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.

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.

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.

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.

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.

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.

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.

Hierarchy of interactions dictating the thermodynamics of real cell membranes: Following the insulin secretory granules paradigm up to fifteen-components vesicles.

A fifteen-components model membrane that reflected the 80 % of phospholipids present in Insulin Secretory Granules was obtained and thermodynamic exploitation was performed, through micro-DSC, in order to assess the synergic contributions to the stability of a mixed complex system very close to real membranes. Simpler systems were also stepwise investigated, to complete a previous preliminary study and to highlight a hierarchy of interactions that can be now summarized as phospholipid tail unsaturation > phospholipid tail length > phospholipid headgroup > membrane curvature. In particular, Small Unilamellar Vesicles (SUVs) that consisted in phospholipids with different headgroups (choline, ethanolamine and serine), was step by step considered, following inclusion of sphingomyelins and lysophosphatidylcholines together with a more complete fatty acids distribution characterizing the phospholipid bilayer of the Insulin Secretory Granules. The inclusion of cholesterol was finally considered and the influence of three FFAs (stearic, oleic and elaidic acids) was investigated in comparison with simpler systems, highlighting the magnitude of the effects on such a detailed membrane in the frame of Type 2 Diabetes Mellitus alterations.

pH-responsive chimeric liposomes: From nanotechnology to biological assessment.

The utilization of liposomes in biomedical applications has greatly benefited the diagnosis and treatment of various diseases. These biomimetic nano-entities have been very useful in the clinical practice as drug delivery systems in their conventional form, comprising lipids as structural components. However, the scientific efforts have recently shifted towards the development of more sophisticated nanotechnological platforms, which apply functional biomaterials, such as stimuli-responsive polymers, in order to aid the drug molecule targeting concept. These nanosystems are defined as chimeric/mixed, because they combine more than one different in nature biomaterials and their development requires intensive study through biophysical and thermodynamic approaches before they may reach in vivo application. Herein, we designed and developed chimeric liposomes, composed of a phospholipid and pH-responsive amphiphilic diblock copolymers and studied their morphology and behavior based on crucial formulation parameters, including biomaterial concentration, dispersion medium pH and polymer composition. Additionally, their interactions with biological components, pH-responsiveness and membrane thermodynamics were assessed. Finally, preliminary in vivo toxicity experiments of the developed nanosystems were carried out, in order to establish a future protocol for full in vivo evaluation. The results have been correlated with the properties of the chimeric nanosystems and highlight the importance of such approaches for designing and developing effective nanocarriers for biomedical applications.

Dissecting the effects of free fatty acids on the thermodynamic stability of complex model membranes mimicking insulin secretory granules.

A stepwise micro-DSC study of Small, Large and Giant Unilamellar Vesicles prepared as pure and mixed systems of DMPC, DPPC, DSPC and DOPC was performed, achieving the preparation of final model membranes whose phospholipid compositions represent the 75% in terms of the phospholipids tails and the 50% headgroups of the Insulin Secretory Granules (vesicles located in the pancreatic Langerhans ß-cells and which are responsible for insulin and amylin storage and secretion in response to nutrient intake). Moreover, the effect of Free Fatty Acids, whose levels are recurrently altered in diabetic and/or obese subjects, on the thermodynamic stability of the final membranes was eventually investigated. The results allowed to discriminate each single thermodynamic contribution among the main factors that dictate the overall thermodynamic stability of these complex unilamellar systems evidencing mainly entropic effects hierarchically summarized as phospholipid unsaturations > phospholipid tail length > membrane curvature. The effect of the Free Fatty Acids highlighted a strong stabilizing effect on the membranes as well as more pronounced phase segregations in the case of saturated acids (palmitic and stearic), whereas the opposite effect was observed in the case of an unsaturated one (oleic).