Electronic effects in profluorescent benzotriazinyl radicals: a combined experimental and theoretical study.

The synthesis, photophysical characterization, and quantum chemical calculations of a series of benzotriazinyl radicals and their styryl radical trapping products are presented. The benzotriazinyl radicals are non-luminescent but surprisingly the corresponding styryl radical trapping products exhibit high fluorescence quantum yields (up to 60% in some cases), making them highly valuable probes or labels. Additionally, the influence of the substitution pattern on the optical properties of the radical trapping products was observed experimentally and interpreted by means of quantum chemical calculations. Specific substitution patterns showed a bathochromic shift compared to the unsubstituted compound. Computationally, it was shown that this substitution pattern leads to a stronger energetic stabilization of the lowest unoccupied molecular orbital than the highest occupied molecular orbital. Analysis of the influence of the substitution pattern on the optical properties showed a bathochromic shift in several examples, which was interpreted by means of quantum chemical calculations.

Biophysical characterization of full-length human phenylalanine hydroxylase provides a deeper understanding of its quaternary structure equilibrium.

Dysfunction of human phenylalanine hydroxylase (hPAH, EC 1.14.16.1) is the primary cause of phenylketonuria, the most common inborn error of amino acid metabolism. The dynamic domain rearrangements of this multimeric protein have thwarted structural study of the full-length form for decades, until now. In this study, a tractable C29S variant of hPAH (C29S) yielded a 3.06 Å resolution crystal structure of the tetrameric resting-state conformation. We used size-exclusion chromatography in line with small-angle X-ray scattering (SEC-SAXS) to analyze the full-length hPAH solution structure both in the presence and absence of Phe, which serves as both substrate and allosteric activators. Allosteric Phe binding favors accumulation of an activated PAH tetramer conformation, which is biophysically distinct in solution. Protein characterization with enzyme kinetics and intrinsic fluorescence revealed that the C29S variant and hPAH are otherwise equivalent in their response to Phe, further supported by their behavior on various chromatography resins and by analytical ultracentrifugation. Modeling of resting-state and activated forms of C29S against SAXS data with available structural data created and evaluated several new models for the transition between the architecturally distinct conformations of PAH and highlighted unique intra- and inter-subunit interactions. Three best-fitting alternative models all placed the allosteric Phe-binding module 8-10 Å farther from the tetramer center than do all previous models. The structural insights into allosteric activation of hPAH reported here may help inform ongoing efforts to treat phenylketonuria with novel therapeutic approaches.

Simulations of the regulatory ACT domain of human phenylalanine hydroxylase (PAH) unveil its mechanism of phenylalanine binding.

Phenylalanine hydroxylase (PAH) regulates phenylalanine (Phe) levels in mammals to prevent neurotoxicity resulting from high Phe concentrations as observed in genetic disorders leading to hyperphenylalaninemia and phenylketonuria. PAH senses elevated Phe concentrations by transient allosteric Phe binding to a protein-protein interface between ACT domains of different subunits in a PAH tetramer. This interface is present in an activated PAH (A-PAH) tetramer and absent in a resting-state PAH (RS-PAH) tetramer. To investigate this allosteric sensing mechanism, here we used the GROMACS molecular dynamics simulation suite on the Folding@home computing platform to perform extensive molecular simulations and Markov state model (MSM) analysis of Phe binding to ACT domain dimers. These simulations strongly implicated a conformational selection mechanism for Phe association with ACT domain dimers and revealed protein motions that act as a gating mechanism for Phe binding. The MSMs also illuminate a highly mobile hairpin loop, consistent with experimental findings also presented here that the PAH variant L72W does not shift the PAH structural equilibrium toward the activated state. Finally, simulations of ACT domain monomers are presented, in which spontaneous transitions between resting-state and activated conformations are observed, also consistent with a mechanism of conformational selection. These mechanistic details provide detailed insight into the regulation of PAH activation and provide testable hypotheses for the development of new allosteric effectors to correct structural and functional defects in PAH.

Plastid stromules are induced by stress treatments acting through abscisic acid.

Stromules are highly dynamic stroma-filled tubules that extend from the surface of all plastid types in all multi-cellular plants examined to date. The stromule frequency (percentage of plastids with stromules) has generally been regarded as characteristic of the cell and tissue type. However, the present study shows that various stress treatments, including drought and salt stress, are able to induce stromule formation in the epidermal cells of tobacco hypocotyls and the root hairs of wheat seedlings. Application of abscisic acid (ABA) to tobacco and wheat seedlings induced stromule formation very effectively, and application of abamine, a specific inhibitor of ABA synthesis, prevented stromule induction by mannitol. Stromule induction by ABA was dependent on cytosolic protein synthesis, but not plastid protein synthesis. Stromules were more abundant in dark-grown seedlings than in light-grown seedlings, and the stromule frequency was increased by transfer of light-grown seedlings to the dark and decreased by illumination of dark-grown seedlings. Stromule formation was sensitive to red and far-red light, but not to blue light. Stromules were induced by treatment with ACC (1-aminocyclopropane-1-carboxylic acid), the first committed ethylene precursor, and by treatment with methyl jasmonate, but disappeared upon treatment of seedlings with salicylate. These observations indicate that abiotic, and most probably biotic, stresses are able to induce the formation of stromules in tobacco and wheat seedlings.

Effective Solid Electrolyte Interphase Formation on Lithium Metal Anodes by Mechanochemical Modification.

Lithium metal batteries are gaining increasing attention due to their potential for significantly higher theoretical energy density than conventional lithium ion batteries. Here, we present a novel mechanochemical modification method for lithium metal anodes, involving roll-pressing the lithium metal foil in contact with ionic liquid-based solutions, enabling the formation of an artificial solid electrolyte interphase with favorable properties such as an improved lithium ion transport and, most importantly, the suppression of dendrite growth, allowing homogeneous electrodeposition/-dissolution using conventional and highly conductive room temperature alkyl carbonate-based electrolytes. As a result, stable cycling in symmetrical Li∥Li cells is achieved even at a high current density of 10 mA cm-2. Furthermore, the rate capability and the capacity retention in NMC∥Li cells are significantly improved.

Manipulation of a cation-π sandwich reveals conformational flexibility in phenylalanine hydroxylase.

Phenylalanine hydroxylase (PAH) is an allosteric enzyme that maintains phenylalanine (Phe) below neurotoxic levels; its failure results in phenylketonuria, an inborn error of amino acid metabolism. Wild type (WT) PAH equilibrates among resting-state (RS-PAH) and activated (A-PAH) conformations, whose equilibrium position depends upon allosteric Phe binding. The RS-PAH conformation of WT rat PAH (rPAH) contains a cation-π sandwich involving Phe80 that cannot exist in the A-PAH conformation. Phe80 variants F80A, F80D, F80L, and F80R were prepared and evaluated using native PAGE, size exclusion chromatography, ion exchange behavior, intrinsic protein fluorescence, enzyme kinetics, and limited proteolysis, each as a function of [Phe]. Like WT rPAH, F80A and F80D show allosteric activation by Phe while F80L and F80R are constitutively active. Maximal activity of all variants suggests relief of a rate-determining conformational change. Limited proteolysis of WT rPAH (minus Phe) reveals facile cleavage within a 4-helix bundle that is buried in the RS-PAH tetramer interface, reflecting dynamic dissociation of that tetramer. This cleavage is not seen for the Phe80 variants, which all show proteolytic hypersensitivity in a linker that repositions during the RS-PAH to A-PAH interchange. Hypersensitivity is corrected by addition of Phe such that all variants become like WT rPAH and achieve the A-PAH conformation. Thus, manipulation of Phe80 perturbs the conformational space sampled by PAH, increasing sampling of on-pathway intermediates in the RS-PAH and A-PAH interchange. The behavior of the Phe80 variants mimics that of disease-associated R68S and suggests a molecular basis for proteolytic susceptibility in PKU-associated human PAH variants.

Effects of antibacterial mineral leachates on the cellular ultrastructure, morphology, and membrane integrity of Escherichia coli and methicillin-resistant Staphylococcus aureus.

BACKGROUND: We have previously identified two mineral mixtures, CB07 and BY07, and their respective aqueous leachates that exhibit in vitro antibacterial activity against a broad spectrum of pathogens. The present study assesses cellular ultrastructure and membrane integrity of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli after exposure to CB07 and BY07 aqueous leachates. METHODS: We used scanning and transmission electron microscopy to evaluate E. coli and MRSA ultrastructure and morphology following exposure to antibacterial leachates. Additionally, we employed Baclight LIVE/DEAD staining and flow cytometry to investigate the cellular membrane as a possible target for antibacterial activity. RESULTS: Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging of E. coli and MRSA revealed intact cells following exposure to antibacterial mineral leachates. TEM images of MRSA showed disruption of the cytoplasmic contents, distorted cell shape, irregular membranes, and distorted septa of dividing cells. TEM images of E. coli exposed to leachates exhibited different patterns of cytoplasmic condensation with respect to the controls and no apparent change in cell envelope structure. Although bactericidal activity of the leachates occurs more rapidly in E. coli than in MRSA, LIVE/DEAD staining demonstrated that the membrane of E. coli remains intact, while the MRSA membrane is permeabilized following exposure to the leachates. CONCLUSIONS: These data suggest that the leachate antibacterial mechanism of action differs for Gram-positive and Gram-negative organisms. Upon antibacterial mineral leachate exposure, structural integrity is retained, however, compromised membrane integrity accounts for bactericidal activity in Gram-positive, but not in Gram-negative cells.

Site preferences of NH4+ in its solid solutions with Cs2WS4 and Rb2WS4 from multinuclear solid-state MAS NMR.

Solid solutions of NH(4)(+) in Cs(2)WS(4) and Rb(2)WS(4) are obtained by precipitation/crystallization from aqueous solutions. By means of (14)N, (87)Rb, and (133)Cs magic angle spinning NMR, compositions and extraordinarily accurate NH(4)(+)-site preferences are established for these materials.

Synthesis and mechanical properties of organic-inorganic hybrid materials from lignin and polysiloxanes.

The preparation of silica-containing organic-inorganic hybrid materials composed of kraft lignin, alkoxysilanes, and organic linkers was investigated. 3-Glycidyloxypropyltrimethoxysilane, 3-(triethoxysilyl)propylisocyanate (IPTES), and bis(trimethoxysilyl)hexane were selected as the most promising linkers. The best materials obtained showed improved mechanical and thermal properties compared with lignin itself. The reaction of the hydroxyl groups with IPTES and the sol-gel reaction between the organic linker molecules were studied by attenuated total reflectance FTIR and solid-state ²9Si magic-angle spinning NMR spectroscopy. The homogeneous composition was demonstrated by electron microscopy and energy-dispersive X-ray spectroscopy mapping. The mechanical properties were investigated by microindentation and dynamic mechanical thermal analysis.