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Protein science 32(2), e4563 (2023). doi:10.1002/pro.4563, Nerve growth factor (NGF), the prototypical neurotrophic factor, is involved in the maintenance and growth of specific neuronal populations, whereas its precursor, proNGF, is involved in neuronal apoptosis. Binding of NGF or proNGF to TrkA, p75NTR, and VP10p receptors triggers complex intracellular signaling pathways that can be modulated by endogenous small-molecule ligands. Here, we show by isothermal titration calorimetry and NMR that ATP binds to the intrinsically disordered pro-peptide of proNGF with a micromolar dissociation constant. We demonstrate that Mg2+, known to play a physiological role in neurons, modulates the ATP/proNGF interaction. An integrative structural biophysics analysis by small angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry unveils that ATP binding induces a conformational rearrangement of the flexible pro-peptide domain of proNGF. This suggests that ATP may act as an allosteric modulator of the overall proNGF conformation, whose likely distinct biological activity may ultimately affect its physiological homeostasis., Published by Protein Society, Bethesda, Md.

Nerve Growth Factor (NGF), the prototypical neurotrophic factor, is involved in the maintenance and growth of specific neuronal populations, whereas its precursor, proNGF, is involved in neuronal apoptosis. Binding of NGF or proNGF to TrkA, p75

A novel black organoammonium iodoplumbate semiconductor, namely phenyl viologen lead iodide C22H18N2(PbI3)2 (PhVPI), was successfully synthesized and characterized. This material showed physical and chemical properties suitable for photovoltaic applications. Indeed, low direct allowed band gap energy (Eg = 1.32 eV) and high thermal stability (up to at least 300 °C) compared to methylammonium lead iodide CH3NH3PbI3 (MAPI, Eg = 1.5 eV) render PhVPI potentially attractive for solar cell fabrication. The compound was extensively characterized by means of X-ray diffraction (performed on both powder and single crystals), UV-Vis diffuse reflectance spectroscopy (UV-Vis DRS), UV-photoelectron spectroscopy (UPS), FT-IR spectroscopy, TG-DTA, and CHNS analysis. Reactivity towards water was monitored through X-ray powder diffraction carried out after prolonged immersion of the material in water at room temperature. Unlike its methyl ammonium counterpart, PhVPI proved to be unaffected by water exposure. The lack of reactivity towards water is to be attributed to the quaternary nature of the nitrogen atoms of the phenyl viologen units that prevents the formation of acid-base equilibria when in contact with water. On the other hand, PhVPI's thermal stability was evaluated by temperature-controlled powder XRD measurements following an hour-long isothermal treatment at 250 and 300 °C. In both cases no signs of decomposition could be detected. However, the compound melted incongruently at 332 °C producing, upon cooling, a mostly amorphous material. PhVPI was found to be slightly soluble in DMF (∼5 mM) and highly soluble in DMSO. Nevertheless, its solubility in DMF can be dramatically increased by adding an equimolar amount of DMSO. Therefore, phenyl viologen lead iodide can be amenable for the fabrication of solar devices by spin coating as actually done for MAPI-based cells. The crystal structure, determined by means of single crystal X-ray diffraction using synchrotron radiation, turned out to be triclinic and consequently differs from the prototypal perovskite structure. In fact, it comprises infinite double chains of corner-sharing PbI6 octahedra along the a-axis direction with phenyl viologen cations positioned between the columns. Finally, the present determination of PhVPI's electronic band structure achieved through UPS and UV-Vis DRS is instrumental in using the material for solar cells.

Perfluorooctanoic acid (PFOA) is a toxic compound that is absorbed and distributed throughout the body by noncovalent binding to serum proteins such as human serum albumin (hSA). Though the interaction between PFOA and hSA has been already assessed using various analytical techniques, a high resolution and detailed analysis of the binding mode is still lacking. We report here the crystal structure of hSA in complex with PFOA and a medium-chain saturated fatty acid (FA). A total of eight distinct binding sites, four occupied by PFOAs and four by FAs, have been identified. In solution binding studies confirmed the 4:1 PFOA-hSA stoichiometry and revealed the presence of one high and three low affinity binding sites. Competition experiments with known hSA-binding drugs allowed locating the high affinity binding site in sub-domain IIIA. The elucidation of the molecular basis of the interaction between PFOA and hSA might provide not only a better assessment of the absorption and elimination mechanisms of these compounds in vivo but also have implications for the development of novel molecular receptors for diagnostic and biotechnological applications.

Self-assembled metal phthalocyanine thin films are receiving considerable interest due to their potential technological applications. In this study, we present a comprehensive study of CoPc and FePc thin films of about 50 nm thickness on technologically relevant substrates such as SiOx/Si, indium tin oxide (ITO) and polycrystalline gold in order to investigate the substrate induced effects on molecular stacking and crystal structure. Raman spectroscopic analysis reveals lower intensity for the vibrational bands corresponding to phthalocyanine macrocycle for the CoPc and FePc thin films grown on ITO as compared to SiOx/Si due to the higher order of phthalocyanine molecules on SiOx/Si. Atomic force microscopy analysis displays higher grain size for FePc and CoPc thin films on ITO as compared to SiOx/Si and polycrystalline gold indicating towards the influence of molecule–substrate interactions on the molecular stacking. Grazing incidence X-ray diffraction reciprocal space maps reveal that FePc and CoPc molecules adopt a combination of herringbone and brickstone arrangement on SiOx/Si and polycrystalline gold substrate, which can have significant implications on the optoelectronic properties of the films due to unique molecular stacking.

Dystroglycan (DG) is a cell adhesion complex composed by two subunits, the highly glycosylated alpha-DG and the transmembrane beta-DG. In skeletal muscle, DG is involved in dystroglycanopathies, a group of heterogeneous muscular dystrophies characterized by a reduced glycosylation of alpha-DG. The genes mutated in secondary dystroglycanopathies are involved in the synthesis of O-mannosyl glycans and in the O-mannosylation pathway of alpha-DG. Mutations in the DG gene (DAG1), causing primary dystroglycanopathies, destabilize the alpha-DG core protein influencing its binding to modifying enzymes. Recently, a homozygous mutation (p.Cys699Phe) hitting the beta-DG ectodomain has been identified in a patient affected by muscle-eye-brain disease with multicystic leucodystrophy, suggesting that other mechanisms than hypoglycosylation of alpha-DG could be implicated in dystroglycanopathies. Herein, we have characterized the DG murine mutant counterpart by transfection in cellular systems and high-resolution microscopy. We observed that the mutation alters the DG processing leading to retention of its uncleaved precursor in the endoplasmic reticulum. Accordingly, small-angle X-ray scattering data, corroborated by biochemical and biophysical experiments, revealed that the mutation provokes an alteration in the beta-DG ectodomain overall folding, resulting in disulfide-associated oligomerization. Our data provide the first evidence of a novel intracellular mechanism, featuring an anomalous endoplasmic reticulum-retention, underlying dystroglycanopathy.

Dystroglycan (DG), composed of and subunits, belongs to the dystrophin-associated glycoprotein complex. alpha-DG is an extracellular matrix protein that undergoes a complex post-translational glycosylation process. The bifunctional glycosyltransferase like-acetylglucosaminyltransferase (LARGE) plays a crucial role in the maturation of alpha-DG, enabling its binding to laminin. We have already structurally analyzed the N-terminal region of murine alpha-DG (alpha-DG-Nt) and of a pathological single point mutant that may affect recognition of LARGE, although the structural features of the potential interaction between LARGE and DG remain elusive. We now report on the crystal structure of the wild-type human alpha-DG-Nt that has allowed us to assess the reliability of our murine crystallographic structure as alpha-DG-Nt general model. Moreover, we address for the first time both structures in solution. Interestingly, small-angle X-ray scattering (SAXS) reveals the existence of two main protein conformations ensembles. The predominant species is reminiscent of the crystal structure, while the less populated one assumes a more extended fold. A comparative analysis of the human and murine alpha-DG-Nt solution structures reveals that the two proteins share a common interdomain flexibility and population distribution of the two conformers. This is confirmed by the very similar stability displayed by the two orthologs as assessed by biochemical and biophysical experiments. These results highlight the need to take into account the molecular plasticity of alpha-DG-Nt in solution, as it can play an important role in the functional interactions with other binding partners.

A novel black organoammonium iodoplumbate semiconductor, namely phenyl viologen lead iodide C22H18N2(PbI3)2 (PhVPI), was successfully synthesized and characterized. This material showed physical and chemical properties suitable for photovoltaic applications. Indeed, low direct allowed band gap energy (Eg = 1.32 eV) and high thermal stability (up to at least 300 °C) compared to methylammonium lead iodide CH3NH3PbI3 (MAPI, Eg = 1.5 eV) render PhVPI potentially attractive for solar cell fabrication. The compound was extensively characterized by means of X-ray diffraction (performed on both powder and single crystals), UV-Vis diffuse reflectance spectroscopy (UV-Vis DRS), UV-photoelectron spectroscopy (UPS), FT-IR spectroscopy, TG-DTA, and CHNS analysis. Reactivity towards water was monitored through X-ray powder diffraction carried out after prolonged immersion of the material in water at room temperature. Unlike its methyl ammonium counterpart, PhVPI proved to be unaffected by water exposure. The lack of reactivity towards water is to be attributed to the quaternary nature of the nitrogen atoms of the phenyl viologen units that prevents the formation of acid-base equilibria when in contact with water. On the other hand, PhVPI's thermal stability was evaluated by temperature-controlled powder XRD measurements following an hour-long isothermal treatment at 250 and 300 °C. In both cases no signs of decomposition could be detected. However, the compound melted incongruently at 332 °C producing, upon cooling, a mostly amorphous material. PhVPI was found to be slightly soluble in DMF (∼5 mM) and highly soluble in DMSO. Nevertheless, its solubility in DMF can be dramatically increased by adding an equimolar amount of DMSO. Therefore, phenyl viologen lead iodide can be amenable for the fabrication of solar devices by spin coating as actually done for MAPI-based cells. The crystal structure, determined by means of single crystal X-ray diffraction using synchrotron radiation, turned out to be triclinic and consequently differs from the prototypal perovskite structure. In fact, it comprises infinite double chains of corner-sharing PbI6 octahedra along the a-axis direction with phenyl viologen cations positioned between the columns. Finally, the present determination of PhVPI's electronic band structure achieved through UPS and UV-Vis DRS is instrumental in using the material for solar cells.

The immobilization of biomolecules at screen printed electrodes for biosensing applications is still an open challenge. To enrich the toolbox of bioelectrochemists, graphite screen printed electrodes (G-SPE) were modified with an electropolymerized film of pyrrole-2-carboxilic acid (Py-2-COOH), a pyrrole derivative rich in carboxylic acid functional groups. These functionalities are suitable for the covalent immobilization of biomolecular recognition layers. The electropolymerization was first optimized to obtain stable and conductive polymeric films, comparing two different electrolytes: sodium dodecyl sulphate (SDS) and sodium perchlorate. The G-SPE modified with Py-2-COOH in 0.1 M SDS solution showed the required properties and were further tested. A proof-of-concept study for the development of an impedimetric sensor for perfluorooctanoic acid (PFOA) was carried out using the delipidated human serum albumin (hSA) as bioreceptor. The data interpretation was supported by size exclusion chromatography and small-angle X-ray scattering (SEC-SAXS) analysis of the bioreceptor-target complex and the preliminary results suggest the possibility to further develop this biosensing strategy for toxicological and analytical studies.

The synthesis of hydroxylammonium lead iodide NH3OHPbI3 was accomplished by means of the reaction between water solutions of HI and NH2OH with PbI2 in sulfolane in conjunction with either crystallization by CH2Cl2 vapor diffusion or sulfolane extraction with toluene. The appropriate choice of the solvent was found to be crucial in order to attain the desired material. The synthesized compound was extensively characterized by single crystal and powder X-ray diffraction, UV-Vis diffuse reflectance spectroscopy, FT-IR spectroscopy, 1H-NMR spectroscopy, TG-DTA-QMS EGA (Evolved Gas Analysis), ESI-MS, and CHNS analysis. NH3OHPbI3 is an extremely reactive, deliquescent solid that easily oxidizes in air releasing iodine. Furthermore, it is the first reported perovskite to melt (m.p. around 80 °C) before decomposing exothermally at 103 °C. Such a chemical behavior, together with its optical absorption properties (i.e. yellow-colored perovskite), renders this material totally unsuitable for photovoltaic applications. The deliquescence of the material is to be ascribed to the strong hydrophilicity of hydroxylammonium ion. On the other hand, the relatively high Brønsted acidity of hydroxylammonium (pKa = 5.97) compared to other ammonium cations, promotes the reduction of atmospheric oxygen to water and the NH3OHPbI3 oxidation. The crystal structure, determined by single crystal X-ray diffraction with synchrotron radiation, is orthorhombic, but differs from the prototypal perovskite structure. Indeed it comprises infinite chains of face-sharing PbI6 octahedra along the c-axis direction with hydroxylammonium cations positioned between the columns, forming layers on the ac plane. The solvent intercalates easily between the layers. The crystal structure is apparently anomalous considering that the expected Goldschmidt's tolerance factor for the system (0.909) lies in the range of a stable prototypal perovskite structure. Therefore, the strong hydrogen bond forming tendency of hydroxylamine is likely to account for the apparent structural anomaly.

The dystroglycan (DG) adhesion complex has a crucial role for muscle stability and is involved in a number of autosomal recessive neuromuscular disorders ranging from severe congenital (Muscle-Eye-Brain and Walker-Warburg syndrome) to milder limb-girdle muscular dystrophies (LGMD2P). The complex undergoes a still poorly understood molecular maturation/targeting pathway, in which a precursor molecule undergoes an early proteolytic event in the endoplasmic reticulum and is cleaved into two subunits, alpha (extracellular) and beta (transmembrane); this is followed by a multistep glycosylation process which mainly takes place within the Golgi apparatus. Most of the currently identified dystroglycanopathies depend on an impaired sugar decoration of the alpha-dystroglycan subunit that, when hypoglycosylated, shows a reduced binding affinity towards laminin-2 (dubbed as secondary dystroglycanopathies). However, lately a novel subgroup of primary dystroglycanopathies, depending on missense mutations of the dystroglycan core protein, is emerging. We have established an array of diverse tools for the analysis of primary dystroglycanopathies. Namely, the production of novel mice lines, the analysis of transfected cellular lines and the recombinant expression of isolated domains for biochemical and crystallization analysis, as well as computational biochemistry. Our cellular, microscopic and biochemical analysis of the murine counterpart (C667F) of the recently identified missense mutation C669F (inducing a severe muscle-eye-brain phenotype) located within the ectodomain of human beta-DG has allowed us to show that i) the processing of the alpha-beta DG precursor is altered; ii) the variant protein is mainly stuck in the ER and iii) there is some degree of oligomerization and/or structural organization within these C667F aggregates. Following the award of an AFM grant to study primary dystroglycanopathies, we have strongly invested into the generation of a first mouse model. The company GenOway is currently taking care of preparing the knocked-in gametes which will be employed at Bonn University for generating the desired heterozygous and homozygous mouse colony that will be used for morphologic and histologic analysis in the brain and muscle.

The synthesis of hydroxylammonium lead iodide NH3OHPbI3 was accomplished by means of the reaction between water solutions of HI and NH2OH with PbI2 in sulfolane in conjunction with either crystallization by CH2Cl2 vapor diffusion or sulfolane extraction with toluene. The appropriate choice of the solvent was found to be crucial in order to attain the desired material. The synthesized compound was extensively characterized by single crystal and powder X-ray diffraction, UV-Vis diffuse reflectance spectroscopy, FT-IR spectroscopy, 1H-NMR spectroscopy, TG-DTA-QMS EGA (Evolved Gas Analysis), ESI-MS, and CHNS analysis. NH3OHPbI3 is an extremely reactive, deliquescent solid that easily oxidizes in air releasing iodine. Furthermore, it is the first reported perovskite to melt (m.p. around 80 °C) before decomposing exothermally at 103 °C. Such a chemical behavior, together with its optical absorption properties (i.e. yellow-colored perovskite), renders this material totally unsuitable for photovoltaic applications. The deliquescence of the material is to be ascribed to the strong hydrophilicity of hydroxylammonium ion. On the other hand, the relatively high Bronsted acidity of hydroxylammonium (pKa = 5.97) compared to other ammonium cations, promotes the reduction of atmospheric oxygen to water and the NH3OHPbI3 oxidation. The crystal structure, determined by single crystal X-ray diffraction with synchrotron radiation, is orthorhombic, but differs from the prototypal perovskite structure. Indeed it comprises infinite chains of face-sharing PbI6 octahedra along the c-axis direction with hydroxylammonium cations positioned between the columns, forming layers on the ac plane. The solvent intercalates easily between the layers. The crystal structure is apparently anomalous considering that the expected Goldschmidt's tolerance factor for the system (0.909) lies in the range of a stable prototypal perovskite structure. Therefore, the strong hydrogen bond forming tendency of hydroxylamine is likely to account for the apparent structural anomaly.

Dystroglycan (DG) is a glycoprotein complex that links the cytoskeleton with the extracellular matrix and it is composed of two subunits: the extracellular ?-DG and the transmembrane ?-DG. ?-DG is a highly glycosylated protein and its hypoglycosylation weakens its interaction with the extracellular matrix proteins, such as laminins, resulting in a functionally compromised protein. The DG complex undergoes a still poorly understood molecular maturation/targeting pathway, where a precursor is cleaved into ? and ? subunits. Furthermore, the ?-DG subunit is decorated with complex glycans in a multistep glycosylation process. Most of the dystroglycanopathies currently identified are due to an impaired functional state of the enzymes involved in ?-DG maturation (secondary dystroglycanopathies). More recently, missense mutations of the dystroglycan core protein has been identified as the cause of distroglycanopathies (primary dystroglycanopathies) of diverse severity. Indeed, a set of point mutations on the N-terminal region of ?-DG (a.a. 50-313 in mouse) determine the hypoglycosylation of the DG complex, due to the impairment of a key step in ?-DG glycosylation operated by the bifunctional glycosyltransferase LARGE1. Moreover, a point mutation affecting ?-DG, namely the mutation of cysteine 667 to phenylalanine results in a pathological retention of ?-DG at the endoplasmic reticulum level leading to a severe pathological state. With the aim of elucidating the structural implications of the pathological mutations leading to primary distroglycanopathies, we have undertaken a multi-technique study, involving biochemical and biophysical methods as well. Indeed, synchrotron radiation (SR) provides an invaluable tool for the elucidation of the molecular structure of biological macromolecules in both crystals and solution. We have used SR-based X-ray crystallography in order to determine the molecular structure, at the atomic resolution, of ?-DG point mutants. Moreover, SR-based Small Angle X-ray Scattering (SAXS) has been used as a low-resolution probe for the structural organization of ? and ? DG mutants at near-physiological conditions. The results of the SR-based experiments, combined with biochemical and microscopic analysis allowed to shed light on the molecular and structural basis of primary dystroglycanopathies.

A better understanding of the interactions between perfluroocatanoic acid (PFOA) and human serum albumin (HSA) can improve the design of protein-based electrochemical biosensors for fluorinated environmental contaminants, namely per- and poly-fluoroalkyl substances (PFAS). In the last decade, the affinity of PFOA for fatted and defatted serum albumin was confirmed by numerous toxicological studies 1-3 . The results showed that PFOA can bind strongly to serum albumins, such as HAS, mimicking fatty acids binding and affecting the functions of these transport proteins. To transpose these findings in the design of bioreceptors, a multi-analytical study was carried out comparing directly the performances of fatted and defatted HSA in terms of stability, number of binding sites and affinity towards PFOA and other PFAS . Isothermal titration calorimetry (ITC) measurements allowed to define the stoichiometry and the affinity constants, while native nano-electronspray ionization MS (nESI-MS) provided additional information about the stability of the proteins and the protein-target complex. To identify the binding site and discriminate between fatted and defatted HSA, vapor diffusion crystallization was performed. All analysis confirmed the higher affinity of PFOA for the defatted HSA even though the fatted HSA showed an higher stability. Both types of HSA were immobilized on the graphite-screen printed electrodes modified with graphene oxide (GO) or electropolymerized o-phenylene diamine (oPD). Impedance electrochemical spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the immobilization strategies performances. Also direct detection by EIS and indirect detection using a redox mediator were compared to evaluate the binding event between PFOA and HAS. The combination of toxicological data and electrochemistry allowed to devise a new detection strategy for environmental contaminants, mimicking a biological process happening in vivo for biosensing purposes.

Nerve Growth Factor (NGF) is the prototype of the neurotrophins family and induces cell growth and differentiation in neuronal cell types. Despite its discovery in the '50s, many molecular and functional properties remain elusive. Small endogenous NGF ligands with biological modulating effect are of increasing interest. Among these, ATP showed to mediate NGF neurotrophic activity through its receptors, although the determinants of the binding mechanism are yet to be unraveled. We thus undertook a biophysical study on NGF/ATP binding. We obtained 15N- and 13C15N-labeled recombinant human NGF (rhNGF), through the optimization of protocols previously established for mouse NGF (rmNGF). Interestingly, rhNGF and rmNGF differ in both their biochemical/biophysical and functional properties, despite their highly similar 3D structures. We performed 2D and 3D NMR experiments in order to complete the backbone and side chains resonance assignment of rhNGF. Differential Scanning Fluorimetry was used to investigate the binding effects of ATP and of a set of divalent ions on rhNGF. We verified by FT-IR that NGF secondary structure is retained in presence of ATP. We then moved to the investigation of the rhNGF/ATP binding mode, using protein-based solution NMR. We recorded 2D HSQC spectra following a titration with increasing amounts of ATP. We identified the likely binding site of ATP on rhNGF dimer and NMR guided molecular docking unveiled a possible interaction mechanism. Finally, we investigated the ATP binding mode of ATP on rhNGF by means of Isothermal Titration Calorimetry. In order to suggest an effect of ATP binding on the NGF/receptors signaling pathways, we undertook a study on the receptor binding by means of SPR. The effect of different divalent cations was also taken into consideration, and the obtained results are interestingly related to the role of NGF and proNGF in health and disease.

Plant cell walls are complex macromolecular composites and their degradation by microorganisms requires a large and diverse panel of hydrolytic enzymes. Moreover, the degradation and fermentation of complex polysaccharides by microbiota is essential for many ecosystem-processes including nutrient cycling and herbivores nutrition. Among the different structural polysaccharides composing the plant cell walls, xylan is the second most abundant one and its degradation in rumen involves the removal of O-acetyl esters decorating the O-2 and O-3 positions of the D-xylanopyranosyl moiety. A transcriptomic analysis of a gene cluster, present in the ruminal anaerobe Prevotella ruminicola 23, revealed a repertoire of xylanolytic enzymes with different functions and cellular localization [1]. Among them Axe7A, an acetylxylan esterase belonging to the CAZy CE7 carbohydrate esterase family, has been established [2]. Interestingly Axe7A amino acidic sequence includes, besides the characteristic catalytic domain, an additional 100 residues long N-terminal domain no previously found in the any other member of the CE7 family members, showing low similarity (around 10% identity) with any other structurally characterized protein annotated in the PDB. In order to gain insights into the functional role of its N-terminal domain, the crystal structure of Axe7XE7A has been solved by SAD phasing and refined at 2.6 Å resolution. Axe7A shares the 32 hexameric doughnut-shape commonly found in with all the CE7 deacetylases [3], while the unique protomer architecture has been disclosed in the CE7 family, for the first time. The N-terminal domain, characterized by a ?-sandwich fold, is actively engaged in the oligomer formation. We are further investigating, by SAXS, the behaviour of Axe7A in solution aiming at validate the crystallographic oligomeric assembly in nearly-physiological conditions.

Dystroglycan (DG) is a glycoprotein complex that links the cytoskeleton with the extracellular matrix. DG is composed of two subunits: alpha-DG and beta-DG. alpha-DG is a highly glycosylated extracellular protein which is essential for high-affinity binding of extracellular matrix proteins such as laminins. The hypoglycosylation of alpha-DG weakens this interaction affinity resulting in severe pathological states. The N-terminal region (aa 50-313 in mouse) of alpha-DG plays a crucial role in the glycosylation of the ?-DG mucin-like domain, being required by the glycosyltransferase LARGE during the extension of the O-glycans implicated in laminin binding. Furthermore, pathological missense mutations, observed at the N-terminal region of alpha-DG, are responsible of alpha-DG hypoglycosylation states resulting in primary distroglycanopathies1. We have been investigating the structures of the three point mutants V72I (V72I-ma-DG), D109N (D109N-ma-DG) and T190M2 (T190M-ma-DG) of mouse alpha-DG (ma-DG), combining X-ray crystallography and in solution Small Angle X-ray Scattering (SAXS). The purpose of this study is to gain evidences about the structural determinants of N-terminal ?-DG that are functionally relevant for its glycosylation pathway. The crystal structures of the three mutants display the same overall structure of WT-ma-DG. In contrast, the solution structural models obtained by SAXS analysis depict a different scenario, where the WT-a-DG is quite flexible in solution, assuming more than one conformation. The comparison of SAXS data from WT-ma-DG and its mutants demonstrates the presence of a perturbation, in both the conformations as well as in the partition among different populations, which is mediated by the flexible peptide linking the Ig-like and S6 small-ribosomal domains, embodied in the alpha-DG N-terminal region. SAXS data thus suggest a more complex and dynamical situation with respect to the crystallographic evidences that may have functional implications for DG post-translational processing and for the interactions with its binding partners.

Dystroglycan (DG) is a glycoprotein complex that links the cytoskeleton with the extracellular matrix. DG is composed of two subunits: alpha-DG and beta-DG. alpha-DG is a highly glycosylated extracellular protein, whereas beta-DG is an integral membrane protein that also interacts with dystrophin in the cytoplasm. alpha-DG's glycosylation is essential for high-affinity binding of extracellular matrix proteins such as laminins. The hypoglycosylation of alpha-DG weakens this interaction affinity resulting in severe pathological states. The N-terminal region (a.a 50-313 in mouse) of alpha-DG plays a crucial role in the glycosylation of the alpha-DG mucin-like domain, being required by the glycosyltransferase LARGE during the extension of the O-glycans implicated in laminin binding. Furthermore, pathological missense mutations, observed at the N-terminal region of alpha-DG, are responsible of alpha-DG hypoglycosylation state. We have been investigating the structures of N-terminal region of the WT human alpha-DG (WT-ha-DG) and of the two point mutants V72I (V72I-ma-DG) and D109N (D109N-ma-DG) of mouse alpha-DG by X-ray crystallography, and in solution by Small Angle X-ray Scattering (SAXS). The purpose of this study is to gain evidences about the structural determinants of N-terminal alpha-DG that are functionally relevant for its glycosylation pathway. The crystal structure of the ha-DG does not significantly deviate from the already determined crystal structure of WT mouse alpha-DG (WT-ma-DG). The overall fold is conserved and differences are restricted to the most flexible part of the protein, i.e. the loop encompassing residues 159-179, which is only partially visible after crystal structure refinement. In addition, the crystal structures of the two mutants V72I-ma-DG and D109N-ma-DG display the same overall structure of WT-ma-DG, suggesting for negligible effects of the point mutations on the overall fold of alpha-DG. The two mutants show limited and local structural dissimilarities with respect to WT-ma-DG, probably influencing the interaction with potential binding partners. In contrast, the solution structural models obtained by SAXS analysis depict a different scenario, where the WT-a-DG is quite flexible in solution, assuming more than one conformation, with the most populated close to the crystal conformation and a second less populated one, which is more extended with respect to the principal conformation. The comparison of SAXS data from WT-ma-DG and its mutants demonstrates the presence of a perturbation, in both the conformations as well as in the partition among different populations. SAXS data thus suggest a more complex and dynamical situation with respect to the crystallographic evidences that may have functional implications for DG post-translational processing and for the interactions with its binding partners.

Trypsins were crystallized by using membrane crystallization techniques, both in static and dynamic configurations. Crystal size distribution analysis revealed the high uniformity in size of the products that is achievable. Crystals produced by using membrane crystallization methods are more uniform than those obtained in conventional batch experiments. Crystals produced in forced solution flow are even more uniform than those obtained in a quiescent membrane system. Typical values obtained of percent standard deviation and Span are comparable to or even better than those reported in the literature. Crystal growth rate is favorably affected by solution convective motions in forced solution flow, by increasing solution velocity up to a maximum, which, in the best case, has been observed to be ca. 1500 μm/s. These high values of suitable velocities are characteristics of the membrane system; as with traditional method, growth rate deceleration was already visible at 250 μm/s. The maximum seems to be shifted...

The production of protein crystals with adequate size and high lattice perfection for X-ray analysis appears as the bottleneck for obtaining diffraction data at a resolution sufficient to establish 3D structure-function correlation. In previous investigations, microporous hydrophobic membranes have emerged as effective tools for macromolecular crystallization: polymeric supports have been used both to generate a controlled supersaturated environment, and to promote heterogeneous nucleation. In this work, experiments have been carried out under forced convection in order to verify if the laminar flow of the mother solution through polypropylene hollow-fibers can induce an ordered growth of hen egg white lysozyme (HEWL) crystals. In the range of flow velocities investigated (210–2100 μm/s), the values of the kinetic Peclet number confirmed a shifting of the operative system point to a regime with significantly faster transport and higher relative weight of interfacial kinetics in the overall control of the crystallization process. Morphological analysis proved that tetragonal lysozyme crystals became convectively-oriented, aligning their c -axis in the flow direction. Kinetic analysis evidenced a maximum in crystal growth rate in correspondence of a flow velocity of about 1100 μm/s for crystallization tests performed at NaCl 3% (w/v), MgCl 2 10% (w/v), and T 5 °C. The enzymatic activity of the crystallized product excluded denaturation of the recirculating protein solution. Finally, crystals have been qualitatively assessed by X-ray crystallographic analysis carried out on a rotating anode X-ray generator (Cu Kα, λ = 1.5418 A) operating at 45 kV and 100 mA.

The homodimer NGF (nerve growth factor) exerts its neuronal activity upon binding to either or both distinct transmembrane receptors TrkA and $p75^{NTR}$. Functionally relevant interactions between NGF and these receptors have been proposed, on the basis of binding and signaling experiments. Namely, a ternary $TrkA/NGF/p75^{NTR}$ complex is assumed to be crucial for the formation of the so-called high-affinity NGF binding sites. However, the existence, on the cell surface, of direct extracellular interactions is still a matter of controversy. Here, supported by a small-angle x-ray scattering solution study of human NGF, we propose that it is the oligomerization state of the secreted NGF that may drive the formation of the ternary heterocomplex. Our data demonstrate the occurrence in solution of a concentration-dependent distribution of dimers and dimer of dimers. A head-to-head molecular assembly configuration of the NGF dimer of dimers has been validated. Overall, these findings prompted us to suggest a new, to our knowledge, model for the transient ternary heterocomplex, i.e., a $TrkA/NGF/p75^{NTR}$ ligand/receptors molecular assembly with a (2:4:2) stoichiometry. This model would neatly solve the problem posed by the unconventional orientation of $p75^{NTR}$ with respect to TrkA, as being found in the crystal structures of the TrkA/NGF and $p75^{NTR}/NGF$ complexes.

Elettra is one of the first 3rd-generation storage rings, recently upgraded to routinely operate in top-up mode at both 2.0 and 2.4 GeV. The facility hosts four dedicated beamlines for crystallography, two open to the users and two under construction, and expected to be ready for public use in 2015. In service since 1994, XRD1 is a general-purpose diffraction beamline. The light source for this wide (4-21 keV) energy range beamline is a permanent magnet wiggler. XRD1 covers experiments ranging from grazing incidence X-ray diffraction to macromolecular crystallography, from industrial applications of powder diffraction to X-ray phasing with long wavelengths. The bending magnet powder diffraction beamline MCX has been open to users since 2009, with a focus on microstructural investigations and studies under non-ambient conditions. A superconducting wiggler delivers a high photon flux to a new fully automated beamline dedicated to macromolecular crystallography and to a branch beamline hosting a high-pressure powder X-ray diffraction station (both currently under construction). Users of the latter experimental station will have access to a specialized sample preparation laboratory, shared with the SISSI infrared beamline. A high throughput crystallization platform equipped with an imaging system for the remote viewing, evaluation and scoring of the macromolecular crystallization experiments has also been established and is open to the user community.

Two kinds of new optically active chelating ligands bearing a chiral sulfinyl functionality, namely the 3,4-bis-(p-tolylsulfinyl)hexanes 4 and the N-mono- and N,N-disubstituted β-p-tolylsulfinyl ethylamines 5 and 2, have been synthesized and their coordination chemistry with Pd(II) and Rh(I) metals has been studied in detail. Complexes formed with ligands 4 featured a homocoordination between the two sulfur donors and the metal atom, in solution as well as in the solid state, as demonstrated by X-ray diffraction of cis-dichloro[3,4-bis-(p-tolylsulfinyl)hexane]palladium(II) ((3R,4R,RS,RS)-7), in which the ligand shows a C2 symmetry. Complexes formed with ligands 2 and 5 showed a heterocoordination of the metal by the sulfur and the nitrogen donors, as confirmed by X-ray diffraction of cis-dichloro[N,N-dimethyl-2-(p-tolylsulfinyl)ethylamine]palladium(II) ((R)-6). All nine complexes 6−11, displaying a monomeric structure, have been synthesized and completely characterized in the solid state (IR, MS-FAB) as w...

A series of adducts of the type [(L # ) y R n SnX 4− n ]· z H 2 O (L # =1-methylimidazole, y =1 or 2, R=Me, Et, Bu n or Ph, n =1, 2 or 3, X=Cl, Br or I, z =0, 1/2 or 1) has been characterized in the solid state and in solution by analyses, spectral (IR, 119 Sn Mossbauer, and 1 H, 13 C and 119 Sn NMR) data and conductivity measurements. The molecular weight determinations and the NMR data indicate that these organotin(IV) complexes partly dissociate in chloroform and acetone solution. The donor L # interacts with [(CH 3 ) 3 SnNO 3 ], yielding the 2:1 ionic complex [(L # ) 2 (CH 3 ) 3 Sn]NO 3 . The derivative [(L # ) 2 (CH 3 ) 2 SnCl 2 ] reacts with NaClO 4 , AgNO 3 , NaBPh 4 and KSCN in ethanol and diethyl ether giving the complexes [(L # ) 2 (CH 3 ) 2 Sn(ClO 4 ) 2 ], [(L # )(CH 3 ) 2 Sn(NO 3 ) 2 (H 2 O)], [(L # )(CH 3 ) 2 SnCl(H 2 O) 2 ]BPh 4 and [(L # ) 2 (CH 3 ) 2 Sn(NCS) 2 ], respectively, whereas when [(L # )(CH 3 ) 3 SnCl] interacts with an equimolar quantity of NaClO 4 , [(L # ) 2 (CH 3 ) 3 Sn]ClO 4 ·1/2H 2 O and (CH 3 ) 3 Sn(ClO 4 ) in 1:1 ratio are obtained. The stability towards self-decomposition of the complexes obtained decreases with increasing number of the Sn-bonded aryl or alkyl groups. The derivative [(L # ) 2 (CH 3 ) 2 SnBr 2 ] reacts with 1,10-phenanthroline (Phen), yielding immediately the complex [(Phen)(CH 3 ) 2 SnBr 2 ], whereas from the reaction between [(L # ) 2 (CH 3 ) 2 Sn(ClO 4 ) 2 ] and Phen, the mixed ligand complex [(L # )(Phen)(CH 3 ) 2 Sn](ClO 4 ) 2 is obtained. A different behaviour has been shown from the diiodide complex [(L # ) 2 (C 2 H 5 ) 2 SnI 2 ] which reacts with Phen, yielding the compound with the 3:2 stoichiometry [(Phen) 3 {(C 2 H 5 ) 2 SnI 2 } 2 ]. Both the crystal structures of [(L # ) 2 (CH 3 ) 2 SnBr 2 ] and [(L # ) 2 (C 2 H 5 ) 2 SnI 2 ] show the tin atom in an all- trans octahedral regular configuration, whereas in [(L # )(C 6 H 5 ) 3 SnCl] the tin atom exhibits a distorted trigonal bipyramidal geometry, with the phenyl groups in the equatorial positions. A comparison was made with structural data of other R 3 SnXN-type derivatives. The molecular parameters of 1-methylimidazole in the tin(IV) complexes were used, together with other structural data in literature, to derive empirical rules concerning the imidazole donor.

X-ray diffraction occurs when an electromagnetic wave of wavelength of the order of magnitude of 1 A ? interacts with an ordered array of atoms like a molecular or ionic crystal. A brief summary of the physical phenomena and its applications to polymer science is given together with the experimental setup required for the experiments.

A brief summary of the principles behind X-ray Synchrotron microtomography is reported. Applications to polymer and membrame science are alo described.

17beta-Hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17beta-HSDcl) is an NADP(H)-dependent enzyme that preferentially catalyses the oxidoreduction of oestrogens and androgens. The enzyme belongs to the short-chain dehydrogenase/reductase superfamily and is the only fungal hydroxysteroid dehydrogenase known to date. 17beta-HSDcl has recently been characterized and cloned and has been the subject of several functional studies. Although several hypotheses on the physiological role of 17beta-HSDcl in fungal metabolism have been formulated, its function is still unclear. An X-ray crystallographic study has been undertaken and the optimal conditions for crystallization of 17beta-HSDcl (apo form) were established, resulting in well shaped crystals that diffracted to 1.7 A resolution. The space group was identified as I4(1)22, with unit-cell parameters a = b = 67.14, c = 266.77 A. Phasing was successfully performed by Patterson search techniques. A catalytic inactive mutant Tyr167Phe was also engineered, expressed, purified and crystallized for functional and structural studies.

New triorganotin(IV) derivatives [(Q)SnR3 · x(H2O)] (x = 0, R  Ph ; x = 1, R  Me and n Bu ) (in general QH  1-R′-3-methyl-4-R″ (CC)-pyrazol-5-one; in detail Q′H: R′ = C6H5, R″ = C6H5; QAH:R′ = C6H5, R″ = p-CH3OC6H4; QNH: R′ C6H5, R″  p-NO2C6H4; QBrH:R′ = C6H5, R″ = p -BrC6H4; Q″H: R′ = C6H5,R″ = CH3; QClH: R′ = C6H5, R″ = CCl3; QFH: R′ = C6H5, R″ = CF3; QMH: R′ = CH3 R″ = C6H5; QDH: R′ = CH3, R″ = CH3) have been synthesized and characterized by analysis and spectral (IR and 1H, 13C and 119Sn NMR) data. The (Q)SnPh3 derivatives are five-coordinated in the solid state, with a likely skewed cis-trigonal bipyramidal (cis-TBP) geometry around the tin center and the ligand (Q)− acting in the bidentate form. In [(Q)SnR3 · (H2O)] derivatives (R = nBu or Me) a coordination site is occupied by water, with the ligand (Q)− coordinating in a monodentate fashion. The crystal structure of [(QASnMe3 · (H2O)] has been determined: the tin atom is found in a distorted TBP environment, with the methyl in the equatorial positions. Two of the SnC bond lengths are normal (2.11(1) and 2.08(2) A) whereas the third is longer (2.18(2) A); the ligand binds the metal atom through one carbonyl oxygen in the apical position (SnO = 2.10(1) A). The bond lenght between H2O and Sn is longer (2.41(2) A), and the OSnO angle is 174.9(5)°. H atoms of water are involved in an intermolecular H-bond network with uncoordinated carbonyl and the pyridinic N atom of the ligand. In chloroform solution the[(Q)Snr3 · (H2O)] derivatives (R = Me or nBu) lost the molecule of water and adopt a tetrahedral arrangement. They also give rise to a slow disproportionation, yielding SnR4 and [(Q)2SnR2] derivatives.

A brief summary is given about X-ray production by synchrotrons. Applications to polymer science are also indicated.

The 17β-HSD (17β-hydroxysteroid dehydrogenase) from the filamentous fungus Cochliobolus lunatus (17β-HSDcl) is a NADP(H)-dependent enzyme that preferentially catalyses the interconversion of inactive 17-oxo-steroids and their active 17β-hydroxy counterparts. 17β-HSDcl belongs to the SDR (short-chain dehydrogenase/reductase) superfamily. It is currently the only fungal 17β-HSD member that has been described and represents one of the model enzymes of the cP1 classical subfamily of NADPH-dependent SDR enzymes. A thorough crystallographic analysis has been performed to better understand the structural aspects of this subfamily and provide insights into the evolution of the HSD enzymes. The crystal structures of the 17β-HSDcl apo, holo and coumestrol-inhibited ternary complex, and the active-site Y167F mutant reveal subtle conformational differences in the substrate-binding loop that probably modulate the catalytic activity of 17β-HSDcl. Coumestrol, a plant-derived non-steroidal compound with oestrogenic activity, inhibits 17β-HSDcl [IC50 2.8 μM; at 100 μM substrate (4-oestrene-3,17-dione)] by occupying the putative steroid-binding site. In addition to an extensive hydrogen-bonding network, coumestrol binding is stabilized further by π–π stacking interactions with Tyr212. A stopped-flow kinetic experiment clearly showed the coenzyme dissociation as the slowest step of the reaction and, in addition to the low steroid solubility, it prevents the accumulation of enzyme–coenzyme–steroid ternary complexes.

6-Trifluoromethyl-12-aryl(vinyl)indolo[1,2-c]quinazolines are prepared in high yield through the palladium-catalyzed reaction of readily available bis(o-trifluoroacetamidophenyl)acetylene with aryl or vinyl halides and triflates, followed by cyclization of the resultant derivatives. The reaction, which tolerates a variety of important functional groups, probably involves the formation of a 3-aryl-2-(o-trifluoroacetamidophenyl)indole intermediate, followed by its cyclization to the indolequinazoline product. Formation of the indoloquinazoline nucleus has been unambiguously determined via X-ray analysis.

The molecule of the zwitterionic title compound, C6H9NO4S, which lies on a mirror plane, shows a puckered chair conformation of the six-membered ring with the S and N atoms out of the mean plane of the other four C atoms by 0.929 (2) and 0.647 (2) Å, respectively. The ionized carboxyl group is equatorially oriented. The hydrogen-bonding network includes very short O—H...O [2.470 (2) Å] and N—H...S [3.471 (2) and 3.416 (2) Å] intermolecular contacts.

The anti-nerve growth factor (NGF) monoclonal antibody alphaD11 is a potent antagonist that neutralizes the biological functions of its antigen in vivo. NGF antagonism is expected to be a highly effective and safe therapeutic approach in many pain states. A comprehensive functional and structural analysis of alphaD11 monoclonal antibody was carried out, showing its ability to neutralize NGF binding to either tropomyosine receptor kinase A (TrkA) or p75 receptors. The 3-D structure of the alphaD11 Fab fragment was solved at 1.7 A resolution. A computational docking model of the alphaD11 Fab-NGF complex, based on epitope mapping using a pool of 44 NGF mutants and experimentally validated by small-angle X-ray scattering, provided the structural basis for identifying the residues involved in alphaD11 Fab binding. The present study pinpoints loop II of NGF to be an important structural determinant for NGF biological activity mediated by TrkA receptor.

The rat monoclonal neuroantibody alphaD11 is a potent antagonist that prevents the binding of nerve growth factor (NGF) to its tyrosine kinase A receptor (TrkA) in a variety of systems, most notably in two in vivo systems linked to crucial pathological states, such as Alzheimer's disease and HIV infection. To provide further insights into the mechanism of action of this potentially therapeutic monoclonal antibody, structural studies of the antigen-binding fragment (Fab) of alphaD11 were performed. alphaD11 IgG2a immunoglobulin was obtained from hybridomas by in vitro tissue culture. The alphaD11 Fab crystallizes in two crystal forms. Form I belongs to space group P1, with unit-cell parameters a = 42.7, b = 50.6, c = 102.7 A, alpha = 82.0, beta = 89.1, gamma = 86.0 degrees. With two molecules in the asymmetric unit, V(M) is 2.3 A(3) Da(-1) and the solvent content is 46%. A complete data set has been collected at 2.7 A resolution on beamline XRD-1 (ELETTRA, Trieste, Italy). Form II belongs to space group C2, with unit-cell parameters a = 114.8, b = 69.4, c = 64.10 A, beta = 117.0 degrees. With one molecule in the asymmetric unit, V(M) is 2.4 A(3) Da(-1) and the solvent content is 48%. A complete data set has been collected at 1.7 A resolution on beamline ID14-1 (ESRF, Grenoble, France). Phasing was successfully performed by Patterson search techniques and refinement of the structures is currently under way. Crystal forms I and II display a close-packing pattern.

The relevant kinetic aspects related to an innovative method of biological macromolecules crystallization based on microporous hydrophobic membranes, used both as active surfaces to promote heterogeneous nucleation and as a mass-transfer apparatus to concentrate macromolecular solutions by solvent removal in vapour phase, have been evaluated. Polypropylene membranes, supplied in the form of hollow fibres, have been aligned in a versatile system, designed for an on-line spectrophotometric monitoring of hen egg white lysozyme crystallizing solutions (experimental conditions: 0.1 M NaAc/HAc Buffer pH 4.6, 0.5–5.8% wt/vol NaCl, 20°C). The turbidity measurements have been exploited in order to follow: (i) the induction time of crystallization, (ii) the early stage nucleation kinetics based on the Rayleigh scattering theory, and (iii) the crystal growth rate (coupled with data evaluated from image-analysis carried out by optical microscopy) under a model hypothesis of exponential growth of clusters. The crystals have been qualitatively assessed by an X-ray crystallographic analysis carried out at the synchrotron light laboratory ELETTRA.

Novel complexes of rhodium(I) [Rh(diolefin)(Q″)] (where HQ″=1-phenyl-3-methyl-4-acetylpyrazol-5-one and diolefin=cycloocta-1,5-diene (COD), bicyclo[2.2.1]hepta-2,5-diene (NBD) or 1,5-hexadiene (HEX)) were synthesized and characterized by analytical and spectral data. [Rh(COD)(Q″)] interacts with 4,5-dimethyl-1,10-phenanthroline (Me 2 Phen) and 2,2′-bipyridil (Bipy) yielding the cationic derivatives [Rh(COD)(Me 2 Phen)](Q″)(H 2 O), [Rh(COD)(Bipy)](Q″)(H 2 O) upon displacement of the (Q″) − donor from the coordination sphere of the metal center. Whereas [Rh(COD)(Q″)] interacts with 2-benzoylpyridine (Bzpy) yielding the 1:1 adduct [Rh(COD)(Bzpy)(Q″)] in which Bzpy acts as N-monodentate donor. On the other hand the monodentate P-donors triphenylphosphine, triphenylphosphite, tricyclohexylphosphine and the bidentate bis(diphenylphosphino)ethane (DPPE) displace the COD ligand from [Rh(COD)(Q″)] giving the neutral derivatives [Rh(PR 3 ) 2 (Q″)] (PR 3 =PPh 3 , or P(OPh) 3 ) and [Rh(DPPE)(Q″)](H 2 O). HQ″ reacts with the dinuclear [Rh(CO) 2 Cl] 2 . The tetradentate cycloocto-tetraene (COT) reacts with [Rh(CO) 2 (Q″)] yielding the derivative [Rh(CO) 2 (HQ″)Cl] in which HQ″ acts as neutral monodentate O-donor ligand. Whereas in presence of NEt 3 HQ″ reacts with [Rh(CO) 2 Cl] 2 yielding [Rh(CO) 2 (Q″)]. In this complex, one molecule of CO can be replaced by one mole of Phen and Bipy or by two moles of PPh 3 and AsPh 3 yielding the derivatives [Rh(CO)(L) n (Q″)]·x(H 2 O) (L=Me 2 Phen or Bipy, n =1; L=PPh 3 or AsPh 3 , n =2) whereas one mole of DPPE displaces both the molecules of CO, yielding [Rh(DPPE)(Q″)] yielding the derivative [Rh(COT)(Q″)]. The X-ray crystal structure determination of [Rh(COD)(Q″)] establishes that the rhodium atom is in a square planar configuration with two adjacent sites occupied by the (Q″) − ligand in the O 2 -bidentate form (Rh–O distances=2.054(2) and 2.061(2) A). The COD ring has a twisted boat conformation with Rh–C distances in the range 2.101(3)–2.110(3) A. Comparison was made with structural data reported for several related tetracoordinated (COD)Rh(I) adducts.

6-Trifluoromethyl-12-aryl(vinyl)indolo[1,2-c]quinazolines are prepared in high yield through the palladium-catalyzed reaction of readily available bis(o-trifluoroacetamidophenyl)acetylene with aryl or vinyl halides and triflates, followed by cyclization of the resultant derivatives. The reaction, which tolerates a variety of important functional groups, probably involves the formation of a 3-aryl-2-(o-trifluoroacetamidophenyl)indole intermediate, followed by its cyclization to the indolequinazoline product. Formation of the indoloquinazoline nucleus has been unambiguously determined via X-ray analysis.