Surface Plasmon Resonance as a Tool for Ligand Binding Investigation of Engineered GPR17 Receptor, a G Protein Coupled Receptor Involved in Myelination

The aim of this study was to investigate the potential of surface plasmon resonance (SPR) spectroscopy for the measurement of real-time ligand-binding affinities and kinetic parameters for GPR17, a G protein-coupled receptor (GPCR) of major interest in medicinal chemistry as potential target in demyelinating diseases. The receptor was directly captured, in a single-step, from solubilized membrane extracts on the sensor chip through a covalently bound anti-6x-His-antibody and retained its ligand binding activity for over 24h. Furthermore, our experimental setup made possible, after a mild regeneration step, to remove the bound receptor without damaging the antibody, and thus to reuse many times the same chip. Two engineered variants of GPR17, designed for crystallographic studies, were expressed in insect cells, extracted from crude membranes and analyzed for their binding with two high affinity ligands: the antagonist Cangrelor and the agonist Asinex 1. The calculated kinetic parameters and binding constants of ligands were in good agreement with those reported from activity assays and highlighted a possible functional role of the N-terminal residues of the receptor in ligand recognition and binding. Validation of SPR results was obtained by docking and molecular dynamics of GPR17-ligands interactions and by functional in vitro studies. The latter allowed us to confirm that Asinex 1 behaves as GPR17 receptor agonist, inhibits forskolin-stimulated adenylyl cyclase pathway and promotes oligodendrocyte precursor cell maturation and myelinating ability

Thermal behaviour of mixtures of an alunite ore with K2CO3, CaCO3 and Ca(OH)2

The thermal behaviour of alunite/K2CO3, alunite/CaCO3 and alunite/Ca(OH)(2) mixtures has been established. DTA coupled with XRD analysis has allowed appraisal of the main reactions that occur when the mixtures are roasted up to about 1000 degrees C. The main products of roasting are K2SO4, CaSO4 and other sulphur compounds and aluminates which retain the SO3 deriving from the alunite. The products are formed between 700 and 800 degrees C, The aim of the work was to assess the feasibility of a thermal process for use in the case of certain kaolin deposits whose exploitation is impossible at the present time because they are contaminated with alunite (KAl3(SO4)(2)(OH)(6)) This mineral causes great difficulty when attempting to use these kaolin ores because kilning drives off SO3 which corrodes the furnaces and is environmentally harmful. The K2SO4 and CaSO4 produced by roasting the indicated mixtures can be leached with water and dilute HCl respectively and the former can be used as fertiliser. The leaching residue, with its reduced sulphur content, can then be employed by the ceramics and building industries. (C) 1997 Elsevier Science B.V

Analytical approaches for the identification of new ligands of Peroxisome Proliferator-Activated Receptors: a complex journey.

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THE STRUCTURE OF THE 2/1 CHANNEL INCLUSION COMPOUND BETWEEN DEOXYCHOLIC-ACID AND PINACOLONE, 2C24H40O4.C6H12O

The title compd., 4C24H40O4.C14H15N3, crystd. in space group P212121 with a 25.676(8), b 13.731(3), and c 7.160(2) .ANG.; d.(exptl.) = 1.17 and d.(calcd.) = 1.18 for Z = 4. The structure was refined to R = 0.093 and RW = 0.099 for 1810 obsd. reflections with I > 2.sigma.(I). At. parameters are given. The crystal packing is characterized by an assembly of pleated antiparallel bilayers, nearly equal to that of the acetophenone-choleic acid complex, which give rise to canals filled by p-dimethylaminoazobenzene mols. The guest mols., located by van der Waals energy calcns., run along c and have their long axes approx. parallel to c. The C(5), C(6), C(19), C(20), C(21), and C(22) atoms, together with their H atoms, are engaged in strong interactions with the atoms of p-dimethylaminoazobenzene

Prochiral selectivity in H2O2-promoted oxidation of arylalkanols catalysed by chloroperoxidase. The role of the interactions between the OH group and the amino-acid residues in the enzyme active site

The H2O2-promoted oxidations of (R)-[alpha-H-2(1)]-and (S)-[alpha-H-2(1)]-arylalkanols catalysed by chloroperoxidase (CPO) from Caldariomyces fumago have been investigated. It has been found that with (R)-[alpha-H-2(1)]-alcohols, the oxidation involves almost exclusively the cleavage of the C-H bond, whereas in the case of the oxidation of (S)-[alpha-H-2(1)]alcohols, the C-D bond is preferentially broken. These results clearly indicate that the reactions of corresponding undeuterated arylalkanols are characterized by a high prochiral selectivity, involving the cleavage of the pro-S C-H bond. This prochiral selectivity is poorly influenced by the electronic effect of ring substituents, whereas it decreases with the length of the carbon lateral chain, in the order: benzyl alcohol > 2-phenylethanol > 3-phenylpropanol. Molecular binding studies showed that the main factor directing the docking of the substrate in such a specific orientation in the enzyme active site is the interaction between the alcoholic OH group and the residue Glu183. This interaction is likely to drive both the stereochemistry and the regiochemistry of these reactions. A bifurcated hydrogen bond involving the OH group, the carboxylate oxygen of Glu183 and the oxoferryl oxygen might also be operating

Design and biophysical characterization of atrazine-sensing peptides mimicking the Chlamydomonas reinhardtii plastoquinone binding niche

The plastoquinone (Q(B)) binding niche of the Photosystem II (PSII) D1 protein is the subject of intense research due to its capability to bind also anthropogenic pollutants. In this work, the Chlamydomonas reinhardtii D1 primary structure was used as a template to computationally design novel peptides enabling the binding of the herbicide atrazine. Three biomimetic molecules, containing the Q(B)-binding site in a loop shaped by two alpha-helices, were reconstituted by automated protein synthesis, and their structural and functional features deeply analysed by biophysical techniques. Standing out among the others, the biomimetic mutant peptide, D1pepMut, showed high ability to mimic the D1 protein in binding both Q(B) and atrazine. Circular dichroism spectra suggested a typical properly-folded alpha-helical structure, while isothermal titration calorimetry (ITC) provided a complete thermodynamic characterization of the molecular interaction. Atrazine binds to the D1pepMut with a high affinity (K-d = 2.84 mu M), and a favourable enthalpic contribution (Delta H = -11.9 kcal mol(-1)) driving the interaction. Fluorescence spectroscopy assays, in parallel to ITC data, provided hyperbolic titration curves indicating the occurrence of a single atrazine binding site. The binding resulted in structural stabilisation of the D1pepMut molecule, as suggested by atrazine-induced cooperative profiles for the fold-unfold transition. The interaction dynamics and the structural stability of the peptides in response to the ligand were particularly considered as mandatory parameters for biosensor/biochip development. These studies paved the way to the set-up of an array of synthetic mutant peptides with a wide range of affinity towards different classes of target analytes, for the development of optical nanosensing platforms for herbicide detection