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Es werden Schwingkreismodelle angegeben, deren Säkulargleichungen mit denen formal identisch sind, die sich bei der Anwendung der Methode der Moleküleigenfunktionen auf das Problem der π -Elektronenzustände in Molekülen ungesättigter und aromatischer Kohlenwasserstoffe ergeben. Damit ergibt sich die Möglichkeit, die quantenmechanischen Säkularprobleme durch Messung der Eigenfrequenzen der Modelle zu bestimmen.
Glial cell line-derived neurotrophic factor (GDNF) is a ligand that activates, through co-receptor GDNF family receptor alpha-1 (GFRα1) and receptor tyrosine kinase “RET”, several signaling pathways crucial in the development and sustainment of multiple neuronal populations. We decided to study whether non-mammalian orthologs of these three proteins have conserved their function: can they activate the human counterparts? Using the baculovirus expression system, we expressed and purified Danio rerio RET, and its binding partners GFRα1 and GDNF, and Drosophila melanogaster RET and two isoforms of co-receptor GDNF receptor-like. Our results report high-level insect cell expression of post-translationally modified and dimerized zebrafish RET and its binding partners. We also found that zebrafish GFRα1 and GDNF are comparably active as mammalian cell-produced ones. We also report the first measurements of the affinity of the complex to RET in solution: at least for zebrafish, the Kd for GFRα1-GDNF binding RET is 5.9 μM. Surprisingly, we also found that zebrafish GDNF as well as zebrafish GFRα1 robustly activated human RET signaling and promoted the survival of cultured mouse dopaminergic neurons with comparable efficiency to mammalian GDNF, unlike E. coli-produced human proteins. These results contradict previous studies suggesting that mammalian GFRα1 and GDNF cannot bind and activate non-mammalian RET and vice versa.
A(syn)-U/T and G(syn)-C+ Hoogsteen (HG) base pairs (bps) are energetically more disfavored relative to Watson–Crick (WC) bps in A-RNA as compared to B-DNA by >1 kcal/mol for reasons that are not fully understood. Here, we used NMR spectroscopy, optical melting experiments, molecular dynamics simulations and modified nucleotides to identify factors that contribute to this destabilization of HG bps in A-RNA. Removing the 2′-hydroxyl at single purine nucleotides in A-RNA duplexes did not stabilize HG bps relative to WC. In contrast, loosening the A-form geometry using a bulge in A-RNA reduced the energy cost of forming HG bps at the flanking sites to B-DNA levels. A structural and thermodynamic analysis of purine-purine HG mismatches reveals that compared to B-DNA, the A-form geometry disfavors syn purines by 1.5–4 kcal/mol due to sugar-backbone rearrangements needed to sterically accommodate the syn base. Based on MD simulations, an additional penalty of 3–4 kcal/mol applies for purine-pyrimidine HG bps due to the higher energetic cost associated with moving the bases to form hydrogen bonds in A-RNA versus B-DNA. These results provide insights into a fundamental difference between A-RNA and B-DNA duplexes with important implications for how they respond to damage and post-transcriptional modifications.
The photochemical cleavage of the endoperoxides of anthradichromene and benzodixanthene into the parent hydrocarbon and oxygen is shown to be an adiabatic photoreaction originating from an upper excited singlet state. This photochemical behaviour is described by a theory for the photochemistry of endoperoxides developed by Kearns and Khan.
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.
The development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350–400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy (SMLM). In a single labeling step, antibodies conjugated with short DNA oligonucleotides visualized multiple targets by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. This approach avoids potential effects on structural integrity when using multiple rounds of immunolabeling and eliminates chromatic aberration, because all targets are imaged using a single excitation laser wavelength. This method proved robust for multi-target imaging in semi-thin tissue sections with a lateral resolution better than 25 nm, paving the way toward structural cell biology with single-molecule SRM.
The neuronal transcriptome changes dynamically to adapt to stimuli from the extracellular and intracellular environment. In this study, we adapted for the first time a click chemistry technique to label the newly synthesized RNA in cultured hippocampal neurons and intact larval zebrafish brain. Ethynyl uridine (EU) was incorporated into neuronal RNA in a time- and concentration-dependent manner. Newly synthesized RNA granules observed throughout the dendrites were colocalized with mRNA and rRNA markers. In zebrafish larvae, the application of EU to the swim water resulted in uptake and labeling throughout the brain. Using a GABA receptor antagonist, PTZ (pentylenetetrazol), to elevate neuronal activity, we demonstrate that newly transcribed RNA signal increased in specific regions involved in neurogenesis.
Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.
For a virtual screening study, we introduce a combination of machine learning techniques, employing a graph kernel, Gaussian process regression and clustered cross-validation. The aim was to find ligands of peroxisome-proliferator activated receptor gamma (PPAR-y). The receptors in the PPAR family belong to the steroid-thyroid-retinoid superfamily of nuclear receptors and act as transcription factors. They play a role in the regulation of lipid and glucose metabolism in vertebrates and are linked to various human processes and diseases. For this study, we used a dataset of 176 PPAR-y agonists published by Ruecker et al. ...
Two methods for the fast, fragment-based combinatorial molecule assembly were developed. The software COLIBREE® (Combinatorial Library Breeding) generates candidate structures from scratch, based on stochastic optimization [1]. Result structures of a COLIBREE design run are based on a fixed scaffold and variable linkers and side-chains. Linkers representing virtual chemical reactions and side-chain building blocks obtained from pseudo-retrosynthetic dissection of large compound databases are exchanged during optimization. The process of molecule design employs a discrete version of Particle Swarm Optimization (PSO) [2]. Assembled compounds are scored according to their similarity to known reference ligands. Distance to reference molecules is computed in the space of the topological pharmacophore descriptor CATS [3]. In a case study, the approach was applied to the de novo design of potential peroxisome proliferator-activated receptor (PPAR gamma) selective agonists. In a second approach, we developed the formal grammar Reaction-MQL [4] for the in silico representation and application of chemical reactions. Chemical transformation schemes are defined by functional groups participating in known organic reactions. The substructures are specified by the linear Molecular Query Language (MQL) [5]. The developed software package contains a parser for Reaction-MQL-expressions and enables users to design, test and virtually apply chemical reactions. The program has already been used to create combinatorial libraries for virtual screening studies. It was also applied in fragmentation studies with different sets of retrosynthetic reactions and various compound libraries.