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The title compound, C12H20N4O, undergoes a phase transition on cooling. The room-temperature structure is tetragonal (P43212, Z′ = 1), with the methoxybornyl group being extremely disordered. Below 213 K the structure is orthorhombic (P212121, Z′ = 2), with ordered molecules. The two independent molecules (A and B) have very similar conformations; significant differences only occur for the torsion angles about the Cbornyl—Ctetrazole bonds. The independent molecules are approximately related by the pseudo-symmetry relation: xB = −1/4 + yA, yB = 3/4 - xA and zB = 1/4 + zA. In the crystal, molecules are connected by N—H⋯N hydrogen bonds between the tetrazole groups, forming a pseudo-43 helix parallel to the c-axis direction. The crystal studied was a merohedral twin with a refined twin fraction value of 0.231 (2).
This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts.
The title compound, [FeZr2(C5H5)4Cl2(C13H18B2)], is a heteronuclear complex that consists of a [3]ferrocenophane moiety substituted at each cyclopentadienyl (Cp) ring by a BH3 group; the BH3 group is bonded via two H atoms to the Zr atom of the zirconocene chloride moiety in a bidentate fashion. The two Cp rings of the [3]ferrocenophane moiety are aligned at a dihedral angle of 8.9 (4)° arising from the strain of the propane-1,3-diyl bridge linking the two Cp rings. [One methylene group is disordered over two positions with a site-occupation factor of 0.552 (18) for the major occupied site.] The dihedral angles between the Cp rings at the two Zr atoms are 50.0 (3) and 51.7 (3)°. The bonding Zr(...)H distances are in the range 1.89 (7)–2.14 (7) Å. As the two Cp rings of the ferrocene unit are connected by an ansa bridge, the two Zr atoms approach each other at 6.485 (1) Å. The crystal packing features C—H(...)Cl interactions.
The crystal structure of the title salt, [Li(CH3CN)4][B(NCS)4], is composed of discrete cations and anions. Both the Li and B atoms show a tetrahedral coordination by four equal ligands. The acetonitrile and isothiocyanate ligands are linear. The bond angles at the B atom are close to the ideal tetrahedral value [108.92 (18)–109.94 (16)°], but the bond angles at the Li atom show larger deviations [106.15 (17)–113.70 (17)°].
High-throughput protein localization studies require multiple strategies. Mass spectrometric analysis of defined cellular fractions is one of the complementary approaches to a diverse array of cell biological methods. In recent years, the protein content of different cellular (sub-)compartments was approached. Despite of all the efforts made, the analysis of membrane fractions remains difficult, in that the dissection of the proteomes of the envelope membranes of chloroplasts or mitochondria is often not reliable because sample purity is not always warranted. Moreover, proteomic studies are often restricted to single (model) species, and therefore limited in respect to differential individual evolution. In this study we analyzed the chloroplast envelope proteomes of different plant species, namely, the individual proteomes of inner and outer envelope (OE) membrane of Pisum sativum and the mixed envelope proteomes of Arabidopsis thaliana and Medicago sativa. The analysis of all three species yielded 341 identified proteins in total, 247 of them being unique. 39 proteins were genuine envelope proteins found in at least two species. Based on this and previous envelope studies we defined the core envelope proteome of chloroplasts. Comparing the general overlap of the available six independent studies (including ours) revealed only a number of 27 envelope proteins. Depending on the stringency of applied selection criteria we found 231 envelope proteins, while less stringent criteria increases this number to 649 putative envelope proteins. Based on the latter we provide a map of the outer and inner envelope core proteome, which includes many yet uncharacterized proteins predicted to be involved in transport, signaling, and response. Furthermore, a foundation for the functional characterization of yet unidentified functions of the inner and OE for further analyses is provided.
Background: Novel microscopic techniques which bypass the resolution limit in light microscopy are becoming routinely established today. The higher spatial resolution of super-resolution microscopy techniques demands for precise correction of drift, spectral and spatial offset of images recorded at different axial planes.
Methods: We employ a hydrophilic gel matrix for super-resolution microscopy of cellular structures. The matrix allows distributing fiducial markers in 3D, and using these for drift correction and multi-channel registration. We demonstrate single-molecule super-resolution microscopy with photoswitchable fluorophores at different axial planes. We calculate a correction matrix for each spectral channel, correct for drift, spectral and spatial offset in 3D.
Results and discussion: We demonstrate single-molecule super-resolution microscopy with photoswitchable fluorophores in a hydrophilic gel matrix. We distribute multi-color fiducial markers in the gel matrix and correct for drift and register multiple imaging channels. We perform two-color super-resolution imaging of click-labeled DNA and histone H2B in different axial planes, and demonstrate the quality of drift correction and channel registration quantitatively. This approach delivers robust microscopic data which is a prerequisite for data interpretation.
The light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum is tightly regulated by the [H+] gradient and transmembrane potential. BR exhibits optoelectric properties, since spectral changes during the photocycle are kinetically controlled by voltage, which predestines BR for optical storage or processing devices. BR mutants with prolonged lifetime of the blue-shifted M intermediate would be advantageous, but the optoelectric properties of such mutants are still elusive. Using expression in Xenopus oocytes and two-electrode voltage-clamping, we analyzed photocurrents of BR mutants with kinetically destabilized (F171C, F219L) or stabilized (D96N, D96G) M intermediate in response to green light (to probe H+ pumping) and blue laser flashes (to probe accumulation/decay of M). These mutants have divergent M lifetimes. As for BR-WT, this strictly correlates with the voltage dependence of H+ pumping. BR-F171C and BR-F219L showed photocurrents similar to BR-WT. Yet, BR-F171C showed a weaker voltage dependence of proton pumping. For both mutants, blue laser flashes applied during and after green-light illumination showed reduced M accumulation and shorter M lifetime. In contrast, BR-D96G and BR-D96N exhibited small photocurrents, with nonlinear current-voltage curves, which increased strongly in the presence of azide. Blue laser flashes showed heavy M accumulation and prolonged M lifetime, which accounts for the strongly reduced H+ pumping rate. Hyperpolarizing potentials augmented these effects. The combination of M-stabilizing and -destabilizing mutations in BR-D96G/F171C/F219L (BR-tri) shows that disruption of the primary proton donor Asp-96 is fatal for BR as a proton pump. Mechanistically, M destabilizing mutations cannot compensate for the disruption of Asp-96. Accordingly, BR-tri and BR-D96G photocurrents were similar. However, BR-tri showed negative blue laser flash-induced currents even without actinic green light, indicating that Schiff base deprotonation in BR-tri exists in the dark, in line with previous spectroscopic investigations. Thus, M-stabilizing mutations, including the triple mutation, drastically interfere with electrochemical H+ gradient generation.
Background: The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane.
Results: To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding.
Conclusions: Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases.