Universitätspublikationen
Refine
Year of publication
Document Type
- Article (651)
- Doctoral Thesis (154)
- Preprint (32)
- Conference Proceeding (11)
- Part of a Book (2)
- Other (1)
Language
- English (851) (remove)
Has Fulltext
- yes (851) (remove)
Is part of the Bibliography
- no (851)
Keywords
- crystal structure (37)
- hydrogen bonding (11)
- NMR spectroscopy (7)
- RNA (7)
- structural biology (7)
- Optogenetics (6)
- Biochemistry (5)
- Bioenergetics (5)
- Membrane proteins (5)
- NMR (5)
Institute
- Biochemie und Chemie (851) (remove)
Das Hauptziel dieser Dissertation lag in der Verbesserung einzelner Schritte im Prozess der automatischen Proteinstrukturbestimmung mittels Kernmagnetischer Resonanz (NMR). Dieser Prozess besteht aus einer Reihe von sequenziellen Schritten, welche zum Teil bereits erfolgreich automatisiert wurden. CYANA ist ein Programmpaket, welches routinemäßig zur automatischen Zuordnung der chemischen Verschiebungen, der Nuclear Overhauser Enhancement (NOE) Signalen und der Strukturrechnung von Proteinen verwendet wird. Einer der Schritte, der noch nicht erfolgreich automatisiert wurde, stellt die Signalidentifizierung von NMR Spektren dar. Dieser Schritt ist besonders wichtig, da Listen von NMR-Signalen Grundlage aller Folgeschritte sind. Fehler in den Signallisten pflanzen sich in allen Folgeschritten der Datenauswertung fort und können am Ende in falschen Strukturen resultieren. Daher war ein Ziel dieser Arbeit, einen robusten und verlässlichen Algorithmus zur Signalidentifizierung von NMR Spektren in CYANA zu implementieren. Dieser Algorithmus sollte mit dem in FLYA implementierten Ansatz zur automatischen Resonanzzuordnung, der automatischen NOE-Zuordnung und der Strukturrechnung mit CYANA kombiniert werden. Der in CYANA implementierte CYPICK Algorithmus ahmt den von Hand durchgeführten Ansatz nach. Bei der manuellen Methode schaut sich der Wissenschaftler zweidimensionale Konturliniendarstellungen der NMR Spektren an und entscheidet anhand verschiedener Geomtrie- und Ähnlichkeitskriterien, ob es sich um ein Signal des Proteins oder um einen Artefakt handelt. Proteinsignale sind ähnlich zu konzentrischen Ellipsen und erfüllen bestimmte geometrische Kriterien, wie zum Beispiel ungefähr kreisförmiges Aussehen nach entsprechender Skalierung der spektralen Achsen und gänzlich konvexe Formen, die Artefakte nicht aufzeigen. CYPICK bewertet die Konturlinien lokaler Extrema nach diesen Bedingungen und entscheidet anhand dieser, ob es sich um ein echtes Signal handelt oder nicht. Das zweite Ziel dieser Arbeit war es ein Maß zur Quantifizierung der Information von strukturellen NMR Distanzeinschränkungen zu entwickeln. Der sogenannte Informationsgehalt (I) ist vergleichbar mit der Auflösung in der Röntgenkristallographie. Ein weiteres Projekt dieser Dissertation beschäftigte sich mit der strukturbasierten Medikamentenentwicklung (SBDD). SBDD wird meist von der Röntgenkristallographie durchgeführt. NMR hat jedoch einige Vorteile gegenüber der Röntgenkristallographie, welche interessant für SBDD sind. Daher wurden Strategien entwickelt, die NMR für SBDD zugänglicher machen sollen.
Understanding the nano-architecture of protein machines in diverse sub-cellular compartments remains a challenge despite rapid progress in super-resolution microscopy. While singlemolecule localization microscopy techniques allow the visualization and identification of cellular structures with near-molecular resolution, multiplex-labeling of tens of target proteins within the same sample has not yet been achieved routinely. However, single sample multiplexing is essential to detect patterns that threaten to get lost in multi-sample averaging. Here, we report maS3TORM (multiplexed automated serial staining stochastic optical reconstruction microscopy), a microscopy approach capable of fully automated 3D dSTORM imaging and solution exchange employing a re-staining protocol to achieve highly multiplexed protein localization within individual biological samples. We demonstrate 3D super-resolution images of 15 target proteins in single cultured cells and 16 targets in individual neuronal tissue samples with <10 nm localization precision. This allowed us to define novel nano-architectural features of protein distribution within the presynaptic nerve terminal.
Understanding the nano-architecture of protein machines in diverse subcellular compartments remains a challenge despite rapid progress in super-resolution microscopy. While single-molecule localization microscopy techniques allow the visualization and identification of cellular structures with near-molecular resolution, multiplex-labeling of tens of target proteins within the same sample has not yet been achieved routinely. However, single sample multiplexing is essential to detect patterns that threaten to get lost in multi-sample averaging. Here, we report maS3TORM (multiplexed automated serial staining stochastic optical reconstruction microscopy), a microscopy approach capable of fully automated 3D direct STORM (dSTORM) imaging and solution exchange employing a re-staining protocol to achieve highly multiplexed protein localization within individual biological samples. We demonstrate 3D super-resolution images of 15 targets in single cultured cells and 16 targets in individual neuronal tissue samples with <10 nm localization precision, allowing us to define distinct nano-architectural features of protein distribution within the presynaptic nerve terminal.
The three-dimensional structure determination of RNAs by NMR spectroscopy relies on chemical shift assignment, which still constitutes a bottleneck. In order to develop more efficient assignment strategies, we analysed relationships between sequence and 1H and 13C chemical shifts. Statistics of resonances from regularly Watson– Crick base-paired RNA revealed highly characteristic chemical shift clusters. We developed two approaches using these statistics for chemical shift assignment of double-stranded RNA (dsRNA): a manual approach that yields starting points for resonance assignment and simplifies decision trees and an automated approach based on the recently introduced automated resonance assignment algorithm FLYA. Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H10/C10 chemical shifts. The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20 nt and 42 nt. The more advanced automated assignment approach was successfully applied to four stemloop RNAs and a 42 nt siRNA, assigning 92–100% of the resonances from dsRNA regions correctly. This is the first automated approach for chemical shift assignment of non-exchangeable protons of RNA and their corresponding 13C resonances, which provides an important step toward automated structure determination of RNAs.
Macrophages ingesting apoptotic cells attenuate inflammatory responses, such as reactive oxygen species (ROS) generation. In atherosclerosis, ongoing inflammation and accumulation of apoptotic/necrotic material are observed, suggesting defects of phagocytes in recognizing or responding to dying cells. Modified lipoproteins such as oxidized LDL (oxLDL) are known to promote inflammation and to interfere with apoptotic cell clearance. Here, we studied the impact of cells exposed to oxLDL on their ability to interfere with the oxidative burst in phagocytes. In contrast to apoptotic cells, cells dying in response to or in the presence of oxLDL failed to suppress ROS generation despite efficiently being taken up by phagocytes. In addition, apoptotic cells, but not oxLDL-treated cells, inhibited phosphorylation of extracellular signal-regulated kinase, which is important for NADPH oxidase activation. oxLDL treatment did not interfere with activation of the antiinflammatory transcriptional regulator peroxisome proliferator-activated receptor gamma by apoptotic cells. Moreover, cells exposed to oxLDL failed to suppress lipopolysaccharide- induced proinflammatory cytokine expression, whereas apoptotic cells attenuated these phagocyte responses. Thus, the presence of oxLDL during cell death impaired the ability of apoptotic cells to act antiinflammatory with regard to oxidative burst inhibition and cytokine expression in phagocytes.
Attenuated NOX2 expression impairs ROS production during the hypoinflammatory phase of sepsis
(2012)
Background: The multicomponent phagocytic NADPH oxidase produces reactive oxygen species (ROS) after activation by microorganisms or inflammatory mediators. In the hypoinflammatory phase of sepsis, macrophages are alternatively activated by contact with apoptotic cells or their secretion products. This inhibits NADPH oxidase and leads to attenuated ROS production and furthermore contributes among others to a hyporeactive host defense. Due to this immune paralysis, sepsis patients suffer from recurrent and secondary infections. We focused on the catalytic subunit of NADPH oxidase, the transmembrane protein NOX2. We assume that after induction of sepsis the expression of NOX2 is reduced and hence ROS production is decreased.
Methods: We induced polymicrobial sepsis in mice by cecal ligation and puncture. The ability of peritoneal macrophages (PMs) to produce ROS was determined by FACS via hydroethidine assay. NOX2 expression of PMs was determined by western blot and qPCR. To elucidate the mechanism causing mRNA destabilization, we performed in vitro experiments using J774 macrophages. To obtain an alternatively activated phenotype, macrophages were stimulated with conditioned medium from apoptotic T cells (CM). By luciferase assays we figured out a 3'UTR-dependent regulation of NOX2 mRNA stability. Assuming that a protein is involved in the mRNA degradation, we performed a RNA pulldown with biotinylated NOX2-3'UTR constructs followed by mass spectrometry. We verified the role of SYNCRIP by siRNA approach. Additionally, we overexpressed NOX2 in J774 cells and analyzed the ROS production (w/wo CM treatment) by FACS.
Results: We found an impaired expression of NOX2 at RNA and protein level along with decreased ROS production after induction of sepsis in mice as well as stimulating J774 macrophages with CM of apoptotic T cells. This is due to a time-dependent NOX2 mRNA degradation depending on SYNCRIP, a RNA-binding protein, which stabilizes NOX2 mRNA through binding to its 3'UTR under normal conditions. In line, knockdown of SYNCRIP also decreases NOX2 mRNA expression. We assume that a CM-dependent modification or degradation of SYNCRIP prevents its stabilizing function. As the overexpression of NOX2 restores ROS production of CM-treated J774 cells, we assume that NOX2 expression is crucial for maintaining NADPH activity during the hypoinflammatory phase of sepsis.
Conclusion: Our data imply a regulatory impact of SYNCRIP on NOX2 stability during the late phase of sepsis. Therefore, further understanding of the regulation of NADPH oxidase could lead to the design of a therapy to reconstitute NADPH oxidase function, finally improving immune function in sepsis patients.
Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing 15N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R2 rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding.
The enantioselective synthesis of 2-aryl-substituted 2,3-dihydroquinolin-4-ones, a class of heterocyclic compounds with interesting biological activities, has been achieved through a Brønsted acidcatalyzed enantioselective intramolecular Michael addition. The products are available in moderate to high yields and with good enantioselectivities.
We have investigated the mechanism responsible for half-of-the-sites activity in the dimeric cytochrome bc(1) complex from Paracoccus denitrificans by characterizing the kinetics of inhibitor binding to the ubiquinol oxidation site at center P. Both myxothiazol and stigmatellin induced a 2-3 nm shift of the visible absorbance spectrum of the b(L) heme. The shift generated by myxothiazol was symmetric, with monophasic kinetics that indicate equal binding of this inhibitor to both center P sites. In contrast, stigmatellin generated an asymmetric shift in the b(L) spectrum, with biphasic kinetics in which each phase contributed approximately half of the total magnitude of the spectral change. The faster binding phase corresponded to a more symmetrical shift of the b(L) spectrum relative to the slower binding phase, indicating that approximately half of the center P sites bound stigmatellin more slowly and in a different position relative to the b(L) heme, generating a different effect on its electronic environment. Significantly, the slow stigmatellin binding phase was lost as the inhibitor concentration was increased. This implies that a conformational change is transmitted from one center P site in the dimer to the other upon stigmatellin binding to one monomer, rendering the second site less accessible to the inhibitor. Because the position that stigmatellin occupies at center P is considered to be analogous to that of the quinol substrate at the moment of electron transfer, these results indicate that the productive enzyme-substrate configuration is prevented from occurring in both monomers simultaneously.
The cytochrome bc1 complex recycles one of the two electrons from quinol (QH2) oxidation at center P by reducing quinone (Q) at center N to semiquinone (SQ), which is bound tightly. We have analyzed the properties of SQ bound at center N of the yeast bc1 complex. The EPR-detectable signal, which reports SQ bound in the vicinity of reduced bH heme, was abolished by the center N inhibitors antimycin, funiculosin, and ilicicolin H, but was unchanged by the center P inhibitors myxothiazol and stigmatellin. After correcting for the EPR-silent SQ bound close to oxidized bH, we calculated a midpoint redox potential (Em) of approximately 90 mV for all bound SQ. Considering the Em values for bH and free Q, this result indicates that center N preferentially stabilizes SQ.bH(3+) complexes. This favors recycling of the electron coming from center P and also implies a >2.5-fold higher affinity for QH2 than for Q at center N, which would potentially inhibit bH oxidation by Q. Using pre-steady-state kinetics, we show that Q does not inhibit the initial rate of bH reduction by QH2 through center N, but does decrease the extent of reduction, indicating that Q binds only when bH is reduced, whereas QH2 binds when bH is oxidized. Kinetic modeling of these results suggests that formation of SQ at one center N in the dimer allows stabilization of SQ in the other monomer by Q reduction after intradimer electron transfer. This model allows maximum SQ.bH(3+) formation without inhibition of Q binding by QH2.
Antigen presentation to cytotoxic T lymphocytes via major histocompatibility complex class I (MHC I) molecules depends on the heterodimeric transporter associated with antigen processing (TAP). For efficient antigen supply to MHC I molecules in the ER, TAP assembles a macromolecular peptide-loading complex (PLC) by recruiting tapasin. In evolution, TAP appeared together with effector cells of adaptive immunity at the transition from jawless to jawed vertebrates and diversified further within the jawed vertebrates. Here, we compared TAP function and interaction with tapasin of a range of species within two classes of jawed vertebrates. We found that avian and mammalian TAP1 and TAP2 form heterodimeric complexes across taxa. Moreover, the extra N-terminal domain TMD0 of mammalian TAP1 and TAP2 as well as avian TAP2 recruits tapasin. Strikingly, however, only TAP1 and TAP2 from the same taxon can form a functional heterodimeric translocation complex. These data demonstrate that the dimerization interface between TAP1 and TAP2 and the tapasin docking sites for PLC assembly are conserved in evolution, whereas elements of antigen translocation diverged later in evolution and are thus taxon specific.
Artificial splitting of a non‐ribosomal peptide synthetase by inserting natural docking domains
(2020)
The interaction in multisubunit non‐ribosomal peptide synthetases (NRPSs) is mediated by docking domains that ensure the correct subunit‐to‐subunit interaction. We introduced natural docking domains into the three‐module xefoampeptide synthetase (XfpS) to create two to three artificial NRPS XfpS subunits. The enzymatic performance of the split biosynthesis was measured by absolute quantification of the products by HPLC‐ESI‐MS. The connecting role of the docking domains was probed by deleting integral parts of them. The peptide production data was compared to soluble protein amounts of the NRPS using SDS‐PAGE. Reduced peptide synthesis was not a result of reduced soluble NRPS concentration but a consequence of the deletion of vital docking domain parts. Splitting the xefoampeptide biosynthesis polypeptide by introducing docking domains was feasible and resulted in higher amounts of product in one of the two tested split‐module cases compared to the full‐length wild‐type enzyme.
Artificial environments for the co-translational stabilization of cell-free expressed proteins
(2013)
An approach for designing individual expression environments that reduce or prevent protein aggregation and precipitation is described. Inefficient folding of difficult proteins in unfavorable translation environments can cause significant losses of overexpressed proteins as precipitates or inclusion bodies. A number of chemical chaperones including alcohols, polyols, polyions or polymers are known to have positive effects on protein stability. However, conventional expression approaches can use such stabilizing agents only post-translationally during protein extraction and purification. Proteins that already precipitate inside of the producer cells cannot be addressed. The open nature of cell-free protein expression systems offers the option to include single chemicals or cocktails of stabilizing compounds already into the expression environment. We report an approach for systematic screening of stabilizers in order to improve the solubility and quality of overexpressed proteins co-translationally. A comprehensive list of representative protein stabilizers from the major groups of naturally occurring chemical chaperones has been analyzed and their concentration ranges tolerated by cell-free expression systems have been determined. As a proof of concept, we have applied the method to improve the yield of proteins showing instability and partial precipitation during cell-free synthesis. Stabilizers that co-translationally improve the solubility and functional folding of human glucosamine 6-phosphate N-acetyltransferase have been identified and cumulative effects of stabilizers have been studied.
Cardiac arrhythmias are often associated with mutations in ion channels or other proteins. To enable drug development for distinct arrhythmias, model systems are required that allow implementing patient-specific mutations. We assessed a muscular pump in Caenorhabditis elegans. The pharynx utilizes homologues of most of the ion channels, pumps and transporters defining human cardiac physiology. To yield precise rhythmicity, we optically paced the pharynx using channelrhodopsin-2. We assessed pharynx pumping by extracellular recordings (electropharyngeograms--EPGs), and by a novel video-microscopy based method we developed, which allows analyzing multiple animals simultaneously. Mutations in the L-type VGCC (voltage-gated Ca(2+)-channel) EGL-19 caused prolonged pump duration, as found for analogous mutations in the Cav1.2 channel, associated with long QT syndrome. egl-19 mutations affected ability to pump at high frequency and induced arrhythmicity. The pharyngeal neurons did not influence these effects. We tested whether drugs could ameliorate arrhythmia in the optogenetically paced pharynx. The dihydropyridine analog Nemadipine A prolonged pump duration in wild type, and reduced or prolonged pump duration of distinct egl-19 alleles, thus indicating allele-specific effects. In sum, our model may allow screening of drug candidates affecting specific VGCCs mutations, and permit to better understand the effects of distinct mutations on a macroscopic level.
Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1
(2011)
The respiratory chain in the inner mitochondrial membrane contains three large multi-enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19-Å 3D map of the 1.7-MDa amphipol-solubilized supercomplex I1III2IV1 from bovine heart obtained by single-particle electron cryo-microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X-ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol-binding sites of complexes I and III, and of 10–11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred.
The thermodynamics of base pairing is of fundamental importance. Fluorinated base analogs are valuable tools for investigating pairing interactions. To understand the influence of direct base–base interactions in relation to the role of water, pairing free energies between natural nucleobases and fluorinated analogs are estimated by potential of mean force calculations. Compared to pairing of AU and GC, pairing involving fluorinated analogs is unfavorable by 0.5–1.0 kcal mol -1. Decomposing the pairing free energies into enthalpic and entropic contributions reveals fundamental differences for Watson–Crick pairs compared to pairs involving fluorinated analogs. These differences originate from direct base–base interactions and contributions of water. Pairing free energies of fluorinated base analogs with natural bases are less unfavorable by 0.5–1.0 kcal mol -1 compared to non-fluorinated analogs. This is attributed to stabilizing C–F…H–N dipolar interactions and stronger N…H–C hydrogen bonds, demonstrating direct and indirect influences of fluorine. 7-methyl-7H-purine and its 9-deaza analog (Z) have been suggested as members of a new class of non-fluorinated base analogs. Z is found to be the least destabilizing universal base in the context of RNA known to date. This is the first experimental evidence for nitrogen-containing heterocylces as bioisosteres of aromatic rings bearing fluorine atoms.
Antigenic and 3D structural characterization of soluble X4 and hybrid X4-R5 HIV-1 Env trimers
(2014)
Background: HIV-1 is decorated with trimeric glycoprotein spikes that enable infection by engaging CD4 and a chemokine coreceptor, either CCR5 or CXCR4. The variable loop 3 (V3) of the HIV-1 envelope protein (Env) is the main determinant for coreceptor usage. The predominant CCR5 using (R5) HIV-1 Env has been intensively studied in function and structure, whereas the trimeric architecture of the less frequent, but more cytopathic CXCR4 using (X4) HIV-1 Env is largely unknown, as are the consequences of sequence changes in and near V3 on antigenicity and trimeric Env structure.
Results: Soluble trimeric gp140 Env constructs were used as immunogenic mimics of the native spikes to analyze their antigenic properties in the context of their overall 3D structure. We generated soluble, uncleaved, gp140 trimers from a prototypic T-cell line-adapted (TCLA) X4 HIV-1 strain (NL4-3) and a hybrid (NL4-3/ADA), in which the V3 spanning region was substituted with that from the primary R5 isolate ADA. Compared to an ADA (R5) gp140, the NL4-3 (X4) construct revealed an overall higher antibody accessibility, which was most pronounced for the CD4 binding site (CD4bs), but also observed for mAbs against CD4 induced (CD4i) epitopes and gp41 mAbs. V3 mAbs showed significant binding differences to the three constructs, which were refined by SPR analysis. Of interest, the NL4-3/ADA construct with the hybrid NL4-3/ADA CD4bs showed impaired CD4 and CD4bs mAb reactivity despite the presence of the essential elements of the CD4bs epitope. We obtained 3D reconstructions of the NL4-3 and the NL4-3/ADA gp140 trimers via electron microscopy and single particle analysis, which indicates that both constructs inherit a propeller-like architecture. The first 3D reconstruction of an Env construct from an X4 TCLA HIV-1 strain reveals an open conformation, in contrast to recently published more closed structures from R5 Env. Exchanging the X4 V3 spanning region for that of R5 ADA did not alter the open Env architecture as deduced from its very similar 3D reconstruction.
Conclusions: 3D EM analysis showed an apparent open trimer configuration of X4 NL4-3 gp140 that is not modified by exchanging the V3 spanning region for R5 ADA.
Phenytoin (PHT), valproic acid, and modern antiepileptic drugs (AEDs), eg, remacemide, loreclezole, and safinamide, are only effective within a maximum of 70%-80% of epileptic patients, and in many cases the clinical use of AEDs is restricted by their side effects. Therefore, a continuous need remains to discover innovative chemical entities for the development of active and safer AEDs. Ligands targeting central histamine H3 receptors (H3Rs) for epilepsy might be a promising therapeutic approach. To determine the potential of H3Rs ligands as new AEDs, we recently reported that no anticonvulsant effects were observed for the (S)-2-(4-(3-(piperidin-1-yl)propoxy)benzylamino)propanamide (1). In continuation of our research, we asked whether anticonvulsant differences in activities will be observed for its R-enantiomer, namely, (R)-2-(4-(3-(piperidin-1-yl)propoxy)benzylamino)propaneamide (2) and analogs thereof, in maximum electroshock (MES)-, pentylenetetrazole (PTZ)-, and strychnine (STR)-induced convulsion models in rats having PHT and valproic acid (VPA) as reference AEDs. Unlike the S-enantiomer (1), the results show that animals pretreated intraperitoneally (ip) with the R-enantiomer 2 (10 mg/kg) were moderately protected in MES and STR induced models, whereas proconvulsant effect was observed for the same ligand in PTZ-induced convulsion models. However, animals pretreated with intraperitoneal doses of 5, 10, or 15 mg/kg of structurally bulkier (R)-enantiomer (3), in which 3-piperidinopropan-1-ol in ligand 2 was replaced by (4-(3-(piperidin-1-yl)propoxy)phenyl)methanol, and its (S)-enantiomer (4) significantly and in a dose-dependent manner reduced convulsions or exhibited full protection in MES and PTZ convulsions model, respectively. Interestingly, the protective effects observed for the (R)-enantiomer (3) in MES model were significantly greater than those of the standard H3R inverse agonist/antagonist pitolisant, comparable with those observed for PHT, and reversed when rats were pretreated with the selective H3R agonist R-(α)-methyl-histamine. Comparisons of the observed antagonistic in vitro affinities among the ligands 1-6 revealed profound stereoselectivity at human H3Rs with varying preferences for this receptor subtype. Moreover, the in vivo anticonvulsant effects observed in this study for ligands 1-6 showed stereoselectivity in different convulsion models in male adult rats.
Subvalent boron compounds contain boron atoms with oxidation numbers lower than +III. Over the last decades, the development of isolable derivatives has relied heavily on the use of specially designed ligands capable of stabilizing the electron‐rich boron centers electronically or through steric protection. Herein, we are exclusively reviewing anionic organo(hydro)boranes largely devoid of stabilizing ligands or heteroatom substituents. The restriction to these subvalent species is intended to minimize the risk of ligand artifacts being included when carving out the characteristic properties of the respective boron centers, such as nucleophilic or carbenoid behavior. The scope of this review encompasses triorganoborane radical monoanions ([·BR3]–) along with closed‐shell dianions ([:BR3]2–), boryl anions ([:BR2]–), as well as B–B single‐bonded diborane(6) dianions ([R3B–BR3]2–) and diborane(5) monoanions ([R2B–BR3]–), and finally B=B double‐bonded diborane(4) dianions ([R2B=BR2]2–). We are showing how these species are related to each other and comment on their bonding situations from an experimentalist's perspective.
Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as substrate selectivity. Non-disruptive methods avoiding critical steps, such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies. The combination of cell-free synthetic biology, nanodisc-technology and non-covalent mass spectrometry provides excellent synergies for the analysis of membrane protein oligomerization within defined membranes. We exemplify our strategy by oligomeric state characterization of various membrane proteins including ion channels, transporters and membrane-integrated enzymes assembling up to hexameric complexes. We further indicate a lipid-dependent dimer formation of MraY translocase correlating with the enzymatic activity. The detergent-free synthesis of membrane protein/nanodisc samples and the analysis by LILBID mass spectrometry provide a versatile platform for the analysis of membrane proteins in a native environment.