weak binding of indole to OBP4 that becomes further impaired at acidic pH. Additional Molecular Dynamic and Differential Scanning Calorimetry studies displayed that the influence of pH on OBP4 stability is significant compared to the modest effect of indole. Furthermore, OBP1-OBP4 heterodimeric models were generated at pH 4.5, 6.5, and 8.5, and compared concerning their interface energy and cross-correlated motions in the absence and presence of indole. The results indicate that the increase in pH may induce the stabilization of OBP4 by increasing its helicity, thereby enabling indole binding at neutral pH that further stabilizes the protein and possibly promotes the creation of a binding site for OBP1. A decrease in interface stability and loss of correlated motions upon transition to acidic pH may provoke the heterodimeric dissociation allowing indole release. Finally, we propose a potential OBP1-OBP4 heterodimer formation/disruption mechanism induced by pH change and indole binding.

High-amylose and Tongil type Korean rice varieties: physical properties, cooking behaviour and starch digestibility.

The National Institute of Crop Science, Rural Development Administration (RDA) of Korea is presently developing new rice varieties suitable for producing Western rice-based foods, such as risotto, a well-known Italian-style product. The study considered different milled rice from five Tongil-type and six Japonica-type varieties. Besides the biometric properties, cooking behaviour, starch properties, and in vitro digestibility of Korean rice samples were compared with those of the 'Carnaroli' Italian variety. The physicochemical traits of the Korean varieties extended over a vast range; the amylose content stood out (from 13.0 to 41.7%), influencing the hardness and stickiness of cooked samples, and their starch digestibility. Although none of the Korean varieties seemed to guarantee cooking performances for risotto similar to the 'Carnaroli' one, 'Saemimyeon' and 'Shingil' cvs were judged the best for this purpose up-to-now.

A new chimeric drug delivery nano system (chi-aDDnS) composed of PAMAM G 3.5 dendrimer and liposomes as doxorubicin's carrier. In vitro pharmacological studies.

Chimeric advanced Drug Delivery nano Systems (chi-aDDnSs) could be defined as mixed nanosystems due to the combination process of nanobiomaterials and can offer advantages as drug carriers. The role of the release modulator from the liposomal system is undertaken by the dendrimer molecules leading to new pharmacokinetic and, probably, pharmacological properties of the chimeric system. In this work, a conventional DOPC/DPPG liposomal system and a new chi-aDDnS composed of liposomes (DOPC/DPPG) incorporating PAMAM G3,5 has been developed, Doxorubicin (Dox) was loaded in the systems and the final formulations were lyophilized. The physicochemical (spectroscopic and calorimetric) investigation concerning the chi-aDDnS, revealed a strong interaction between both lipophilic and hydrophilic parts of the liposomal membrane and the dendrimer, with the induction of multiple energetic states. These states are probably the basis of higher Dox encapsulation and slower release rate compared to the respective conventional liposome. These results, in conjunction with the increase in TI observed in two investigated cancer cell lines (i.e., MB231 and MCF7), compared to the respective conventional liposomal system and to the free Dox, make this new chi-aDDnS the basic candidate for further in vivo investigations.

Grapevine stilbenoids as natural food preservatives: calorimetric and spectroscopic insights into the interaction with model cell membranes.

Food contamination with pathogenic microorganisms, such as Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus and Bacillus cereus, is a common health concern. Natural products, which have been the main source of antimicrobials for centuries, may represent a turning point in alleviating the antibiotic crisis, and plant polyphenolic compounds are considered a promising source for new antibacterial agents. Resveratrol and resveratrol-derived monomers and oligomers (stilbenoids) have been shown to exert a variegated pattern of efficacy as antimicrobials depending on both the polyphenols' structure and the nature of the microorganisms, and the bacterial cell membrane seems to be one of their primary targets.In this scenario and based on the thermodynamic information reported in the literature about cell membranes, this study aimed at the investigation of the direct interaction of selected stilbenoids with a simple but informative model cell membrane. Three complete stilbenoid "monomer/dimer/dehydro-dimer" sets were chosen according to different geometries and substitution patterns. Micro-DSC was performed on 2 : 3 DPPC : DSPC small unilamellar vesicles with incorporated polyphenols at physiological pH and the results were integrated using complementary NMR data. The study highlighted the molecular determinants and mechanisms involved in the stilbenoid-membrane interaction, and the results were well correlated with the microbiological evidence previously assessed.

Chicken ileal bile-acid-binding protein: a promising target of investigation to understand binding co-operativity across the protein family.

Protein-bile acid interactions are crucial microscopic events at the basis of both physiological and pathological biochemical pathways. BABPs (bile-acid-binding proteins) are intracellular transporters able to bind ligands with different stoichiometry, selectivity and co-operativity. The molecular determinants and energetics of interaction are the observables that connect the microscopic to the macroscopic frameworks. The present paper addresses the study and proposes a mechanism for the multi-site interaction of bile acids with chicken I-BABP (ileal BABP) with the aim of elucidating the determinants of ligand binding in comparison with homologous proteins from different species and tissues. A thermodynamic binding model describing two independent consecutive binding sites is derived from isothermal titration calorimetry experiments and validated on the basis of both protein-observed and ligand-observed NMR titration data. It emerges that a singly bound protein is relatively abundant at low ligand/protein molar ratios assessing the absence of strong co-operativity. Both the measured energetics of binding and the distributed protein chemical-shift perturbations are in agreement with a first binding event triggering a global structural rearrangement. The enthalpic and entropic contributions associated with binding of the first ligand indicate that the interaction increases stability and order of the bound protein. The results described in the present study point to the presence of a protein scaffold which is able to establish long-range communication networks, but does not manifest positive-binding co-operativity, as observed for the human protein. We consider chicken I-BABP a suitable model to address the molecular basis for a gain-of-function on going from non-mammalian to mammalian species.