Michaelis-Menten Quantification of Ligand Signaling Bias Applied to the Promiscuous Vasopressin V2 Receptor.

Activation of G protein-coupled receptors by agonists may result in the activation of one or more G proteins and recruitment of arrestins. The extent of the activation of each of these pathways depends on the intrinsic efficacy of the ligand. Quantification of intrinsic efficacy relative to a reference compound is essential for the development of novel compounds.

High-throughput Site-directed Scanning Mutagenesis Using a Two-fragment PCR Approach.

Site-directed scanning mutagenesis is a useful tool applied in studying protein function and designing proteins with new properties, such as increased stability or enzymatic activity. Creating a systematic library of hundreds of site-directed mutants is still a demanding and expensive task. The established protocols for making such libraries include PCR amplification of the recombinant DNA using a pair of primers carrying a target mutation in the same PCR.

Soluble and membrane-bound protein carrier mediate direct copper transport to the ethylene receptor family.

The plant hormone ethylene is a key regulator of plant growth, development and stress adaption. Ethylene perception and response are mediated by a family of integral membrane receptors (ETRs) localized at the ER-Golgi network. The biological function of these receptors relies on a protein-bound copper cofactor.

Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1.

Synthetic peptides derived from ethylene-insensitive protein 2 (EIN2), a central regulator of ethylene signalling, were recently shown to delay fruit ripening by interrupting protein-protein interactions in the ethylene signalling pathway. Here, we show that the inhibitory peptide NOP-1 binds to the GAF domain of ETR1 - the prototype of the plant ethylene receptor family. Site-directed mutagenesis and computational studies reveal the peptide interaction site and a plausible molecular mechanism for the ripening inhibition.

High-throughput mutagenesis using a two-fragment PCR approach.

Site-directed scanning mutagenesis is a powerful protein engineering technique which allows studies of protein functionality at single amino acid resolution and design of stabilized proteins for structural and biophysical work. However, creating libraries of hundreds of mutants remains a challenging, expensive and time-consuming process. The efficiency of the mutagenesis step is the key for fast and economical generation of such libraries.

Large-scale production and protein engineering of G protein-coupled receptors for structural studies.

Structural studies of G protein-coupled receptors (GPCRs) gave insights into molecular mechanisms of their action and contributed significantly to molecular pharmacology. This is primarily due to technical advances in protein engineering, production and crystallization of these important receptor targets. On the other hand, NMR spectroscopy of GPCRs, which can provide information about their dynamics, still remains challenging due to difficulties in preparation of isotopically labeled receptors and their low long-term stabilities.

Crystal structures and magnetic properties of a set of dihalo-bridged oxalamidato copper(II) dimers.

A set of four copper(ii) complexes, and (X = Cl, Br; = N-(l-leucine methyl ester)-N'-((2-pyridin-2-yl)methyl)oxalamide and = N-benzyl-N'-((2-pyridin-2-yl)methyl)oxalamide), have been synthesized and characterized by X-ray structural analysis, electron paramagnetic resonance (EPR) spectroscopy on single crystals and by SQUID magnetization measurements. X-ray diffraction studies show one-dimensional hydrogen bonded networks of dimeric copper(ii)-complexes bridged by two halide ions and with the two metal centers 3.44-3.

Cyclometalated heteronuclear Pt/Ag and Pt/Tl complexes: a structural and photophysical study.

To investigate the factors influencing the luminescent properties of polymetallic cycloplatinated complexes a detailed study of the photophysical and structural properties of the heteronuclear complexes [Pt2Me2(bhq)2(μ-dppy)2Ag2(μ-acetone)](BF4)2, 2, [PtMe(bhq)(dppy)Tl]PF6, 3, and [Pt2Me2(bhq)2(dppy)2Tl]PF6, 4, [bhq = benzo[h]quinoline, dppy = 2-(diphenylphosphino)pyridine] was conducted. Complexes 3 and 4 synthesized by the reaction of [PtMe(bhq)(dppy)], 1, with TlPF6 (1 or 1/2 equiv.) and stabilized by unsupported Pt-Tl bonds as revealed by multinuclear NMR spectroscopy and confirmed by X-ray crystallography for 3.

The formation of CuCl2-specific metallogels of pyridyloxalamide derivatives in alcohols.

Isomeric pyridyloxalamide derivatives 1-3, which differed in the position of the nitrogen atom on the pyridyl ring, showed remarkably different gel-forming aptitudes in the presence of CuCl2 salt in alcohols. Whilst derivatives 1 and 3 formed a soluble complex and a solid precipitate, respectively, ligand 2 generated a remarkably metal- and anion-specific metallogel.

Crystallographic snapshots of tyrosine phenol-lyase show that substrate strain plays a role in C-C bond cleavage.

The key step in the enzymatic reaction catalyzed by tyrosine phenol-lyase (TPL) is reversible cleavage of the Cβ-Cγ bond of L-tyrosine. Here, we present X-ray structures for two enzymatic states that form just before and after the cleavage of the carbon-carbon bond. As for most other pyridoxal 5'-phosphate-dependent enzymes, the first state, a quinonoid intermediate, is central for the catalysis.

Organoplatinum(II) complexes containing chelating or bridging bis(N-heterocyclic carbene) ligands: formation of a platinum(II) carbonate complex by aerial CO2 fixation.

The preparation of two new bis(N-heterocyclic carbene) platinum(II) complexes, in which NHC rings are joined by a CH(2) linker group, is described. While, the chelate complex [PtMe(2)(bis-NHC1)], 1, was formed with large tert-butyl wingtips, the iso-propyl N-substituent analogue favors formation of the cluster complex [Pt(2)Me(4)(μ-SMe(2))(μ-bis-NHC2)](2)(μ-Ag(2)Br(2)), 2, in which two binuclear platinum(II) complexes are linked together by an Ag(2)Br(2) unit. The chelating platinum complex 1 undergoes aerial CO(2) fixation and forms platinum(II) carbonate complex [Pt(CO(3))(bis-NHC1)], 3.

Cyclometalated cluster complex with a butterfly-shaped Pt2Ag2 core.

The cyclometalated platinum complex [PtMe(bhq)(dppy)] (1), in which bhq = benzo{h}quinoline and dppy = 2-(diphenylphosphino)pyridine, was prepared by the reaction of [PtMe(SMe(2))(bhq)] with 1 equiv of dppy at room temperature. Complex 1 contains one free pyridyl unit and was readily characterized by multinuclear NMR spectroscopy and elemental microanalysis. The reaction of complex 1 with 1 equiv of [Ag(CH(3)CN)(4)]BF(4) gave the cyclometalated cluster complex [Pt(2)Me(2)(bhq)(2)(mu-dppy)(2)Ag(2)(mu-acetone)](BF(4))(2) (2) in 70% yield.

Vanadium-induced formation of thiadiazole and thiazoline compounds. Mononuclear and dinuclear oxovanadium(v) complexes with open-chain and cyclized thiosemicarbazone ligands.

Reactions of the salicylaldehyde 4-phenylthiosemicarbazone (H(2)L) with selected vanadium(iv) and vanadium(v) precursors ([VO(acac)(2)], [VO(OAc)(2)], VOSO(4), [V(2)O(4)(acac)(2)]) were investigated under aerobic conditions in different alcohols (methanol, ethanol, propanol). In all examined cases mononuclear alkoxo vanadium(v) complexes [VOL(OR)] (1) (OR = OMe, OEt, OPr) were isolated as major products. On prolonged standing, mother liquids afforded dinuclear vanadium(v) complexes [V(2)O(3)(L(cycl))(2)(OR)(2)] (3) (OR = OMe, OEt, OPr), where L(cycl)(-) represents 1,3,4-thiadiazole ligand, formed by vanadium-induced oxidative cyclization of H(2)L.

Three quinolone compounds featuring O...I halogen bonding.

Ethyl 1-ethyl-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylate, C14H14INO3, (I), and ethyl 1-cyclopropyl-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylate, C15H14INO3, (II), have isomorphous crystal structures, while ethyl 1-dimethylamino-6-iodo-4-oxo-1,4-dihydroquinoline-3-carboxylate, C14H15IN2O3, (III), possesses a different solid-state supramolecular architecture. In all three structures, O..

Insights into the catalytic mechanism of tyrosine phenol-lyase from X-ray structures of quinonoid intermediates.

Amino acid transformations catalyzed by a number of pyridoxal 5'-phosphate (PLP)-dependent enzymes involve abstraction of the Calpha proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. Despite the key role played by the quinonoid intermediates in the catalysis by PLP-dependent enzymes, limited accurate information is available about their structures. We trapped the quinonoid intermediates of Citrobacter freundii tyrosine phenol-lyase with L-alanine and L-methionine in the crystalline state and determined their structures at 1.

Structures of apo- and holo-tyrosine phenol-lyase reveal a catalytically critical closed conformation and suggest a mechanism for activation by K+ ions.

Tyrosine phenol-lyase, a tetrameric pyridoxal 5'-phosphate dependent enzyme, catalyzes the reversible hydrolytic cleavage of L-tyrosine to phenol and ammonium pyruvate. Here we describe the crystal structure of the Citrobacter freundii holoenzyme at 1.9 A resolution.