X-ray powder diffraction was combined, for the first time, with Nuclear Magnetic Resonance spectroscopy and direct infusion mass spectrometry to characterise fresh and brined grape leaves. Covariance analysis of data generated by the three techniques was performed with the aim to correlate information deriving from the solid part with those obtained for soluble metabolites. The results obtained indicate that crystalline components can be correlated to the metabolites contained in the grape leaves, paving the way to the use of X-ray diffraction analysis for food fingerprinting purposes. Moreover it was ascertained that, differently from most of the metabolites present in the fresh vine leaves, linolenic acid (an omega-3-fatty acid) and quercetin-3-O-glucuronide (a polyphenol metabolite) do not undergo sensible degradation during the brining process, which is used as preservative method for the grape leaves. (C) 2013 Elsevier Ltd. All rights reserved.

Multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy provides valuable structural information about adducts between naturally unfolded proteins and their ligands. These are often highly pharmacologically relevant. Unfortunately, the determination of the contributions to observed chemical shifts changes upon ligand binding is complicated. Here we present a tool that uses molecular dynamics (MD) trajectories to help interpret two-dimensional (2D) NMR data. We apply this tool to the naturally unfolded protein human a-synuclein interacting with dopamine, an inhibitor of fibril formation, and with its oxidation products in water solutions. By coupling 2D NMR experiments with MD simulations of the adducts in explicit water, the tool confirms with experimental data that the ligands bind preferentially to (YEMPS129)-Y-123 residues in the C-terminal region and to a few residues of the so-called NAC region consistently. It also suggests that the ligands might cause conformational rearrangements of distal residues located at the N-terminus. Hence, the performed analysis provides a rationale for the observed changes in chemical shifts in terms of direct contacts with the ligand and conformational changes in the protein.

Introduction: Prothrombin deficiency is a very rare disorder caused by mutations in the F2 gene that generate hypoprothrombinemia or dysprothrombinemia and is characterized by bleeding manifestations that can vary from clinically irrelevant to life-threatening.

New methods have been recently developed to improve the structure solution of macromolecules by ab initio (Patterson or Direct Methods) and non ab initio (Molecular Replacement) approaches. Phasing proteins at non-atomic resolution is still a challenge for any ab initio method. The combined use of different algorithms [Patterson deconvolution and superposition techniques, cross-correlation function (C-Map), the VLD (Vive la Difference) approach included in the Direct Space Refinement (DSR) procedure, a new probabilistic formula estimating triplet invariants and capable of exploiting a model electron density maps, the FREE LUCH extrapolation method, a new FOM to identify the correct solution] allow to overcome the lack of experimental information. The new methods have been applied to a large number of protein diffraction data with resolution up to 2.1Å, under the condition that Ca or heavier atoms are in the structure. Results show that solving proteins at limited resolution is a feasible task, achievable even by new Direct Methods algorithms, against the traditional common believe that atomic resolution is a necessary condition for the success of a direct ab initio phasing process.A new procedure (REVAN) , aiming at solving protein structures via Molecular Replacement and density guided optimization algorithms, has been assembled. It combines a variety of programs (REMO09, REFMAC, COOT) and algorithms (Cowtan-EDM, DSR, VLD, FREE LUNCH), and can successfully lead to the structure solution also when the sequence identity between target and model structures is smaller than 0.30 and data resolution up to ~ 3Å. The application to a wide set of test structures (including difficult cases proposed by DiMaio et al. (2011), solved by using MR procedures together with energy guided programs) suggests that REVAN is quite effective even far from atomic resolution and, in combination with EDM techniques and sequence mutation algorithms, it is able to efficiently extend and refine the set of phases, reducing its average error.The final step of the automatic solving process (ab initio or MR approaches) is the application of an Automated Model Building program (i.e. Buccaneer, Nautilus, ARP-wARP or Phenix-Autobuild) in order to recover the correct structure. Results suggest that the quality of the phases at the end of the phasing process is good enough to lead the AMB program to success.These new efficient procedures are implemented in the current version of the software package SIR2014.

Humankind is in great need of new energy sources. The use of solar radiation for powering the planet would fulfil the energy requirements of Earth's inhabitants as well as greatly mitigate tension flares arising from the uneven distribution of fossil fuels and environmental problems associated to their extraction procedures. How to proceed than? Easy to say! Mother Nature is inspiring: all life on earth is based on the conversion of the solar radiation into high energy molecules, including gas and oil human beings are consuming these days, by mean of the so-called primary photoconverters, i.e. the photosynthetic organisms, plants, algae and some kind of bacteria. So, let's learn from Nature and assemble in our laboratories artificial systems capable of exploiting solar energy for photocatalysis and electrical energy production, i.e. mimic photosynthesis. Not an easy task of course, but a large number of laboratory are heavily involved since the last 25 years in the field of artificial photosynthesis and are obtaining encouraging results. The photosynthetic apparatus used by photosynthetic organisms to convert solar energy and drive their metabolism is the photochemical core where photoconversion takes place, and is constituted by a protein portion allocating several pigments directly involved in the harvesting of solar light and in the subsequent sequence of electron transfer reactions which eventually lead to the formation of an electron-hole couple to be used for any energy requiring process. In artificial photosynthesis the role of the protein scaffold in often ignored and attention is devoted to assembly molecular system for optimising light harvest and electron-transfer reactions, focussing to the "less-complex" portion of the photosynthetic apparatus. What would be a different paradigm in artificial photosynthesis? Assemble artificial photoconverters using genuine natural components formed by hybrid organic-biologic systems. The hybrids have a central protein, the so-called photosynthetic reaction center (RC) that converts sunlight into a charge-separated state having a lifetime sufficient to allow ancillary chemistry to take place. The RCs can be eventually garnished with opportune organic moieties to be used for different applications.The state of the art of these hybrid organic-biologic photosynthetic assemblies will be reviewed.

alpha-Helices are peculiar atomic arrangements characterizing protein structures. Their occurrence can be used within crystallographic methods as minimal a priori information to drive the phasing process towards solution. Recently, brute-force methods have been developed which search for all possible positions of alpha-helices in the crystal cell by molecular replacement and explore all of them systematically. Knowing the alpha-helix orientations in advance would be a great advantage for this kind of approach. For this purpose, a fully automatic procedure to find alpha-helix orientations within the Patterson map has been developed. The method is based on Fourier techniques specifically addressed to the identification of helical shapes and operating on Patterson maps described in spherical coordinates. It supplies a list of candidate orientations, which are then refined by using a figure of merit based on a rotation function calculated for a template polyalanine helix oriented along the current direction. The orientation search algorithm has been optimized to work at 3 A resolution, while the candidates are refined against all measured reflections. The procedure has been applied to a large number of protein test structures, showing an overall efficiency of 77% in finding alpha-helix orientations, which decreases to 48% on limiting the number of candidate solutions (to 13 on average). The information obtained may be used in many aspects in the framework of molecular-replacement phasing, as well as to constrain the generation of models in computational modelling programs. The procedure will be accessible through the next release of IL MILIONE and could be decisive in the solution of new unknown structures.

A method is presented to determine the helix orientation starting from X ray diffraction data. Our method is based on the periodicity properties of alpha helix and on the analysis of the properties of the corresponding Patterson function, obtained directly from raw crystallographic intensities. Knowledge of helix orientation is a useful information within the structure solution process of proteins. © 2011 IEEE.

Hydrogel composite membranes (HCMs) are used as novel mineralization platforms for the bioinspired synthesis of CaCO3 superstructures. A comprehensive statistical analysis of experimental results revealed quantitative relationships between crystallization conditions and crystal texture and the strong selectivity toward complex morphologies when monomers bearing carboxyl and hydroxyl groups are used together in the hydrogel synthesis in HCMs.

Gold nanoparticles exhibit unique electronic, optical, and catalytic properties that are different from those of bulk metal and have several applications in optoelectronics, imaging technology, catalysis, and drug delivery. Currently, there is a growing need to develop eco-friendly nanoparticle synthesis processes using living organisms, such as bacteria, fungi and algae. In particular, microorganisms are well known to protect themselves from metal ion stress either by intracellular-segregation mechanism or by secreting them into the external medium. This defensive behaviour can be exploited to obtain a more efficient fabrication of advanced functional nanomaterials than chemical synthesis routes: biological syntheses do not require hazardous organic solvents and surfactants , and can work at environmental temperature and pressure, preserving high selectivity and reproducibility.Rhodobacter sphaeroides is a facultative phototrophic anoxygenic proteobacterium known for its capacity to grow under a wide range of environmental conditions, with promising applications in bioremediation [1, 2].The response of the photosynthetic bacterium Rhodobacter sphaeroides to gold exposure and its reducing capability of Au(III) to produce stable Au(0) nanoparticles is reported in this study. The properties of prepared nanoparticles were characterized by UV-Visible (UV-Vis) spectroscopy, Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy, Transmission Electron Microscopy (TEM), X-ray Photoelectron Spectroscopy (XPS), X-ray Fluorescence Spectrometry (XRF) and X-ray Absorption Spectroscopy (XAS) measurements. Gold nanoparticles (AuNPs) were spherical in shape with an average size of 10±3 nm. Based on our experiments, the particles were likely fabricated by the aid of reducing sugars present in the bacterial cell membrane and were capped by a protein/peptide coat. The nanoparticles were hydrophilic and resisted to aggregation for several months. Gold nanoparticles were also positively tested for their catalytic activity in nitroaromatic compounds degradation.

The approach of combining pyrolysis or pyrogasification processes for energy production with biochar addition to soil takes advantage of biochar's proven ability to retain cations, remove CO2 from atmosphere, regenerate degraded lands and reduce environmental pollution. To evaluate the full potential of biochar as soil amendment, precise knowledge of its structural features, related to the thermochemical process conditions, and adsorptive properties, related to the minerals absorbed during crop cycles, is needed. To this aim, X­ray powder diffraction has been used to analyze sub­micrometric properties of biochar and biochar added soil. The structural properties have been monitored during the growing cycle of zucchini crop (Cucurbita pepo L.) in an alkaline soil from South of Italy. Diffraction patterns taken before, during and after the zucchini crop cycle have been analyzed by qualitative, quantitative and morphological analyses. The crystalline content of zucchini leaves and fruits has been also determined. X­ray powder diffraction profiles of soil and soil/biochar mixtures have been compared by using multivariate analysis.

X-ray diffraction methods in general provide a representation of the average structure, thus allowing only limited chemical selectivity. As recently shown [D. Chernyshov, et al., Acta Crystallogr., Sect. A: Found. Crystallogr., 2011, 67, 327], some structural information on a subset of atoms can be obtained using the modulation enhanced diffraction (MED), thus providing a new tool that is able to enhance selectivity in diffraction. MED uses a periodic stimulus supplied in situ on a crystal while diffraction data are collected continuously during one or more stimulation periods. Such large data sets can then be treated by different methods. Herein, we present and compare phase sensitive detection (PSD) and principal component analysis (PCA) for in situ X-ray powder diffraction (XRPD) data treatment. The application of PCA to MED data is described for the first time in the present paper. Simulated and experimental MED powder data were produced using an MFI zeolite as a static spectator in which Xe, acting as the active species, is adsorbed and desorbed in a periodic manner. By demodulating the simulated and experimental data, MED allowed the powder diffraction pattern of the responding scattering density to be obtained and enabled the selective extraction of crystallographic information on Xe by solving the crystal structure of the active species independently of the static zeolite framework. The "real world" experiments indicated that the PSD-MED approach has some limitations related to the degree of fulfilment of some theoretical assumptions. When applied to in situ XRPD data, PCA, despite being based on blind statistical analysis, gave results similar to those obtained by PSD (based on Fourier analysis) for simulated data. Moreover, PCA is complementary to PSD thanks to its capability of gathering information on the Xe substructure even in the presence of a non-periodic stimulus, i.e. using the most simple stimulus shape as a single temperature ramp. In particular, PC1 results are able to perfectly reproduce the corresponding 1? signal from a traditional PSD analysis. Moreover PCA can be applied directly to raw non periodic XRPD data, opening the possibility of using it during an "in situ" experiment. PCA can thus be envisaged as a very useful, fast and efficient tool to improve data collection and maximize data quality. To date, however, PSD remains superior for substructure solution from the analysis of 2? demodulated data.

Cobalt is an important oligoelement required for bacteria; if present in high concentration, exhibits toxic effects that, depending on the microor-ganism under investigation, may even result in growth inhibition. The photosynthetic bacterium Rhodobacter (R.) sphaeroides tolerates high cobalt concentration and bioaccumulates Co+2 ion, mostly on the cellular surface. Very little is known on the chemical fate of the bioaccumulated cobalt, thus an X-ray absorption spectroscopy investigation was conducted on R. sphaeroides cells to gain structural insights into the Co+2 binding to cellular components. X-ray absorption near-edge spectroscopy and extended X-ray absorption fine structure measurements were performed on R. sphaeroides samples containing whole cells and cell-free fractions obtained from cultures exposed to 5 mM Co+2. An octahedral coordination geometry was found for the cobalt ion, with six oxygen-ligand atoms in the first shell. In the soluble portion of the cell, cobalt was found bound to carboxylate groups, while a mixed pattern containing equivalent amount of two sulfur and two carbon atoms was found in the cell envelope fraction, suggesting the presence of carboxylate and sulfonate metal-binding functional groups, the latter arising from sulfolipids ofthe cell envelope.

Human (Hu) familial prion diseases are associated with about 40 point mutations of the gene coding for the prion protein (PrP). Most of the variants associated with these mutations are located in the globular domain of the protein. We performed 50 ns of molecular dynamics for each of these mutants to investigate their structure in aqueous solution. Overall, 1.6 ?s of molecular dynamics data is presented. The calculations are based on the AMBER(parm99) force field, which has been shown to reproduce very accurately the structural features of the HuPrP wild type and a few variants for which experimental structural information is available. The variants present structural determinants different from those of wild-type HuPrP and the protective mutation HuPrP(E219K-129M). These include the loss of salt bridges in ?(2)-?(3) regions and the loss of ?-stacking interactions in the ?(2)-?(2) loop. In addition, in the majority of the mutants, the ?(3) helix is more flexible and Y169 is more solvent exposed. The presence of similar traits in this large spectrum of mutations hints to a role of these fingerprints in their known disease-causing properties. Overall, the regions most affected by disease-linked mutations in terms of structure and/or flexibility are those involved in the pathogenic conversion to the scrapie form of the protein and in the interaction with cellular partners. These regions thus emerge as optimal targets for antibody- and ligand-binding studies

Previously we presented the purification, biochemical characterization, and cloning of a cationic peroxidase isoenzyme (CysPrx) from artichoke (Cynara cardunculus subsp scolymus (L.) Hegi) leaves. The protein was shown to have some interesting properties, suggesting that CysPrx could be a considered as a potential candidate for industrial application. In addition, from the CysPrx sequence, two full-lengh cDNAs: CysPrx1 and CysPrx2, differing for three amino acids, were isolated. A three-dimensional model was predicted from CysPrx1 by homology modeling, using two different computational tools. Herein we discuss the roles of particular amino acid residues and structural motifs or regions of both deduced sequences with the aim to find new understandings between the new plant peroxidase isoenzymes and their physiological substrates. Additionally, the obtained information may lead to new methods for improving the stability of the enzyme in several processes of biotechnological interest for peroxidase applications.

SIR2014 is the latest program of the SIR suite for crystal structure solution of small, medium and large structures. A variety of phasing algorithms have been implemented, both ab initio (standard or modern direct methods, Patterson techniques, Vive la Différence) and non-ab initio (simulated annealing, molecular replacement). The program contains tools for crystal structure refinement and for the study of three-dimensional electron-density maps via suitable viewers.

The metal-binding ability of human ubiquitin (hUb) has been probed towards a selection of biologically relevant metal-ions and complexes. Different techniques have been used to obtain crystals suitable for crystallographic analysis. In a first type of experiments, crystals of hUb have been soaked in solutions containing copper(II) acetate and two metallodrugs: Zeise's salt and cisplatin. Zeise's salt is used in a test for hepatitis while cisplatin is one of the most powerful anticancer drugs in clinical use. Zeise's salt reacts smoothly with hUb crystals affording an adduct with three platinum residues per protein molecule: Pt3-hUb. In contrast, copper(II) acetate and cisplatin were found to be unreactive for contact times up to one hour and to cause degradation of the hUb crystals for longer times. In a second type of experiments, human ubiquitin was co-crystallized with solutions of copper(II) and zinc(II) acetate and with cisplatin. Zinc(II) acetate gives, at low metal-to-protein molar ratio, crystals containing one metal ion per three molecules of protein, Zn-hUb3 (already reported in a previous work), while at high metal-to-protein ratio (70:1) gives crystals containing three Zn(II) ions per protein molecule: Zn3-hUb. In contrast, once again, copper(II) acetate and cisplatin, even at low metal-to-protein ratio, do not give crystalline material. In the soaking experiment, Zeise's anion leads to simultaneous platination of His68, Met1 and Lys6. Present and previous results of co-crystallization experiments performed with zinc(II) and other group-12 metal ions allow to reach a comprehensive understanding of the metal-ion binding properties of hUb with His68 as main anchoring site, followed by Met1 and Glu18. The amount of metal ion, with respect to that of the protein, appears to be a relevant parameter influencing the crystal packing.

The authenticity and quality of productions is an area of priority interest that involves safety of consumers and potential economic damages deriving from frauds on origin, adulteration and labeling of products. Several investigation techniques are currently used to characterize food matrices from physical-chemical-biological point of view using different methods in order to limit possible adulterations. In this work, we have developed an experimental and computational framework to improve the potentialities of sensitive crystallization: an experimental technique known since 1936, but never used for quantitative assessment of food quality. As a test case, it has been applied to investigate the geographical traceability and quality of coffee samples. An extensive statistical analysis associated with a careful choice of advanced image descriptors allows gathering quantitative information about the samples, which can constitute a digital fingerprint of their composition. With this new tool we are able to distinguish with blind tests high-quality coffee brands from low-quality mixtures, different coffee species, green from toasted condition of beans and, to a lesser extent, the macro-geographical provenience. A powder X-ray diffraction analysis reinforces the results obtained by sensitive crystallization for the case where crystalline domains are present in the coffee sample.

This communication reports on the possibility to achieve the direct production of either carbamazepine crystals or carbamazepine–saccharin cocrystals from water/ethanol solvent mixtures by using membrane-based crystallization technology, by choosing the opportune initial conditions.

Two examples of anionic complexes having vapochromic behavior are investigated: [K(H2O)][Pt(ppy)(CN)2] "Pt(ppy)" and [K(H2O)][Pt(bzq)(CN)2] "Pt(bzq)", where ppy = 2-phenylpyridinate and bzq = 7,8-benzoquinolate. These monohydrate-potassium salts exhibit a change in color from purple to yellow [Pt(ppy)] and from red to yellow [Pt(bzq)] upon heating to 110 °C, and they transform back into the original color upon absorption of water molecules from the environment. Available only in the form of polycrystalline samples, no structural information on such compounds is accessible, due to highly overlapping peaks in powder diffraction profiles. We use in situ Pair Distribution Function measurements on powder samples to investigate the dynamics of the structural changes induced by temperature variations. By means of a multivariate approach, we were able to extract dynamic structural information from collected profiles without using prior knowledge on the static crystal structure of the compounds. The critical temperature and the characteristics of the vapochromic transition have been identified, as well as the main structural changes causing it.t]

Members of the fungal genus Fusarium can produce numerous secondary metabolites, including the nonribosomal mycotoxins beauvericin (BEA) and enniatins (ENNs). Both mycotoxins are synthesized by the multifunctional enzyme enniatin synthetase (ESYN1) that contains both peptide synthetase and S-adenosyl-l-methionine-dependent N-methyltransferase activities. Several Fusarium species can produce ENNs, BEA or both, but the mechanism(s) enabling these differential metabolic profiles is unknown. In this study, we analyzed the primary structure of ESYN1 by sequencing esyn1 transcripts from different Fusarium species. We measured ENNs and BEA production by ultra-performance liquid chromatography coupled with photodiode array and Acquity QDa mass detector (UPLC-PDA-QDa) analyses. We predicted protein structures, compared the predictions by multivariate analysis methods and found a striking correlation between BEA/ENN-producing profiles and ESYN1 three-dimensional structures. Structural differences in the ? strand's Asn789-Ala793 and His797-Asp802 portions of the amino acid adenylation domain can be used to distinguish BEA/ENN-producing Fusarium isolates from those that produce only ENN.

In this work, the antisolvent membrane crystallization process has been used to influence the polymorphic composition in the crystallization of L-histidine. When finely dosing the amount of ethanol in the crystallizing solution, by generating the proper trans-membrane flux, the composition of the precipitate shifts regularly from the prevalence of the thermodynamic stable polymorph A toward the predominance of the metastable phase B. Form A crystals are always the first to nucleate, thus following an “anti-Ostwald rule” behavior and in accordance with classical nucleation theory previsions.However, as the antisolvent content exceeds a threshold limit, the number of phase A crystals progressively reduces and the first weak nucleation is followed by a second massive production of a powder of the polymorph B. The elapsed time between the two events reduces as the antisolvent dosing flux increases. This result might be due to a combined effect on the nucleation and growth rates when the amount of ethanol exceeds the critical value. No significant membrane fouling was observed over the duration of the experiments, and stable fluxes were observed. A slight increase in membrane permeability, due to the reduced hydrophobicity when using a high initial amount of ethanol, was observed.

The beta-d-glucose-containing compound 3, bearing 2-chlorothiophene and 1-isopropylpiperidine moieties as binders of the S1 and S4 pockets, respectively, proved to be potent competitive inhibitor of factor Xa (fXa, K-i = 0.090 nM) and thrombin (fIIa, Ki = 100 nM). The potency of 3 increases, over the parent compound 1, against fIIa (110-fold), much more than against fXa (7-fold). Experimental deconstruction of 3 into smaller fragments revealed a binding cooperativity of the P3/P4 and propylene-linked beta-d-glucose fragments, stronger in fIIa (15.5 kJ center dot mol(-1)) than in fXa (2.8 kJ center dot mol(-1)). The crystal structure of human fIIa in complex with 3 revealed a binding mode including a strong H-bond network between the glucose O1', O3', and O5' and two critical residues, namely R221a and K224, belonging to the Na+-binding site which may allosterically perturb the specificity sites. The potential of 3 as antithrombotic agent was supported by its ability to inhibit thrombin generation and to stimulate fibrinolysis at submicromolar concentration.

Combination of polymeric hydrogel and porous membranes, which leads to composites displaying the synergic properties of both membrane and gel layer, has been increasingly prevalent in the field of membrane separations [1]. Among different preparation methods, photo-initiated grafting of hydrogel onto a porous support has a much interest due to mild reaction conditions and selectivity to absorb UV light without affecting the bulk polymer [2]. In this work, preparation of hydrogel composites based on polymeric membranes, via photo-initiated graft polymerization, has been investigated, with the aim to develop novel membranes comprising an optimized porous support and a thin hydrogel layer with tailored chemical composition, controlled mesh size, and engineered surface morphology [3]. Several combination of monomers and cross-linkers, displaying variable chemical functionalities, were investigated for hydrogel preparation; furthermore, hydrophobic or hydrophilic membranes, like polypropylene and polyethersulfone, were used as support. The effect of monomer concentration, monomer mixture composition and UV irradiation time on composites preparation has been studied. The performance of prepared hydrogel membranes were evaluated in membrane distillation (MD) and membrane crystallization (MCr) applications for water desalination and for both proteins crystallization and in the biomimetic synthesis of advanced materials. In MD, composite membranes were characterized in terms of transmembrane flux, rejection, and stability over long time operation; in MCr of proteins, hydrogel composite membranes were used as heterogeneous growing media to provide the suitable environment for the formation of crystals with greater size, fewer effects and enhanced diffraction properties; furthermore the same composites were used as platform for the biomimetic synthesis of calcium based mineral layers to develop new routes conductive to hybrid structures with hierarchical architectures.In addition to the general performances of hydrogel membranes in both MD and MCr applications, these composites show the potentiality to tailor the chemical functionalization of the gel layer and its surface nanostructure and morphology, thus providing new routes to adapt its application to special cases.

In this work we studied glycine crystallization with two main objectives: (i) to get improved control of crystal growth and polymorphic selectivity of organic molecules; (ii) to achieve additional insights into the nucleation mechanisms of glycine polymorphs. To reach these goals, membrane crystallization technology, a tool which allows improved control of supersaturation in solution crystallization, was used under different operating conditions: the variable solvent removal rate, acidic and almost neutral pH, the presence of a pulsed electric field. The traditional explanation for the crystallization of a and g glycine polymorphs from aqueous solution is based on the general cyclic dimer hypothesis and the self-poisoning mechanism. In contrast with both the conventional theories, experimental results suggest that the relative nucleation rates with respect to the relative growth kinetics of the two forms under the different conditions play a dominant role in determining the polymorphic outcome. Our results instead support a molecular nucleation route where open chain dimers can behave as building units for both g- and a-glycines in the rate determining structuring step of the two-step nucleation mechanism.

Co-crystallization brings new opportunities for improving the solubility and dissolution rate of drugs with the chance of finely tuning some relevant chemical-physical properties of mixtures containing bioactive compounds. As co-crystallization process involves several molecular species, which are generally solid at room conditions, its control requires accurate knowledge and monitoring of the different phase that might appear during the formulation stage. In the present study the suitability of X-ray powder diffraction (XRPD) and Fourier-transformed infrared (FTIR) spectroscopy in quantifying mixtures of carbamazepine polymorphs (forms I and III), saccharin, and carbamazepine-saccharin cocrystals (form I) is assessed. Quaternary crystalline mixtures typically produced in the process of co-crystal production were analyzed by multivariate methods. Principal component analysis (PCA) was used for the identification of the crystal phases, while unsupervised simultaneous fitting of the spectra from pure phases, or supervised partial least squares (PLS) methods were used for their quantitative determination. The performance of data analysis was enhanced by applying peculiar pre-processing methods, such as SNIP filtering in case of FTIR and PCA filtering in case of XRPD. It was found that, for XRPD data, the automatic multi-fitting procedures and PLS models developed in this study are able to quantify single phases in mixtures to an accuracy level comparable to that obtained by the widely used Rietveld method, which, however, requires knowledge of the crystal structures. For FTIR data the results here obtained prove that this technique can be used as a fast method for polymorph characterization.

Two new computational methods dedicated to neutron crystallography, called n-FreeLunch and DNDM-NDM, have been developed and successfully tested. The aim in developing these methods is to determine hydrogen and deuterium positions in macromolecular structures by using information from neutron density maps. Of particular interest is resolving cases in which the geometrically predicted hydrogen or deuterium positions are ambiguous. The methods are an evolution of approaches that are already applied in X-ray crystallography: extrapolation beyond the observed resolution (known as the FreeLunch procedure) and a difference electron-density modification (DEDM) technique combined with the electron-density modification (EDM) tool (known as DEDM-EDM). It is shown that the two methods are complementary to each other and are effective in finding the positions of H and D atoms in neutron density maps.

Copper trafficking proteins have been implicated in the cellular response to platinum anticancer drugs. We investigated the reaction of the chaperone Atox1 with an activated form of oxaliplatin, the third platinum drug to reach worldwide approval. Unlike cisplatin, which contains mono dentate ammines, oxaliplatin contains chelated 1,2-diaminocyclohexane (DACH), which is more resistant to displacement by nucleophiles. In solution, one or two {Pt(DACH)(2+)} moieties bind to the conserved CXXC metal-binding motif of Atox1; in the latter case the two sulfur atoms likely bridging the two platinum units. At longer reaction times, a dimeric species is formed whose composition, Atox1(2)center dot Pt-2(2+), indicates complete loss of the diamine ligands. Such a dimerization process is accompanied by partial unfolding of the protein. Crystallization experiments aiming at the characterization of the monomeric species have afforded, instead, a dimeric species resembling that already obtained by Boal and Rosenzweig in a similar reaction performed with cisplatin. However, while in the latter case there was only one Pt-binding site (0.4 occupancy) made of four sulfur atoms of the CXXC motifs of the two Atox1 chains in a tetrahedral arrangement, we found, in addition, a secondary Pt-binding site involving Cys41 of the B chain (0.25 occupancy). Moreover, both platinum atoms have lost their diamines. Thus, there appears to be little relationship between what is observed in solution and what is formed in the solid state. Since full occupancy of the tetrahedral cavity is a common feature of all Atox1 dimeric structures obtained with other metal ions (Cu+, Cd2+, and Hg2+), we propose that in the case of platinum, where the occupancy is only 0.4, the remaining cavities are occupied by Cu+ ions. Experimental evidence is reported in support of the latter hypothesis. Our proposal represents a meeting point between the initial proposal of Boal and Rosenzweig (0.4 Pt occupancy) and the reinterpretation of the original crystallographic data put forward by Shabalin et al. (1 Cu occupancy), and could apply to other cases.

Crystallography is a major tool for structure-driven drug design, as it allows knowledge of the 3D structure of protein targets and protein-ligand complexes. However, the route for crystal structure determination involves many steps, some of which may hamper its high-throughput use. Recent efforts have produced significant advances in experimental and computational tools and protocols. They include automatic crystallization tools, faster data collection devices, more efficient phasing methods and improved ligand-fitting procedures. The timescales of drug-discovery processes have been also reduced by using a fragment-based screening approach. Herein, the achievements in protein crystallography over the last 5 years are reviewed, and advantages and disadvantages of the fragment-based approaches to drug discovery that make use of X-ray crystallography as a primary screening method are examined. In particular, in some detail, five recent case studies pertaining to the development of new hits or leads in relevant therapeutic areas, such as cancer, immune response, inflammation, metabolic syndrome and neurology are described. © 2013 Future Science Ltd.

Phasing proteins at non-atomic resolution is still a challenge for any ab initio method. A variety of algorithms [Patterson deconvolution, superposition techniques, a cross-correlation function (C map), the VLD (vive la difference) approach, the FF function, a nonlinear iterative peak-clipping algorithm (SNIP) for defining the background of a map and the free lunch extrapolation method] have been combined to overcome the lack of experimental information at non-atomic resolution. The method has been applied to a large number of protein diffraction data sets with resolutions varying from atomic to 2.1 Å, with the condition that S or heavier atoms are present in the protein structure. The applications include the use of ARP/wARP to check the quality of the final electron-density maps in an objective way. The results show that resolution is still the maximum obstacle to protein phasing, but also suggest that the solution of protein structures at 2.1 Å resolution is a feasible, even if still an exceptional, task for the combined set of algorithms implemented in the phasing program. The approach described here is more efficient than the previously described procedures: e.g. the combined use of the algorithms mentioned above is frequently able to provide phases of sufficiently high quality to allow automatic model building. The method is implemented in the current version of SIR2014. © 2014 International Union of Crystallography.

A cationic soluble peroxidase isoenzyme (CysPrx) has been purified and characterized from artichoke (Cynara cardunculus subsp. scolymus (L.) Hegi) leaves by combination of aqueous two phase extraction, ion exchange chromatography, and gel filtration. The purification fold was 149 and the activity recovery 5.5%. CysPrx was stable from 5 to 45 °C with a pH optimum around 5.5; the pI was 8.3 and the MW of 37.7 ± 1.5 kDa. MALDI-TOF MS analysis provided partial peptide sequences and resolved CysPrx isoenzyme into two putative isoforms. The presence of these isoforms was confirmed by the isolation of full-length cDNA encoding CysPrx that generate two slightly different sequences coding for two putative CysPrx: CysPrx1 and CysPrx2. The obtained MS peptides showed a 35% coverage with 100% identity with the two CysPrx deduced protein sequences. A molecular modeling analysis was carried out to predict in silico the protein structure and compare it with other plant Prx structures. Considering that CysPrx is quite stable, the study carried out in this paper will offer new insights for the production of the recombinant protein for utilization of CysPrx as an alternative Prx for food technology, biomedical analysis and bioremediation.

The possibility of exploiting a thermally-induced solid-state reaction to obtain the formation of a fluorene and TCNQ molecular complex was proposed by some of us (Kumar et al., Cryst. Growth Des., 2009, 9(8), 3396-3404). In this paper, we generalize and rationalize the approach to other charge transfer (CT) molecular complexes with naphthalene and anthracene, changing the size of the donor moiety to propose a general approach for the preparation of this class of materials by exploiting the solid-state synthesis method. Moreover, the kinetic features of the solid state reactions were fully elucidated by Raman spectroscopy and high resolution X-ray Powder Diffraction analysis (in situ Raman/XRPD), exploiting the Avrami equation in isothermal and non-isothermal conditions; rate constants, reaction orders and activation energies were obtained. All the three tested solid-state charge transfer (SS-CT) reactions obeyed a general rule, whereby the most apt reaction temperature was predicted. Finally, a method based on principal component analysis (PCA) for a fast kinetic analysis of in situ XRD synchrotron data was successfully developed, employing a formalism suitable for the analysis of non-isothermal reaction data, thus allowing a high throughput approach for the fast screening of the kinetics of parent reactions. PCA analysis was exploited as an alternative technique to obtain kinetic information in a faster and more efficient way, which can be used for online monitoring and/or in all those cases where Rietveld analysis is not feasible.

Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat) and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential.

RootProf is a multi-purpose program which implements multivariate analysis of unidimensional profiles. Series of measurements, performed on related samples or on the same sample by varying some external stimulus, are analysed to find trends in data, classify them and extract quantitative information. Qualitative analysis is performed by using principal component analysis or correlation analysis. In both cases the data set is projected in a latent variable space, where a clustering algorithm classifies data points. Group separation is quantified by statistical tools. Quantitative phase analysis of a series of profiles is implemented by whole-profile fitting or by an unfolding procedure, and relies on a variety of pre-processing methods. Supervised quantitative analysis can be applied, provided a priori information on some samples is provided. RootProf can be applied to measurements from different techniques, which can be combined by means of a covariance analysis. A specific analysis for powder diffraction data allows estimation of the average size of crystal domains. RootProf borrows its graphics and data analysis capabilities from the Root framework, developed for high-energy physics experiments.

This work aims to describe a systematic study on the conditions promoting the selective formation of carbamazepine-saccharin cocrystals or single component crystals from water/ethanol solvent mixtures, by using a membrane crystallization process. Results revealed the ability to operate in the proper zone of the phase diagram of the system when opportunely choosing the initial solution conditions and limiting the maximum level of supersaturation by using the membrane-based technology. Control in the selective crystallization of a specific solid form can be achieved by adjusting the solvent evaporation through the micropores of the membrane. Furthermore, the direct correlation between transmembrane flow and polymorphic composition in the case of carbamazepine precipitation confirmed the possibility to produce particular metastable phases upon increasing the supersaturation rate. © 2012 American Chemical Society.

A facile and fast microwave-assisted hydrothermal method is proposed for the synthesis of magnetitenanoparticles. The addition of different surfactants (polyvinylpyrrolidone, oleic acid, or trisodium citrate)was studied to investigate the effect on size distribution, morphology, and functionalization of themagnetite nanoparticles. Microwave irradiation at 150 °C for 2 h of aqueous ferrous chloride andhydrazine without additives results in hexagonal magnetite nanoplatelets with a facet-to-facet distance of116 nm and a thickness of 40 nm having a saturation magnetization of ~65 Am2 kg-1. The use ofpolyvinylpyrrolidone led to hexagonal nanoparticles with a facet-to-facet distance of 120 nm and athickness of 53 nm with a saturation magnetization of ~54 Am2 kg-1. Additives such as oleic acid andtrisodium citrate yielded quasi-spherical nanoparticles of 25 nm in size with a saturation magnetization of~70 Am2 kg-1 and spheroidal nanoparticles of 60 nm in size with a saturation magnetization up to~82 Am2 kg-1, respectively. A kinetic control of the crystal growth is believed to be responsible for thehexagonal habit of the nanoparticles obtained without additive. Conversely, a thermodynamic control ofthe crystal growth, leading to spheroidal nanoparticles, seems to occur when additives which stronglyinteract with the nanoparticle surface are used. A thorough characterization of the materials wasperformed. Magnetic properties were investigated by Superconducting Quantum Interference Device andVibrating Sample Magnetometer. Based on the observed magnetic properties, the magnetite obtained usingcitrate appears to be a promising support for magnetically transportable catalysts.

SIR2011, the successor of SIR2004, is the latest program of the SIR suite. It can solve ab initio crystal structures of small- and medium-size molecules, as well as protein structures, using X-ray or electron diffraction data. With respect to the predecessor the program has several new abilities: e. g. a new phasing method (VLD) has been implemented, it is able to exploit prior knowledge of the molecular geometry via simulated annealing techniques, it can use molecular replacement methods for solving proteins, it includes new tools like free lunch and new approaches for electron diffraction data, and it visualizes three-dimensional electron density maps. The graphical interface has been further improved and allows the straightforward use of the program even in difficult cases.

The role of protein Z (PZ) in the etiology of human disorders is unclear. A number of PZ gene variants, sporadic or polymorphic and found exclusively in the serine protease domain, have been observed. Crystal structures of PZ in complex with the PZ-dependent inhibitor (PZI) have been recently obtained. The aim of this study was a structural investigation of the serine protease PZ domain, aiming at finding common traits across disease-linked mutations. We performed 10-20 ns molecular dynamics for each of the observed PZ mutants to investigate their structure in aqueous solution. Simulation data were processed by novel tools to analyse the residue-by-residue backbone flexibility. Results showed that sporadic mutations are associated with anomalous flexibility of residues belonging to specific regions. Among them, the most important is a loop region which is in contact with the longest I helix of PZI. Other regions have been identified, which hold anomalous flexibility associated with potentially protective gene variants. In conclusion, a possible interpretation of effects associated with observed gene variants is provided. The exploration of PZ/PZI interactions seems essential in explaining these effects.

The mammalian bombesin receptor family comprises three G protein-coupled receptors: the neuromedin B receptor, the gastrin-releasing peptide receptor (BB2), and the bombesin receptor subtype 3. BB2 receptor plays a role in gastrointestinal functions; however, at present the role of this subtype in physiological and pathological conditions is unknown due to the lack of specific binders for all subclasses of bombesin receptors. Here, we present a study focused on the properties of the peptoid bombesin antagonist called PD176252, and other structural analogues with the aim to elucidate causes of their different affinity towards the BB2 receptor. By means of computational techniques, based on QSAR, docking and homology building, supported by experimental data (X-ray diffraction and NMR spectroscopy) fresh insights on binding modes of this class of biological targets were achieved.

Fusarium genus is able to produce several metabolites including the emerging mycotoxins beauvericin (BEA) and enniatins (ENs). Due to their ionophoric property, BEA and ENs exert many biological properties, including antimicrobial, insecticidal, and cytotoxic activity in human cell lines, so that they are recently proposed as novel anticancer drugs. BEA and ENs are cyclic hexadepsipeptides with an alternating sequence of three N-metyl-L-amino acids and three D-?-hydroxyisovaleric acids; in BEA the amino acid residues are aromatic N-metyl-pheylalanines, whereas in ENs the amino acid residues are aliphatic N-metyl-valine, -leucine or -isoleucine. Both mycotoxins are synthesized by the multifunctional enzyme enniatin synthetase (ESYN1) representing hybrid system of peptide synthetase and S-adenosyl-L-methionine-dependent N-methyltransferase. Several Fusarium species have been reported to produce ENs, BEA or both, and our hypothesis is that the different production profile depends on the esyn1 sequence. Our aim was to investigate this relation by mean of a bioinformatics approach, based on structural investigations.The esyn1 sequences of 18 Fusarium isolates belonging to 8 different species were extracted from published and unpublished genomes by BLASTN search using as query the available sequences of Fusarium scirpi and Fusarium proliferatum. The protein sequences were predicted using the Sequence Translation tools of the EMBOSS Programs (EMBL-EBI), manually curated using exon/intron boundary predictions from SpliceView (http://bioinfo4.itb.cnr.it/), and confirmed by sequencing the RT-PCR products.The selected sequences have been processed by the web server RaptorX to obtain structural predictions by homology modelling and threading methods. While the full sequences have been submitted for prediction, it has been accomplished only for part of the whole enzyme. In particular, the region containing the N-methyltransferase activity has been not completely structured. A comprehensive analysis of the predicted structures has allowed identifying common structural domains, which have been compared by considering their dihedral backbone angles and by using the tool T-PAD, developed to characterize the protein flexibility and identify hot spot residues responsible for hinge motions. The residue-by-residue flexibility profiles have been analysed by multivariate analysis, to produce a classification by Principal Component Analysis, followed by hierarchical clustering.We have thus identified the conserved structural regions and the pivotal residues responsible for the structural variability. The common domains for each of the 18 esyn1 sequences have been grouped in clusters, and the residues responsible for the classification have been singled out. Overall, we have achieved a comprehensive view of the structural features of the analysed esyn1 sequences, where the structural variability has been related to the sequence variability, and interpreted in terms

An X-ray investigation has been performed with the aim of characterizing the binding sites of a platinum-based inhibitor (K[PtCl3(DMSO)]) of matrix metalloproteinase-3 (stromelysin-1). The platinum complex targets His224 in the S1' specificity loop, representing the first step in the selective inhibition process (PDB ID code 4JA1).

The influence of drying temperature on the starch crystallites and its impact on durum wheat pasta sensory properties is addressed in this work. In particular, spaghetti were produced by means of a pilot plant using 5 different drying temperature profiles. The sensory properties, as well as the cooking quality of pasta were assessed. X-ray powder diffraction was used for investigating changes in the crystallinity content of the samples. Starch crystallinity, size and density of the starch crystallites were determined from the analysis of the diffraction profiles. As expected, spaghetti sensory properties improved as the drying temperatures increased. In particular, attributes as resistance to break for uncooked samples and firmness, elasticity, bulkiness and stickiness for cooked samples, all benefit from drying temperature increase. The spaghetti cooking quality was also positively affected by the drying temperature increase. Diffraction analysis suggested that the improvement of sensory properties and cooking quality of pasta were directly related to the increase in density of both physical crosslink of starch granules and chemical crosslink of protein matrix. (C) 2016 Elsevier Ltd. All rights reserved.

The design and synthesis of a new class of nonpeptide direct thrombin inhibitors, built on the structure of 1-(pyridin-4-yl)piperidine-4-carboxamide, are described. Starting from a strongly basic 1-amidinopiperidine derivative (6) showing poor thrombin (fIIa) and factor Xa (fXa) inhibition activities, anti-ilia activity and artificial membrane permeability were considerably improved by optimizing the basic PI and the X-substituted phenyl P4 binding moieties. Structure-activity relationship studies, usefully complemented with molecular modeling results, led us to identify compound 13b, which showed excellent fIIa inhibition (K-i = 6 nM), weak anti-Xa activity (K-i = 5.64 mu M), and remarkable selectivity over other serine proteases (e.g., trypsin). Compound 13b showed in vitro anticoagulant activity in the low micromolar range and significant membrane permeability. In mice (ex vivo), 13b demonstrated anticoagulant effects at 2 h after oral dosing (100 mg.kg(-1)), with a significant 43% prolongation of the activated partial thromboplastin time (aPTT), over controls (P < 0.05).

Crystallization still represents the bottleneck in the process of protein structure determination at high resolution, despite high-throughput structural genomics programs require optimized crystallization strategies regarding crystal quality, time, success rate, reproducibility and used protein amount. On the other side, the development of suitable materials for controlled heterogeneous nucleation might facilitate biomacromolecular crystallization in a variety of experimental conditions which are not conventionally fruitful. Here we show the possibility to fabricate hydrogel membranes displaying controlled chemical composition and nanostructure and to use them as heterogeneous supports for biomacromolecular crystallization. Diverse gel morphologies were obtained by controlling phase separation kinetics during gel layer formation on membrane support. These composite materials were found to increase the efficiency of the crystallization process so that crystals with enhanced diffraction properties were produced at lower protein concentration than conventional technique, thus affording the possibility to improve current approaches to protein crystallization and to be adapted to specific targets.

Modulated enhanced diffraction (MED) is a technique allowing the dynamicstructural characterization of crystalline materials subjected to an externalstimulus, which is particularly suited for in situ and operando structuralinvestigations at synchrotron sources. Contributions from the (active) part of thecrystal system that varies synchronously with the stimulus can be extracted by anoffline analysis, which can only be applied in the case of periodic stimuli andlinear system responses. In this paper a new decomposition approach based onmultivariate analysis is proposed. The standard principal component analysis(PCA) is adapted to treat MED data: specific figures of merit based on theirscores and loadings are found, and the directions of the principal componentsobtained by PCA are modified to maximize such figures of merit. As a result, ageneral method to decompose MED data, called optimum constrainedcomponents rotation (OCCR), is developed, which produces very preciseresults on simulated data, even in the case of nonperiodic stimuli and/ornonlinear responses. The multivariate analysis approach is able to supply in oneshot both the diffraction pattern related to the active atoms (through the OCCRloadings) and the time dependence of the system response (through the OCCRscores). When applied to real data, OCCR was able to supply only the latterinformation, as the former was hindered by changes in abundances of differentcrystal phases, which occurred besides structural variations in the specific caseconsidered. To develop a decomposition procedure able to cope with thiscombined effect represents the next challenge in MED analysis.

The joint probability distribution function P(E, E(p)), where E and E(p) are the normalized structure factors of the target and of a model structure, respectively, is a fundamental tool in crystallographic methods devoted to crystal structure solution. It plays a central role in any attempt for improving phase estimates from a given structure model. More recently the difference electron density P(q) = rho - rho(p) has been revisited and methods based on its modifications have started to play an important role in combination with electron density modification approaches. In this paper new coefficients for the difference electron density have been obtained by using the joint probability distribution function P(E, E(p), E(q)) and by taking into account both errors in the model and in measurements. The first applications show the correctness of our theoretical approach and the superiority of the new difference Fourier synthesis, particularly when the model is a rough approximation of the target structure. The new and the classic difference syntheses coincide when the model represents the target structure well.