Combined computational and experimental study about the incorporation of phosphorus into the structure of graphene oxide.

Phosphorus-containing graphene-based hybrids are materials with outstanding properties for diverse applications. In this work, an easy route to produce phosphorus-graphene oxide hybrid materials is described, involving the use of variable amounts of H3PO4 and H2SO4 during the reaction of oxidation of a graphitic precursor. The physical and chemical features of the hybrids change significantly with the variation in the acid amounts used in the syntheses. XPS and solid-state 13C and 31P NMR results show that the hybrids contain large amounts of oxygen functional groups, with the phosphorus incorporation proceeding mostly through the formation of phosphate-like linkages and other functions with C-O-P bonds. The experimental findings are supported by DFT calculations, which allow the assessment of the energetics and the geometry of the interaction between phosphate groups and graphene-based models; these calculations are also used to predict the chemical shifts in the 31P and 13C NMR spectra of the models, which show good agreement with the experimentally observed solid-state NMR spectra.

Mechanistic insights into the recycling machine of the SNARE complex.

Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.

Nanostructured faujasite zeolite as metal ion adsorbent: kinetics, equilibrium adsorption and metal recovery studies.

The hydrothermal synthesis of nano-faujasite has been successfully performed and the effects of some crystallization parameters were investigated, along with the use of this material as a heavy-metal ion adsorbent. X-ray diffraction patterns have shown that the structure of the nano-faujasite is strongly dependent on both the crystallization time and the alkalinity of the synthesis medium. According to N2 physisorption, X-ray fluorescence, SEM/EDS, and solid state 29Si and 27Al NMR data, the produced nano-faujasite consists of a solid with low molar Si/Al ratio (1.7), with high availability of ion exchange sites and high surface area/small particle size, allowing easy diffusion of metal ions to adsorbent active sites. As a consequence, an excellent performance on removal of Cd2+, Zn2+ and Cu2+ ions was found for this solid. The adsorption capacity followed the order Cd2+ (133 mg·g-1) > Zn2+ (115 mg·g-1) > Cu2+ (99 mg·g-1), which agrees with the order of increasing absolute values of the hydration energy of the metal ions. Kinetic studies and adsorption isotherms showed that the metal ion removal takes place by ion exchange on the monolayer surface of the nano-faujasite. The electrochemical recovery of copper in metallic form exhibited an efficiency of 80.2% after 120 min, which suggests that this process can be adequately implemented for full-scale metal removal.

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes.

In presynaptic nerve terminals, complexin regulates spontaneous "mini" neurotransmitter release and activates Ca2+-triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using single-particle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca2+-independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents.

Towards reconstitution of membrane fusion mediated by SNAREs and other synaptic proteins.

Proteoliposomes have been widely used for in vitro studies of membrane fusion mediated by synaptic proteins. Initially, such studies were made with large unsynchronized ensembles of vesicles. Such ensemble assays limited the insights into the SNARE-mediated fusion mechanism that could be obtained from them. Single particle microscopy experiments can alleviate many of these limitations but they pose significant technical challenges. Here we summarize various approaches that have enabled studies of fusion mediated by SNAREs and other synaptic proteins at a single-particle level. Currently available methods are described and their advantages and limitations are discussed.

Munc18a does not alter fusion rates mediated by neuronal SNAREs, synaptotagmin, and complexin.

Sec1/Munc18 (SM) proteins are essential for membrane trafficking, but their molecular mechanism remains unclear. Using a single vesicle-vesicle content-mixing assay with reconstituted neuronal SNAREs, synaptotagmin-1, and complexin-1, we show that the neuronal SM protein Munc18a/nSec1 has no effect on the intrinsic kinetics of both spontaneous fusion and Ca(2+)-triggered fusion between vesicles that mimic synaptic vesicles and the plasma membrane. However, wild type Munc18a reduced vesicle association ∼50% when the vesicles bearing the t-SNAREs syntaxin-1A and SNAP-25 were preincubated with Munc18 for 30 min. Single molecule experiments with labeled SNAP-25 indicate that the reduction of vesicle association is a consequence of sequestration of syntaxin-1A by Munc18a and subsequent release of SNAP-25 (i.e. Munc18a captures syntaxin-1A via its high affinity interaction). Moreover, a phosphorylation mimic mutant of Munc18a with reduced affinity to syntaxin-1A results in less reduction of vesicle association. In summary, Munc18a does not directly affect fusion, although it has an effect on the t-SNARE complex, depending on the presence of other factors and experimental conditions. Our results suggest that Munc18a primarily acts at the prefusion stage.

Disassembly of all SNARE complexes by N-ethylmaleimide-sensitive factor (NSF) is initiated by a conserved 1:1 interaction between α-soluble NSF attachment protein (SNAP) and SNARE complex.

Vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase NSF (N-ethylmaleimide-sensitive factor) and the adaptor protein α-SNAP (soluble NSF attachment protein) disassemble all SNARE complexes formed throughout different pathways, but the effect of SNARE sequence and domain variation on the poorly understood disassembly mechanism is unknown. By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering. We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding. Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.

Processive ATP-driven substrate disassembly by the N-ethylmaleimide-sensitive factor (NSF) molecular machine.

SNARE proteins promote membrane fusion by forming a four-stranded parallel helical bundle that brings the membranes into close proximity. Post-fusion, the complex is disassembled by an AAA+ ATPase called N-ethylmaleimide-sensitive factor (NSF). We present evidence that NSF uses a processive unwinding mechanism to disassemble SNARE proteins. Using a real-time disassembly assay based on fluorescence dequenching, we correlate NSF-driven disassembly rates with the SNARE-activated ATPase activity of NSF. Neuronal SNAREs activate the ATPase rate of NSF by ∼26-fold. One SNARE complex takes an average of ∼5 s to disassemble in a process that consumes ∼50 ATP. Investigations of substrate requirements show that NSF is capable of disassembling a truncated SNARE substrate consisting of only the core SNARE domain, but not an unrelated four-stranded coiled-coil. NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism. We further investigated processivity using single-turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF. We propose a processive helicase-like mechanism for NSF in which ∼1 residue is unwound for every hydrolyzed ATP molecule.

Mannans: Structural carbohydrates produced during seed maturation in Euterpe edulis Martius, an Atlantic Forest species vulnerable to extinction.

Palm seedlings are visually selected from mature fruits in a slow process that leads to nonuniform germination and high embryo mortality. In this study, we determined the levels of monosaccharides, their crystallinity, and their role in the formation of Euterpe edulis endosperm during seed maturation. Seeds harvested from 108 to 262 days after anthesis (DAA) were analyzed morphologically, physiologically, and chemically to measure soluble and insoluble lignins, ashes, structural carbohydrates, degree of crystallinity, and endo-ß-mannanase. The seeds achieved maximum germination and vigor at 164 DAA. During the early stages, only compounds with a low structural order were formed. The contents of soluble and insoluble lignins, ashes, glucans, and galactans decreased during maturation. Those of mannans, the main structural carbohydrate in the endosperm, increased along with the degree of crystallinity, as suggested by a mannan-I-type X-ray diffraction pattern. Similarly, endo-ß-mannanase activity peaked at 262 DAA. The superior physiological outcome of seeds and seedlings at 164 DAA implies a 98-day shorter harvesting time. The state of mannans during seed maturation could be used as a marker to improve seedling production by E. edulis.

Coumarin/ß-Cyclodextrin Inclusion Complexes Promote Acceleration and Improvement of Wound Healing.

Coumarins have great pharmacotherapeutic potential, presenting several biological and pharmaceutical applications, like antibiotic, fungicidal, anti-inflammatory, anticancer, anti-HIV, and healing activities, among others. These molecules are practically insoluble in water, and for biological applications, it became necessary to complex them with cyclodextrins (CDs), which influence their bioavailability in the target organism. In this work, we studied two coumarins, and it was possible to conclude that there were structural differences between 4,7-dimethyl-2H-chromen-2-one (DMC) and 7-methoxy-4-methyl-2H-chromen-2-one (MMC)/ß-CD that were solubilized in ethanol, frozen, and lyophilized (FL) and the mechanical mixtures (MM). In addition, the inclusion complex formation improved the solubility of DMC and MMC in an aqueous medium. According to the data, the inclusion complexes were formed and are more stable at a molar ratio of 2:1 coumarin/ß-CD, and hydrogen bonds along with π-π stacking interactions are responsible for the better stability, especially for (MMC)2@ß-CD. In vivo wound healing studies in mice showed faster re-epithelialization and the best deposition of collagen with the (DMC)2@ß-CD (FL) and (MMC)2@ß-CD (FL) inclusion complexes, demonstrating clearly that they have potential in wound repair. Therefore, (DMC)2@ß-CD (FL) deserves great attention because it presented excellent results, reducing the granulation tissue and mast cell density and improving collagen remodeling. Finally, the protein binding studies suggested that the anti-inflammatory activities might exert their biological function through the inhibition of MEK, providing the possibility of development of new MEK inhibitors.

Complexin-1 enhances the on-rate of vesicle docking via simultaneous SNARE and membrane interactions.

In synaptic terminals, complexin is thought to have inhibitory and activating roles for spontaneous "mini" release and evoked synchronized neurotransmitter release, respectively. We used single vesicle-vesicle microscopy imaging to study the effect of complexin-1 on the on-rate of docking between vesicles that mimic synaptic vesicles and the plasma membrane. We found that complexin-1 enhances the on-rate of docking of synaptic vesicle mimics containing full-length synaptobrevin-2 and full-length synaptotagmin-1 to plasma membrane-mimicking vesicles containing full-length syntaxin-1A and SNAP-25A. This effect requires the C-terminal domain of complexin-1, which binds to the membrane, the presence of PS in the membrane, and the core region of complexin-1, which binds to the SNARE complex.

36 degrees step size of proton-driven c-ring rotation in FoF1-ATP synthase.

Synthesis of adenosine triphosphate ATP, the 'biological energy currency', is accomplished by F(o)F(1)-ATP synthase. In the plasma membrane of Escherichia coli, proton-driven rotation of a ring of 10 c subunits in the F(o) motor powers catalysis in the F(1) motor. Although F(1) uses 120 degrees stepping during ATP synthesis, models of F(o) predict either an incremental rotation of c subunits in 36 degrees steps or larger step sizes comprising several fast substeps. Using single-molecule fluorescence resonance energy transfer, we provide the first experimental determination of a 36 degrees sequential stepping mode of the c-ring during ATP synthesis.

Inclusion complex of ketoconazole and p-sulfonic acid calix[6]arene improves antileishmanial activity and selectivity against Leishmania amazonensis and Leishmania infantum.

Many previous studies presented the effectiveness of ketoconazole (KTZ) against leishmaniasis. However, the bioavailability and therapeutic efficacy of free KTZ are limited due to its low aqueous solubility. In this study, an inclusion complex (IC6HKTZ) was prepared with p-sulfonic acid calix[6]arene (CX6SO3H) to improve the solubility and efficacy of KTZ against Leishmania amazonensis and Leishmania infantum promastigotes. A linear increase in KTZ solubility as a function of CX6SO3H concentration was verified using the phase-solubility diagram. The resulting diagram was classified as AL-type and a 1:1 host-guest stoichiometry was assumed to prepare IC6HKTZ by freeze-drying. FTIR, TG/DSC, XRD, and solid-state 13C NMR spectroscopy analyses were performed to confirm the formation of IC6HKTZ. The solubility enhancement of KTZ by 120.00 µM CX6SO3H was about 95 times. The IC50 values of IC6HKTZ and free KTZ were 3.95 and 14.35 µM for Leishmania amazonensis and 6.74 and 17.47 µM for Leishmania infantum, respectively. The viability of DH82 macrophages was not affected by CX6SO3H. These results show that CX6SO3H is a new supramolecular carrier system that improves antileishmanial activities to KTZ for the treatment of cutaneous and visceral leishmaniasis.

Heterogeneous Fenton-like surface properties of oxygenated graphitic carbon nitride.

The photo-Fenton activity of graphitic carbon nitride (g-C3N4) has been widely studied, nevertheless, its Fenton-like catalytic behavior in the dark has not yet been demonstrated. In the present work, it is shown that oxygenated g-C3N4 obtained at different temperatures (500-600 °C) can degrade indigo carmine with hydrogen peroxide in the dark by a reaction similar to a conventional Fenton's reaction. Based on an extensive characterization of g-C3N4, we conclude that Fenton-like activity is directly related to the oxygenated functional groups on g-C3N4 structure, mainly by -OH functional groups. Oxygenated functional groups (e.g., hydroquinone-like groups) can reduce the H2O2 and generate oxidizing hydroxyl radicals, just like in the Fenton reaction performed by metals. In addition to new information on g-C3N4 surface reactivity revealed by this study, the metal-free oxygenated g-C3N4 catalyst may be an alternative to traditional metal catalysts used in Fenton-like reactions for advanced oxidation.

Cryo-EM structures of inhibitory antibodies complexed with arginase 1 provide insight into mechanism of action.

Human Arginase 1 (hArg1) is a metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and modulates T-cell-mediated immune response. Arginase-targeted therapies have been pursued across several disease areas including immunology, oncology, nervous system dysfunction, and cardiovascular dysfunction and diseases. Currently, all published hArg1 inhibitors are small molecules usually less than 350 Da in size. Here we report the cryo-electron microscopy structures of potent and inhibitory anti-hArg antibodies bound to hArg1 which form distinct macromolecular complexes that are greater than 650 kDa. With local resolutions of 3.5 Å or better we unambiguously mapped epitopes and paratopes for all five antibodies and determined that the antibodies act through orthosteric and allosteric mechanisms. These hArg1:antibody complexes present an alternative mechanism to inhibit hArg1 activity and highlight the ability to utilize antibodies as probes in the discovery and development of peptide and small molecule inhibitors for enzymes in general.

Physicochemical characterization and in vitro biological evaluation of solid compounds from furazolidone-based cyclodextrins for use as leishmanicidal agents.

The discovery of new drugs and dosage forms for the treatment of neglected tropical diseases, such as human and animal leishmaniasis, is gaining interest in the chemical, biological, pharmaceutical, and medical fields. Many pharmaceutical companies are exploring the use of old drugs to establishing new drug dosage forms and drug delivery systems, in particular for use in neglected diseases. The formation of complexes with cyclodextrins is widely used to improve the stability, solubility, and bioavailability of pharmaceutical drugs, as well as reduce both the toxicity and side effects of many of these drugs. The aim of this study was to characterize solid compounds obtained from the association between furazolidone (FZD) and ß-cyclodextrin (ß-CD) or hydroxypropyl-ß-cyclodextrin (HP-ß-CD). The solid compounds were prepared in molar ratios of 1:1 and 1:2 (drug:CD) by kneading and lyophilization. Molecular docking was used to predict the preferred relative orientation of FZD when bound in both studied cyclodextrins. The resulting solid compounds were qualitatively characterized by scanning electron microscopy (SEM), thermal analysis (DSC and TG/DTG), X-ray diffraction (XRD), Raman spectroscopy with image mapping (Raman mapping), and 13C nuclear magnetic resonance spectroscopy (13C NMR) in the solid state. The cytotoxicity of the compounds against THP-1 macrophages and the 50% growth inhibition (IC50) against Leishmania amazonensis promastigote forms were subsequently investigated using in vitro techniques. For all of the solid compounds obtained, the existence of an association between FZD and CD were confirmed by one or more characterization techniques (TG/DTG, DSC, SEM, XRD, RAMAN, and 13C NMR), particularly by a significant decrease in the crystallinity of these materials and a reduction in the melting enthalpy associated with furazolidone thermal events. The formation of more effective interactions occurred in the compounds prepared by lyophilization, in a 1:2 molar ratio of the two CDs studied. However, the formation of an inclusion complex was confirmed only for the solid compound obtained from HP-ß-CD prepared by lyophilization (LHFZD1:2). The absence of cytotoxicity on the THP-1 macrophage lineages and the leishmanicidal activity were confirmed for all compounds. MHFZD1:2 and LHFZD1:2 were found to be very active against promastigote forms of L. amazonensis, while all others were considered only active. These results are in line with the literature, demonstrating the existence of biological activity for associations between drugs and CDs in the form of complexes and non-complexes. All solid compounds obtained were found to be promising for use as leishmanicidal agents against promastigote forms of L. amazonensis.

Structure and regulation of the vacuolar ATPases.

The vacuolar (H(+))-ATPases (V-ATPases) are ATP-dependent proton pumps responsible for both acidification of intracellular compartments and, for certain cell types, proton transport across the plasma membrane. Intracellular V-ATPases function in both endocytic and intracellular membrane traffic, processing and degradation of macromolecules in secretory and digestive compartments, coupled transport of small molecules such as neurotransmitters and ATP and in the entry of pathogenic agents, including envelope viruses and bacterial toxins. V-ATPases are present in the plasma membrane of renal cells, osteoclasts, macrophages, epididymal cells and certain tumor cells where they are important for urinary acidification, bone resorption, pH homeostasis, sperm maturation and tumor cell invasion, respectively. The V-ATPases are composed of a peripheral domain (V(1)) that carries out ATP hydrolysis and an integral domain (V(0)) responsible for proton transport. V(1) contains eight subunits (A-H) while V(0) contains six subunits (a, c, c', c'', d and e). V-ATPases operate by a rotary mechanism in which ATP hydrolysis within V(1) drives rotation of a central rotary domain, that includes a ring of proteolipid subunits (c, c' and c''), relative to the remainder of the complex. Rotation of the proteolipid ring relative to subunit a within V(0) drives active transport of protons across the membrane. Two important mechanisms of regulating V-ATPase activity in vivo are reversible dissociation of the V(1) and V(0) domains and changes in coupling efficiency of proton transport and ATP hydrolysis. This review focuses on recent advances in our lab in understanding the structure and regulation of the V-ATPases.

Production of high-purity cellulose, cellulose acetate and cellulose-silica composite from babassu coconut shells.

Lignocellulosic biomass has been widely studied as a source of cellulose- and related products, attracting the great interest of researchers dealing with renewable energy sources, vegetable waste recycling and biomaterials. In this work, the babassu coconut shells (epicarp and endocarp) were used for the achievement of products such as cellulose, cellulose acetate and cellulose-silica composite, which were chemically and structurally characterized by solid-state 13C nuclear magnetic resonance spectroscopy and scanning electron microscopy, among other techniques. As this precursor also naturally contains a significant amount of silica, a composite containing cellulose fibers mixed with amorphous silica particles (with rosette-like shape) was also produced. Finally, the possibility of synthesis of cellulose acetate was also demonstrated, illustrating the plethora of potential applications of this important lignocellulosic residue for the production of cellulose-based materials of high technological interest.

ß-Cyclodextrin inclusion complexes with essential oils: Obtention, characterization, antimicrobial activity and potential application for food preservative sachets.

The current study aimed obtaining antimicrobial sachets that could be used as preservatives for foods. Basil (BEO) and Pimenta dioica (PDEO) essential oils (EOs) were analyzed by GC-FID and GC-MS and tested against the foodborne bacteria S. aureus, E. coli, L. monocytogenes, P. aeruginosa, S. Enteritidis, and the food-spoilage mold B. nivea. Then, inclusion complexes (ICs) with EOs and ß-cyclodextrin (ß-CD) were prepared as a strategy to reduce volatility and increase the release time of EOs. Eight ICs were prepared by kneading and freeze-drying methods, in two molar ratios, and have been characterized by complementary methods: FT-IR, thermal analysis (DSC and TG/DTG), powder XRD, and solid state 13C NMR. In vitro antimicrobial activities of ICs, both dispersed in agar and loaded in sachets, have also been investigated. Complexation was confirmed for all samples. PDEO-based ICs prepared by kneading method, at both molar ratios, displayed better in vitro antimicrobial activity. The obtained results strongly suggest a potential application of these ICs as natural antimicrobials.

Tethering polypeptides through bifunctional PEG cross-linking agents to probe protein function: application to ATP synthase.

Chemical crosslinking mediated by short bifunctional reagents has been widely used for determining physical relationships among polypeptides in multisubunit proteins, but less often for functional studies. Here we introduce the approach of tethering polypeptides by using bifunctional reagents containing a lengthy, flexible PEG linker as a form of crosslinking especially suited to functional analyses. The rotary molecular motor ATP synthase was used as a model subject. Single cysteine residues were introduced into selected positions of ATP synthase epsilon subunit, a component of the rotor subcomplex of the enzyme, and the unrelated maltose binding protein (MBP), then the two purified recombinant proteins were crosslinked by means of a dimaleimido-PEG cross-linking agent. Following purification, the epsilon-PEG-MBP was incorporated into membrane-bound ATP synthase by reconstitution with epsilon-depleted F(1)-ATPase and membrane vesicles that had been stripped of endogenous F(1). ATP synthase reconstituted using epsilon-PEG-MBP had reduced ATP hydrolytic activity that was uncoupled from the pumping of H(+), indicating the physical blockage of rotation of the gammaepsilonc(10) rotor by the conjugated MBP, whereas enzyme reconstituted with epsilon-PEG was normal. These results directly demonstrate the feasibility of studying mechanistic features of molecular motors through PEG-based conjugation of unrelated proteins. Since tethering polypeptides provides a means of maintaining proximity without directly specifying or modifying interactions, application of the general method to other types of protein functional studies is envisioned.