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The group of neurodegenerative diseases, Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) all exhibit inclusions containing amyloid-type α-synuclein (α-syn) aggregates within degenerating brain cells. α-syn also exists as soluble oligomeric species that are hypothesized to represent intermediates between its native and aggregated states. These oligomers are present in brain extracts from patients suffering from synucleinopathies and hold great potential as biomarkers. Although easily prepared in vitro, oligomers are metastable and dissociate over time, thereby complicating α-syn oligomer research. Using the small amine-reactive cross-linker, formaldehyde (FA), we successfully stabilized α-syn oligomers without affecting their size, overall structure or antigenicity towards aggregate-conformation specific α-syn antibodies FILA and MJFR-14-6-4-2. Further, cross-linked α-syn oligomers show resistance towards denaturant like urea and SDS treatment and remain fully functional as internal standard in an aggregation-specific enzyme-linked immunosorbent assay (ELISA) despite prior incubation with urea. We propose that FA cross-linked α-syn oligomers could serve as important calibrators to facilitate comparative and standardized α-syn biomarker studies going forward.
The combination of high-throughput sequencing and in vivo crosslinking approaches leads to the progressive uncovering of the complex interdependence between cellular transcriptome and proteome. Yet, the molecular determinants governing interactions in protein-RNA networks are not well understood. Here we investigated the relationship between the structure of an RNA and its ability to interact with proteins. Analysing in silico, in vitro and in vivo experiments, we find that the amount of double-stranded regions in an RNA correlates with the number of protein contacts. This relationship —which we call structure-driven protein interactivity— allows classification of RNA types, plays a role in gene regulation and could have implications for the formation of phase-separated ribonucleoprotein assemblies. We validate our hypothesis by showing that a highly structured RNA can rearrange the composition of a protein aggregate. We report that the tendency of proteins to phase-separate is reduced by interactions with specific RNAs.
De novo fatty acid biosynthesis in humans is accomplished by a multidomain protein, the Type I fatty acid synthase (FAS). Although ubiquitously expressed in all tissues, fatty acid synthesis is not essential in normal healthy cells due to sufficient supply with fatty acids by the diet. However, FAS is overexpressed in cancer cells and correlates with tumor malignancy, which makes FAS an attractive selective therapeutic target in tumorigenesis. Herein, we present a crystal structure of the condensing part of murine FAS, highly homologous to human FAS, with octanoyl moieties covalently bound to the transferase (MAT—malonyl‐/acetyltransferase) and the condensation (KS—β‐ketoacyl synthase) domain. The MAT domain binds the octanoyl moiety in a novel (unique) conformation, which reflects the pronounced conformational dynamics of the substrate‐binding site responsible for the MAT substrate promiscuity. In contrast, the KS binding pocket just subtly adapts to the octanoyl moiety upon substrate binding. Besides the rigid domain structure, we found a positive cooperative effect in the substrate binding of the KS domain by a comprehensive enzyme kinetic study. These structural and mechanistic findings contribute significantly to our understanding of the mode of action of FAS and may guide future rational inhibitor designs.
Current metabolomics approaches utilize cellular metabolite extracts, are destructive, and require high cell numbers. We introduce here an approach that enables the monitoring of cellular metabolism at lower cell numbers by observing the consumption/production of different metabolites over several kinetic data points of up to 48 hours. Our approach does not influence cellular viability, as we optimized the cellular matrix in comparison to other materials used in a variety of in‐cell NMR spectroscopy experiments. We are able to monitor real‐time metabolism of primary patient cells, which are extremely sensitive to external stress. Measurements are set up in an interleaved manner with short acquisition times (approximately 7 minutes per sample), which allows the monitoring of up to 15 patient samples simultaneously. Further, we implemented our approach for performing tracer‐based assays. Our approach will be important not only in the metabolomics fields, but also in individualized diagnostics.
Current metabolomics approaches utilize cellular metabolite extracts, are destructive, and require high cell numbers. We introduce here an approach that enables the monitoring of cellular metabolism at lower cell numbers by observing the consumption/production of different metabolites over several kinetic data points of up to 48 hours. Our approach does not influence cellular viability, as we optimized the cellular matrix in comparison to other materials used in a variety of in‐cell NMR spectroscopy experiments. We are able to monitor real‐time metabolism of primary patient cells, which are extremely sensitive to external stress. Measurements are set up in an interleaved manner with short acquisition times (approximately 7 minutes per sample), which allows the monitoring of up to 15 patient samples simultaneously. Further, we implemented our approach for performing tracer‐based assays. Our approach will be important not only in the metabolomics fields, but also in individualized diagnostics.
Remote control of the synthesis of a [2]rotaxane and its shuttling via metal‐ion translocation
(2019)
Remote control in an eight‐component network commanded both the synthesis and shuttling of a [2]rotaxane via metal‐ion translocation, the latter being easily monitored by distinct colorimetric and fluorimetric signals. Addition of zinc(II) ions to the red colored copper‐ion relay station rapidly liberated copper(I) ions and afforded the corresponding zinc complex that was visualized by a bright sky blue fluorescence at 460 nm. In a mixture of all eight components of the network, the liberated copper(I) ions were translocated to a macrocycle that catalyzed formation of a rotaxane by a double‐click reaction of acetylenic and diazide compounds. The shuttling frequency in the copper‐loaded [2]rotaxane was determined to k298=30 kHz (ΔH≠=62.3±0.6 kJ mol−1, ΔS≠=50.1±5.1 J mol−1 K−1, ΔG≠298=47.4 kJ mol−1). Removal of zinc(II) ions from the mixture reversed the system back generating the metal‐free rotaxane. Further alternate addition and removal of Zn2+ reversibly controlled the shuttling mode of the rotaxane in this eight‐component network where the ion translocation status was monitored by the naked eye.
A novel method for the highly stereoselective synthesis of tetrahydropyrans is reported. This domino reaction is based on a twofold addition of enamides to aldehydes followed by a subsequent cyclization and furnishes fully substituted tetrahydropyrans in high yields. Three new σ‐bonds and five continuous stereogenic centers are formed in this one‐pot process with a remarkable degree of diastereoselectivity. In most cases, the formation of only one out of 16 possible diastereomers is observed. Two different stereoisomers can be accessed in a controlled fashion starting either from an E‐ or a Z‐configured enamide.
The lysosomal polypeptide transporter TAPL belongs to the superfamily of ATP-binding cassette transporters. TAPL forms a homodimeric transport complex, which translocates oligo- and polypeptides into the lumen of lysosomes driven by ATP hydrolysis. Although the structure and the function of ABC transporters were intensively studied in the past, details about the single steps of the transport cycle are still elusive. Therefore, we analyzed the coupling of peptide binding, transport and ATP hydrolysis for different substrate sizes. Although longer and shorter peptides bind with the same affinity and are transported with identical Km values, they differ significantly in their transport rates. This difference can be attributed to a higher activation energy for the longer peptide. TAPL shows a basal ATPase activity, which is inhibited in the presence of longer peptides. Uncoupling between ATP hydrolysis and peptide transport increases with peptide length. Remarkably, also the type of nucleotide determines the uncoupling. While GTP is hydrolyzed as good as ATP, peptide transport is significantly reduced. In conclusion, TAPL does not differentiate between transport substrates in the binding process but during the following steps in the transport cycle, whereas, on the other hand, not only the coupling efficiency but also the activation energy varies depending on the size of peptide substrate.
The on-surface synthesis of bisheptahelicene by Ullmann coupling of 9-bromoheptahelicene on Au(111) and its temperature-induced dehydrogenation is studied using temperature-programmed reaction spectroscopy and time-of-flight secondary ion mass spectrometry. Specific dehydrogenation products of bisheptahelicene after loss of 6, 8 and 10 hydrogen atoms are identified, corresponding to molecules having undergone Diels–Alder transformations and intramolecular C–C coupling reactions. By combining with atomic hydrogen produced by dehydrogenation, the Ullmann coupling side-product bromine desorbs as HBr. H2 desorption emerges only after all Br has desorbed. Such characteristic behavior is explained by a kinetic model which explicitly considers the coverage of transient atomic H on the surface. Heating experiments performed with saturated layers of different Br-containing molecules reveal that the onset of HBr desorption depends strictly on the dehydrogenation step and therefore on the structure of the molecules.
EDTA is commonly used as an efficient chelator of metal ion enzyme cofactors. It is highly soluble, optically inactive and does not interfere with most chemicals used in standard buffers making EDTA a common choice to generate metal-free conditions for biochemical and biophysical investigations. However, the controversy in the literature on metal-free enzyme activities achieved using EDTA or by other means called our attention to a putative effect of EDTA beyond chelation. Here, we show that EDTA competes for the nucleotide binding site of the nucleotide hydrolase dUTPase by developing an interaction network within the active site similar to that of the substrate. To achieve these findings, we applied kinetics and molecular docking techniques using two different dUTPases. Furthermore, we directly measured the binding of EDTA to dUTPases and to two other dNTPases, the Taq polymerase and MutT using isothermal titration calorimetry. EDTA binding proved to be exothermic and mainly enthalpy driven with a submicromolar dissociation constant considerably lower than that of the enzyme:substrate or the Mg:EDTA complexes. Control proteins, including an ATPase, did not interact with EDTA. Our findings indicate that EDTA may act as a selective inhibitor against dNTP hydrolyzing enzymes and urge the rethinking of the utilization of EDTA in enzymatic experiments.
Formation of the anteroposterior and dorsoventral body axis in Caenorhabditis elegans depends on cortical flows and advection of polarity determinants. The role of this patterning mechanism in tissue polarization after formation of cell-cell contacts is not fully understood. Here, we demonstrate that planar asymmetries are established during left-right symmetry breaking: Centripetal cortical flows asymmetrically and differentially advect anterior polarity determinants (aPARs) from contacts to the medial cortex, resulting in their unmixing from apical myosin. Contact localization and advection of PAR-6 requires balanced CDC-42 activation, while asymmetric retention and advection of PAR-3 can occur independently of PAR-6. Concurrent asymmetric retention of PAR-3, E-cadherin/HMR-1 and opposing retention of antagonistic CDC-42 and Wnt pathway components leads to planar asymmetries. The most obvious mark of planar asymmetry, retention of PAR-3 at a single cell-cell contact, is required for proper cytokinetic cell intercalation. Hence, our data uncover how planar polarity is established in a system without the canonical planar cell polarity pathway through planar asymmetric retention of aPARs.
(Coumarin‐4‐yl)methyl (c4m) and p‐hydroxyphenacyl (pHP)‐based compounds are well known for their highly efficient photoreactions, but often show limited solubility in aqueous media. To circumvent this, we synthesized and characterized the two new c4m and pHP‐based photoacid generators (PAGs), 7‐[bis(carboxymethyl)amino]‐4‐(acetoxymethyl)coumarin (c4m‐ac) and p‐hydroxyphenacyl‐2,5,8,11‐tetraoxatridecan‐13‐oate (pHP‐t), and determined their solubilities, stabilities and photolysis in aqueous media. The two compounds showed high solubilities in water of 2.77 mmol L−1±0.07 mmol L−1 (c4m‐ac) and 124.66 mmol L−1±2.1 mmol L−1 (pHP‐t). In basic conditions at pH 9, solubility increased for c4m‐ac to 646.46 mmol L−1±0.63 mmol L−1, for pHP‐t it decreased to 34.68 mmol L−1±0.62 mmol L−1. Photochemical properties of the two PAGs, such as the absorption maxima, the maximum molar absorption coefficients and the quantum yields, were found to be strongly pH‐dependent. Both PAGs showed high stabilities s24h ≥95 % in water for 24 h, but decreasing stability with increasing pH value due to hydrolysis. The present study contributes to a clearer insight into the synthesis, solubilities, stabilities, and photolysis of c4m and pHP‐based PAGs for further photochemical applications when high PAG concentrations are required, such as in polymeric foaming.
Background: Esophageal cancer (EC) is one of the deadliest cancers worldwide. The contemporary strong increase of the adenocarcinomas in Western countries and the high mortality rates require the intensification of prospective multinational studies.
Methods: Therefore, this global health issue has been chosen for the bibliometric review of the global publication output. As source for meta and citation data, the Web of Science has been used and Density Equalizing Maps were applied for visualization.
Results: 17,387 articles on EC could be identified. The years with publication and citation maxima correspond to the appearance of the most prolific articles. China is the most publishing country, followed by Japan and the USA. Germany and the UK ranked 4th and 5th. The analysis of the ratios articles and socio-economic parameters emphasizes the leading position of the Scandinavian countries and Japan. Here, the high-income countries come out on top. The high incidence regions are mainly represented by Chinese and Japanese research. The association of the publication output and the overall research funding could be shown.
Conclusions: A strengthened international network increasingly consisting of the scientifically best positioned countries as well as more of the high incidence countries worldwide is mandatory for future research. The findings deliver scientists, clinicians and decision makers backgrounds for future decisions all over the world.
Nanopores are key in portable sequencing and research given their ability to transport elongated DNA or small bioactive molecules through narrow transmembrane channels. Transport of folded proteins could lead to similar scientific and technological benefits. Yet this has not been realised due to the shortage of wide and structurally defined natural pores. Here we report that a synthetic nanopore designed via DNA nanotechnology can accommodate folded proteins. Transport of fluorescent proteins through single pores is kinetically analysed using massively parallel optical readout with transparent silicon-on-insulator cavity chips vs. electrical recordings to reveal an at least 20-fold higher speed for the electrically driven movement. Pores nevertheless allow a high diffusive flux of more than 66 molecules per second that can also be directed beyond equillibria. The pores may be exploited to sense diagnostically relevant proteins with portable analysis technology, to create molecular gates for drug delivery, or to build synthetic cells.
The nuclear farnesoid X receptor (FXR) and the enzyme soluble epoxide hydrolase (sEH) are validated molecular targets to treat metabolic disorders such as non‐alcoholic steatohepatitis (NASH). Their simultaneous modulation in vivo has demonstrated a triad of anti‐NASH effects and thus may generate synergistic efficacy. Here we report dual FXR activators/sEH inhibitors derived from the anti‐asthma drug Zafirlukast. Systematic structural optimization of the scaffold has produced favorable dual potency on FXR and sEH while depleting the original cysteinyl leukotriene receptor antagonism of the lead drug. The resulting polypharmacological activity profile holds promise in the treatment of liver‐related metabolic diseases.
Thiophenylazobenzene: an alternative photoisomerization controlled by lone‐pair⋅⋅⋅π interaction
(2019)
Azoheteroarene photoswitches have attracted attention due to their unique properties. We present the stationary photochromism and ultrafast photoisomerization mechanism of thiophenylazobenzene (TphAB). It demonstrates impressive fatigue resistance and photoisomerization efficiency, and shows favorably separated (E)‐ and (Z)‐isomer absorption bands, allowing for highly selective photoconversion. The (Z)‐isomer of TphAB adopts an unusual orthogonal geometry where the thiophenyl group is perfectly perpendicular to the phenyl group. This geometry is stabilized by a rare lone‐pair⋅⋅⋅π interaction between the S atom and the phenyl group. The photoisomerization of TphAB occurs on the sub‐ps to ps timescale and is governed by this interaction. Therefore, the adoption and disruption of the orthogonal geometry requires significant movement along the inversion reaction coordinates (CNN and NNC angles). Our results establish TphAB as an excellent photoswitch with versatile properties that expand the application possibilities of AB derivatives.
Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 from Krokinobacter eikastus was discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na+ pumping is not yet understood. Here, we present a structure of the cationic (non H+) light-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics.
Archaea are motile by the rotation of the archaellum. The archaellum switches between clockwise and counterclockwise rotation, and movement along a chemical gradient is possible by modulation of the switching frequency. This modulation involves the response regulator CheY and the archaellum adaptor protein CheF. In this study, two new crystal forms and protein structures of CheY are reported. In both crystal forms, CheY is arranged in a domain-swapped conformation. CheF, the protein bridging the chemotaxis signal transduction system and the motility apparatus, was recombinantly expressed, purified and subjected to X-ray data collection.
From a global viewpoint, a lot of time is spent within the indoor air compartment of vehicles. A German study on mobility has revealed that, on average, people spend 45 minutes per day inside vehicles. In recent years the number of cars has increased to around 43 million vehicles in private households. This means that more than one car can be used in every household. The ratio has been growing, especially in eastern Germany and rural areas. "Overall and especially outside the cities, the car remains by far number one mode of transport, especially in terms of mileage". Therefore, numerous international studies have addressed different aspects of indoor air hygiene, in the past years. In this paper, meaningful original studies on car indoor air pollution, related to VOCs, COx, PMs, microbials, BFRs, OPFRs, cigarettes, electronic smoking devices, high molecular weight plasticizer, and NOx are summarized in the form of a review. This present review aimed to summarize recently published studies in this important field of environmental medicine and points to the need for further studies with special recommendations for optimizing the interior air hygiene.
Resonance assignments are challenging for membrane proteins due to the size of the lipid/detergent-protein complex and the presence of line-broadening from conformational exchange. As a consequence, many correlations are missing in the triple-resonance NMR experiments typically used for assignments. Herein, we present an approach in which correlations from these solution-state NMR experiments are supplemented by data from 13C unlabeling, single-amino acid type labeling, 4D NOESY data and proximity of moieties to lipids or water in combination with a structure of the protein. These additional data are used to edit the expected peaklists for the automated assignment protocol FLYA, a module of the program package CYANA. We demonstrate application of the protocol to the 262-residue proton pump from archaeal bacteriorhodopsin (bR) in lipid nanodiscs. The lipid-protein assembly is characterized by an overall correlation time of 44 ns. The protocol yielded assignments for 62% of all backbone (H, N, Cα, Cβ, C′) resonances of bR, corresponding to 74% of all observed backbone spin systems, and 60% of the Ala, Met, Ile (δ1), Leu and Val methyl groups, thus enabling to assign a large fraction of the protein without mutagenesis data. Most missing resonances stem from the extracellular half, likely due intermediate exchange line-broadening. Further analysis revealed that missing information of the amino acid type of the preceding residue is the largest problem, and that 4D NOESY experiments are particularly helpful to compensate for that information loss.
Advanced colorectal carcinoma is currently incurable, and new therapies are urgently needed. We report that phosphotyrosine-dependent Eph receptor signaling sustains colorectal carcinoma cell survival, thereby uncovering a survival pathway active in colorectal carcinoma cells. We find that genetic and biochemical inhibition of Eph tyrosine kinase activity or depletion of the Eph ligand EphrinB2 reproducibly induces colorectal carcinoma cell death by autophagy. Spautin and 3-methyladenine, inhibitors of early steps in the autophagic pathway, significantly reduce autophagy-mediated cell death that follows inhibition of phosphotyrosine-dependent Eph signaling in colorectal cancer cells. A small-molecule inhibitor of the Eph kinase, NVP-BHG712 or its regioisomer NVP-Iso, reduces human colorectal cancer cell growth in vitro and tumor growth in mice. Colorectal cancers express the EphrinB ligand and its Eph receptors at significantly higher levels than numerous other cancer types, supporting Eph signaling inhibition as a potential new strategy for the broad treatment of colorectal carcinoma.
Objective: Inhaled particulate matter (PM) in secondhand smoke (SHS) is deleterious for smokers and non-smokers. Different additives in cigarettes might effect the amount of PM. This study aimed to assess the influence of additives on the PM emissions from different cigarette types in SHS.
Design: An experimental study of PM measuring in SHS of cigarettes without exposition of any person.
Method: The concentrations of PM (PM10, PM2.5 and PM1) in SHS of four different types of cigarettes of the brand Lucky Strike, two types with additives (Original Red, Original Blue) and two types without additives (Straight Red, Straight Blue), in comparison to the reference cigarette 3R4F were analysed. An automatic environmental tobacco smoke emitter generated SHS in an enclosed space with a volume of 2.88 m3. PM was measured with a laser aerosol spectrometer (Grimm model 1.109). Afterwards, the measuring values of the four Lucky Strike brands and the reference cigarette were statistically evaluated and visualised.
Results: Lucky Strike Straight Blue, a cigarette type without additives and lower tar amount, showed 10% to 25% lower PM mean values compared with the other tested Lucky Strike products, but 21% (PM1) respectively 27% (PM2.5,PM10) higher mean values than the reference cigarette. The PM mean of all measured smoke-free baseline values (clean air) was 1.6 µg/m³. It increased up to about 1800 µg/m³ for the reference cigarette and up to about 3070 µg/m³ for the Lucky Strike Original Blue.
Conclusions: The findings of this study show the massive increase of PM amount by smoking cigarettes in enclosed spaces and suggest that additives in tobacco products increase the PM amount in SHS. For validation, further comparative studies are necessary focusing on the comparison of the PM concentration of cigarettes with and without additives.
Implications: Due to the exposure to SHS, 890 000 people die each year worldwide. PM in SHS endangers the health of both non-smokers and smokers. This study considers the effect of additives like aromatics and humectant agents in cigarettes on PM in SHS. Do additives in tobacco products increase the amount of PM?
Corneal topometric, aberrometric and biomechanical parameters in mucopolysaccharidosis patients
(2019)
Aims: To report corneal topometric and aberrometric values in mucopolysaccharidosis (MPS) and to investigate their correlation with biomechanical corneal parameters.
Methods: One randomly chosen eye of 20 MPS patients with no to moderate corneal clouding and one eye of 23 healthy controls with comparable age were prospectively included into this study. Corneal surface regularity was assessed by index of surface variance (ISV), -vertical asymmetry (IVA), -height asymmetry (IHA), -height decentration (IHD); keratoconus index (KI), central keratoconus index (CKI) and Zernike indices of anterior and posterior corneal surface using Scheimpflug imaging (Pentacam). Corneal resistance factor (CRF) and corneal hysteresis (CH) were assessed by Ocular Response Analyzer. Statistical analyses were performed using Mann-Whitney-Test and Spearman Correlation Coefficients.
Results: IVA, ISV, IHD, IHA, but not KI and CKI were significantly higher in MPS patients compared to age matched healthy controls. Spherical aberration and asphericity coefficients either at the anterior or at the posterior corneal surface differed significantly between both groups. The grade of the MPS-associated corneal opacity correlated significantly with ISV (rho = 0.52), IVA (rho = 0.54), IHA (rho = 0.57) and IHD (rho = 0.48). Density of the MPS-affected corneas correlated significantly with ISV (rho = 0.52), IVA (rho = 0.72), IHA (rho = 0.57), IHD (rho = 0.69), 3rd order horizontal trefoil aberration at the posterior (rho = 0.62) and anterior surface (rho = 0.48) as well as with CH (rho = 0.55) and CRF (rho = 0.57). Spherical aberration at the back surface correlated with CRF and CH in MPS and in healthy controls.
Conclusions: This is the first study analyzing shape of the corneal surface in MPS patients. Topometric indices of corneal asymmetry are significantly increased and correlate with MPS-related corneal opacity and density. Spherical aberration and asphericity coefficient at the front and at the back corneal surface differ significantly between MPS and healthy controls.
The inhalation of particulate matter (PM) in second-hand smoke (SHS) is hazardous to health of smokers and non-smokers. Tobacco strength (amount of tar, nicotine, and carbon monoxide) and different additives might have an effect on the amount of PM. This study aimed to investigate the influence of tobacco strength or additives on PM. Four cigarette types of the brand Marlboro with different strengths and with or without additives were analyzed in comparison to the 3R4F reference cigarette. SHS was generated by an automatic environmental tobacco smoke emitter (AETSE) in an enclosed space with a volume of 2.88 m³. PM concentrations (PM10, PM2.5, PM1) were measured with a laser aerosol spectrometer followed by statistical analysis. The two strongest Marlboro brands (Red and Red without additives) showed the highest PM concentrations of all tested cigarettes. The measured mean concentrations Cmean of PM10 increased up to 1458 µg/m³ for the Marlboro Red without additives (PM2.5: 1452 µg/m³, PM1: 1263 µg/m³). The similarly strong Marlboro Red showed very similar PM values. The second strongest type Marlboro Gold showed 36% (PM10, PM2.5) and 32% (PM1) lower values, respectively. The “lightest” type Marlboro Silver Blue showed 54% (PM10, PM2.5) or 50% (PM1) lower PM values. The results indicate that the lower the tar, nicotine, and carbon monoxide amounts, as well as the longer the cigarette filter, the lower are the PM levels. An influence of additives could not be determined.
Site-specific cleavage of RNAs derived from the PIM1 3′-UTR by a metal-free artificial ribonuclease
(2019)
Oligonucleotide conjugates of tris(2-aminobenzimidazole) have been reported previously to cleave complementary RNA strands with high levels of sequence and site specificity. The RNA substrates used in these studies were oligonucleotides not longer than 29-mers. Here we show that ~150–400-mer model transcripts derived from the 3′-untranslated region of the PIM1 mRNA reacted with rates and specificities comparable to those of short oligonucleotide substrates. The replacement of DNA by DNA/LNA mixmers further increased the cleavage rate. Tris(2-aminobenzimidazoles) were designed to interact with phosphates and phosphate esters. A cell, however, contains large amounts of phosphorylated species that may cause competitive inhibition of RNA cleavage. It is thus important to note that no loss in reaction rates was observed in phosphate buffer. This opens the way to in-cell applications for this type of artificial nuclease. Furthermore, we disclose a new synthetic method giving access to tris(2-aminobenzimidazoles) in multigram amounts.
High-resolution cryo-EM structures of respiratory complex I: Mechanism, assembly, and disease
(2019)
Respiratory complex I is a redox-driven proton pump, accounting for a large part of the electrochemical gradient that powers mitochondrial adenosine triphosphate synthesis. Complex I dysfunction is associated with severe human diseases. Assembly of the one-megadalton complex I in the inner mitochondrial membrane requires assembly factors and chaperones. We have determined the structure of complex I from the aerobic yeast Yarrowia lipolytica by electron cryo-microscopy at 3.2-Å resolution. A ubiquinone molecule was identified in the access path to the active site. The electron cryo-microscopy structure indicated an unusual lipid-protein arrangement at the junction of membrane and matrix arms that was confirmed by molecular simulations. The structure of a complex I mutant and an assembly intermediate provide detailed molecular insights into the cause of a hereditary complex I-linked disease and complex I assembly in the inner mitochondrial membrane.
Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions face the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.
To date, in-cell NMR has elucidated various aspects of protein behaviour by associating structures in physiological conditions. Meanwhile, current studies of this method mostly have deduced protein states in cells exclusively based on ‘indirect’ structural information from peak patterns and chemical shift changes but not ‘direct’ data explicitly including interatomic distances and angles. To fully understand the functions and physical properties of proteins inside cells, it is indispensable to obtain explicit structural data or determine three-dimensional (3D) structures of proteins in cells. Whilst the short lifetime of cells in a sample tube, low sample concentrations, and massive background signals make it difficult to observe NMR signals from proteins inside cells, several methodological advances help to overcome the problems. Paramagnetic effects have an outstanding potential for in-cell structural analysis. The combination of a limited amount of experimental in-cell data with software for ab initio protein structure prediction opens an avenue to visualise 3D protein structures inside cells. Conventional nuclear Overhauser effect spectroscopy (NOESY)-based structure determination is advantageous to elucidate the conformations of side-chain atoms of proteins as well as global structures. In this article, we review current progress for the structure analysis of proteins in living systems and discuss the feasibility of its future works.
1H-detected solid-state NMR experiments feasible at fast magic-angle spinning (MAS) frequencies allow accessing 1H chemical shifts of proteins in solids, which enables their interpretation in terms of secondary structure. Here we present 1H and 13C-detected NMR spectra of the RNA polymerase subunit Rpo7 in complex with unlabeled Rpo4 and use the 13C, 15N, and 1H chemical-shift values deduced from them to study the secondary structure of the protein in comparison to a known crystal structure. We applied the automated resonance assignment approach FLYA including 1H-detected solid-state NMR spectra and show its success in comparison to manual spectral assignment. Our results show that reasonably reliable secondary-structure information can be obtained from 1H secondary chemical shifts (SCS) alone by using the sum of 1Hα and 1HN SCS rather than by TALOS. The confidence, especially at the boundaries of the observed secondary structure elements, is found to increase when evaluating 13C chemical shifts, here either by using TALOS or in terms of 13C SCS.
Genetic code expansion facilitates position‐selective labeling of rna for biophysical studies
(2019)
Nature relies on reading and synthesizing the genetic code with high fidelity. Nucleic acid building blocks that are orthogonal to the canonical A‐T and G‐C base‐pairs are therefore uniquely suitable to facilitate position‐specific labeling of nucleic acids. Here, we employ the orthogonal kappa‐xanthosine‐base‐pair for in vitro transcription of labeled RNA. We devised an improved synthetic route to obtain the phosphoramidite of the deoxy‐version of the kappa nucleoside in solid phase synthesis. From this DNA template, we demonstrate the reliable incorporation of xanthosine during in vitro transcription. Using NMR spectroscopy, we show that xanthosine introduces only minor structural changes in an RNA helix. We furthermore synthesized a clickable 7‐deaza‐xanthosine, which allows to site‐specifically modify transcribed RNA molecules with fluorophores or other labels.
PfEMP1 (erythrocyte membrane protein 1) adhesins play a pivotal role in the pathophysiology of falciparum malaria, by mediating sequestration of Plasmodium falciparum-infected erythrocytes in the microvasculature. PfEMP1 variants are expressed by var genes and are presented on membrane elevations, termed knobs. However, the organization of PfEMP1 on knobs is largely unclear. Here, we use super-resolution microscopy and genetically altered parasites expressing a modified var2csa gene in which the coding sequence of the photoactivatable mEOS2 was inserted to determine the number and distribution of PfEMP1 on single knobs. The data were verified by quantitative fluorescence-activated cell sorting analysis and immuno-electron microscopy together with stereology methods. We show that knobs contain 3.3 ± 1.7 and 4.3 ± 2.5 PfEMP1 molecules, predominantly placed on the knob tip, in parasitized erythrocytes containing wild type and sickle haemoglobin, respectively. The ramifications of our findings for cytoadhesion and immune evasion are discussed.
By running a temperature series of molecular dynamics (MD) simulations starting from the known low-temperature phase, the experimentally observed phase transition in a `jumping crystal' was captured, thereby providing a prediction of the unknown crystal structure of the high-temperature phase and clarifying the phase-transition mechanism. The phase transition is accompanied by a discontinuity in two of the unit-cell parameters. The structure of the high-temperature phase is very similar to that of the low-temperature phase. The anisotropic displacement parameters calculated from the MD simulations readily identified libration as the driving force behind the phase transition. Both the predicted crystal structure and the phase-transition mechanism were verified experimentally using TLS (translation, libration, screw) refinement against X-ray powder diffraction data.
We have encountered two polymorphs of the title compound, C24H16B2OS2, both of which display almost the same unit-cell parameters. Compound (I) crystallizes in the non-centrosymmetric space group P21 with four molecules in the asymmetric unit. These molecules are related by pseudosymmetry. As a result, the space group looks like P21/c, but the structure cannot be refined successfully in that space group. Compound (II) on the other hand crystallizes in the centrosymmetric space group P21/c with only two molecules in the asymmetric unit. The crystals studied for (I) and (II) were both non-merohedral twins.
Iodo(triphenyl)silane
(2019)
The molecular structure of the title compound, C18H15ISi, which crystallizes in the space group C2/c, does not exhibit any unusual features. Two weak C—H⋯π interactions may help to consolidate the packing. The present structure is not isostructural with the known Ph3SiX (X = F, Cl or Br) compounds.
The title co-crystal, 1,3,5,7-tetraazatricyclo[3.3.1.13,7]decane (HMTA, 1)–4-fluorophenol (4-FP) (1/1), C6H12N4·C6H5FO, shows an unusual asymmetric unit that comprises eight independent molecules (Z′′ = 8), four for each component, with four formula units per asymmetric unit (Z′ = 4). In the molecular packing, each HMTA molecule bridges one 4-FP molecule via an O−H···N hydrogen bond to form a two-molecule aggregate. Differences can be observed between the bond lengths and angles of the independent HMTA and 4-FP molecules and those of the molecules in the aggregate. The C−N bonds exhibit different bond lengths in the tetrahedral cage-like structure of the HMTA molecules, but the largest differences between the molecular aggregates are in the bond lengths in the 4-fluorophenol ring. In the crystal, the HMTA and 4-FP molecules form two hydrogen-bonded (O−H···N, C−H···F and C−H···O) dimers of HMTA and 4-FP molecules, A···D and B···C inversion dimers, which generate enlarged R88(34) ring motifs in both supramolecular structures. In both structures, the crystal packing also features additional C−H···F and C−H···O interactions. The A···D and B···C dimers are linked by additional C−H···F and C−H···O hydrogen bonds, forming columns along the a and b axes, respectively. The importance of the C−H···F interaction to the structure and crystal packing has been demonstrated.
Global response of diacylglycerol kinase towards substrate binding observed by 2D and 3D MAS NMR
(2019)
Escherichia coli diacylglycerol kinase (DGK) is an integral membrane protein, which catalyses the ATP-dependent phosphorylation of diacylglycerol (DAG) to phosphatic acid (PA). It is a unique trimeric enzyme, which does not share sequence homology with typical kinases. It exhibits a notable complexity in structure and function despite of its small size. Here, chemical shift assignment of wild-type DGK within lipid bilayers was carried out based on 3D MAS NMR, utilizing manual and automatic analysis protocols. Upon nucleotide binding, extensive chemical shift perturbations could be observed. These data provide evidence for a symmetric DGK trimer with all of its three active sites concurrently occupied. Additionally, we could detect that the nucleotide substrate induces a substantial conformational change, most likely directing DGK into its catalytic active form. Furthermore, functionally relevant interprotomer interactions are identified by DNP-enhanced MAS NMR in combination with site-directed mutagenesis and functional assays.
Amorphous formulation technologies to improve oral absorption of poorly soluble active pharmaceutical ingredients (APIs) have become increasingly prevalent. Currently, polymer-based amorphous formulations manufactured by spray drying, hot melt extrusion (HME), or co-precipitation are most common. However, these technologies have challenges in terms of the successful stabilization of poor glass former compounds in the amorphous form. An alternative approach is mesoporous silica, which stabilizes APIs in non-crystalline form via molecular adsorption inside nano-scale pores. In line with these considerations, two poor glass formers, haloperidol and carbamazepine, were formulated as polymer-based solid dispersion via HME and with mesoporous silica, and their stability was compared under accelerated conditions. Changes were monitored over three months with respect to solid-state form and dissolution. The results were supported by solid-state nuclear magnetic resonance spectroscopy (SS-NMR) and scanning electron microscopy (SEM). It was demonstrated that mesoporous silica was more successful than HME in the stabilization of the selected poor glass formers. While both drugs remained non-crystalline during the study using mesoporous silica, polymer-based HME formulations showed recrystallization after one week. Thus, mesoporous silica represents an attractive technology to extend the formulation toolbox to poorly soluble poor glass formers.
Mitochondrial ATP synthases form dimers, which assemble into long ribbons at the rims of the inner membrane cristae. We reconstituted detergent-purified mitochondrial ATP synthase dimers from the green algae Polytomella sp. and the yeast Yarrowia lipolytica into liposomes and examined them by electron cryotomography. Tomographic volumes revealed that ATP synthase dimers from both species self-assemble into rows and bend the lipid bilayer locally. The dimer rows and the induced degree of membrane curvature closely resemble those in the inner membrane cristae. Monomers of mitochondrial ATP synthase reconstituted into liposomes do not bend membrane visibly and do not form rows. No specific lipids or proteins other than ATP synthase dimers are required for row formation and membrane remodelling. Long rows of ATP synthase dimers are a conserved feature of mitochondrial inner membranes. They are required for cristae formation and a main factor in mitochondrial morphogenesis.
Hydride transfers play a crucial role in a multitude of biological redox reactions and are mediated by flavin, deazaflavin or nicotinamide adenine dinucleotide cofactors at standard redox potentials ranging from 0 to –340 mV. 2-Naphthoyl-CoA reductase, a key enzyme of oxygen-independent bacterial naphthalene degradation, uses a low-potential one-electron donor for the two-electron dearomatization of its substrate below the redox limit of known biological hydride transfer processes at E°’ = −493 mV. Here we demonstrate by X-ray structural analyses, QM/MM computational studies, and multiple spectroscopy/activity based titrations that highly cooperative electron transfer (n = 3) from a low-potential one-electron (FAD) to a two-electron (FMN) transferring flavin cofactor is the key to overcome the resonance stabilized aromatic system by hydride transfer in a highly hydrophobic pocket. The results evidence how the protein environment inversely functionalizes two flavins to switch from low-potential one-electron to hydride transfer at the thermodynamic limit of flavin redox chemistry.
Rhodopsin-based voltage imaging tools for use in muscles and neurons of Caenorhabditis elegans
(2019)
Genetically encoded voltage indicators (GEVIs) based on microbial rhodopsins utilize the voltage-sensitive fluorescence of all-trans retinal (ATR), while in electrochromic FRET (eFRET) sensors, donor fluorescence drops when the rhodopsin acts as depolarization-sensitive acceptor. In recent years, such tools have become widely used in mammalian cells but are less commonly used in invertebrate systems, mostly due to low fluorescence yields. We systematically assessed Arch(D95N), Archon, QuasAr, and the eFRET sensors MacQ-mCitrine and QuasAr-mOrange, in the nematode Caenorhabditis elegans ATR-bearing rhodopsins reported on voltage changes in body wall muscles (BWMs), in the pharynx, the feeding organ [where Arch(D95N) showed approximately 128% ΔF/F increase per 100 mV], and in neurons, integrating circuit activity. ATR fluorescence is very dim, yet, using the retinal analog dimethylaminoretinal, it was boosted 250-fold. eFRET sensors provided sensitivities of 45 to 78% ΔF/F per 100 mV, induced by BWM action potentials, and in pharyngeal muscle, measured in simultaneous optical and sharp electrode recordings, MacQ-mCitrine showed approximately 20% ΔF/F per 100 mV. All sensors reported differences in muscle depolarization induced by a voltage-gated Ca2+-channel mutant. Optogenetically evoked de- or hyperpolarization of motor neurons increased or eliminated action potential activity and caused a rise or drop in BWM sensor fluorescence. Finally, we analyzed voltage dynamics across the entire pharynx, showing uniform depolarization but compartmentalized repolarization of anterior and posterior parts. Our work establishes all-optical, noninvasive electrophysiology in live, intact C. elegans.
Much of the research on Na+/H+ exchange has been done in prokaryotic models, mainly on the NhaA Na+/H+-exchanger from Escherichia coli (EcNhaA). Two conserved aspartate residues, Asp-163 and Asp-164, are essential for transport and are candidates for possible binding sites for the two H+ that are exchanged for one Na+ to make the overall transport process electrogenic. More recently, a proposed mechanism of transport for EcNhaA has suggested direct binding of one of the transported H+ to the conserved Lys-300 residue, a salt bridge partner of Asp-163. This contention is supported by a study reporting that substitution of the equivalent residue, Lys-305, of a related Na+/H+ antiporter, NapA from Thermus thermophilus, renders the transporter electroneutral. In this work, we sought to establish whether the Lys-300 residue and its partner Asp-163 are essential for the electrogenicity of EcNhaA. To that end, we replaced Lys-300 with Gln, either alone or together with the simultaneous substitution of Asp-163 with Asn, and characterized these transporter variants in electrophysiological experiments combined with H+ transport measurements and stability analysis. We found that K300Q EcNhaA can still support electrogenic Na+/H+ antiport in EcNhaA, but has reduced thermal stability. A parallel electrophysiological investigation of the K305Q variant of TtNapA revealed that it is also electrogenic. Furthermore, replacement of both salt bridge partners in the ion-binding site of EcNhaA produced an electrogenic variant (D163N/K300Q). Our findings indicate that alternative mechanisms sustain EcNhaA activity in the absence of canonical ion-binding residues and that the conserved lysines confer structural stability.
In fungi, the mitochondrial respiratory chain complexes (complexes I–IV) are responsible for oxidative phosphorylation, as in higher eukaryotes. Cryo-EM was used to identify a 200 kDa membrane protein from Neurospora crassa in lipid nanodiscs as cytochrome c oxidase (complex IV) and its structure was determined at 5.5 Å resolution. The map closely resembles the cryo-EM structure of complex IV from Saccharomyces cerevisiae. Its ten subunits are conserved in S. cerevisiae and Bos taurus, but other transmembrane subunits are missing. The different structure of the Cox5a subunit is typical for fungal complex IV and may affect the interaction with complex III in a respiratory supercomplex. Additional density was found between the matrix domains of the Cox4 and Cox5a subunits that appears to be specific to N. crassa.
The industry‐scale production of methylchloromonosilanes in the Müller–Rochow Direct Process is accompanied by the formation of a residue, the direct process residue (DPR), comprised of disilanes MenSi2Cl6‐n (n=1–6). Great research efforts have been devoted to the recycling of these disilanes into monosilanes to allow reintroduction into the siloxane production chain. In this work, disilane cleavage by using alkali and alkaline earth metal salts is reported. The reaction with metal hydrides, in particular lithium hydride (LiH), leads to efficient reduction of chlorine containing disilanes but also induces disproportionation into mono‐ and oligosilanes. Alkali and alkaline earth chlorides, formed in the course of the reduction, specifically induce disproportionation of highly chlorinated disilanes, whereas highly methylated disilanes (n>3) remain unreacted. Nearly quantitative DPR conversion into monosilanes was achieved by using concentrated HCl/ether solutions in the presence of lithium chloride.
The title compound, C4H9N5O2+·SO42−·H2O, is the monohydrate of the commercially available compound `C4H7N5O·H2SO4·xH2O'. It is obtained by reprecipitation of C4H7N5O·H2SO4·xH2O from dilute sodium hydroxide solution with dilute sulfuric acid. The crystal structure of anhydrous 2,4,5-triamino-1,6-dihydropyrimidin-6-one sulfate is known, although called by the authors 5-amminium-6-amino-isocytosinium sulfate [Bieri et al. (1993[Bieri, J. H., Prewo, R. & Linden, A. (1993). Private communication (refcode HACDEU). CCDC, Cambridge, England]). Private communication (refcode HACDEU). CCDC, Cambridge, England]. In the structure, the sulfate group is deprotonated, whereas one of the amino groups is protonated (R2C—NH3+) and one is rearranged to a protonated imine group (R2C=NH2+). This arrangement is very similar to the known crystal structure of the anhydrate. Several tautomeric forms of the investigated molecule are possible, which leads to questionable proton attributions. The measured data allowed the location of all hydrogen atoms from the residual electron density. In the crystal, ions and water molecules are linked into a three-dimensional network by N—H⋯O and O—H⋯O hydrogen bonds.
Gram‐negative bacteria are intrinsically resistant against cytotoxic substances by means of their outer membrane and a network of multidrug efflux systems, acting in synergy. Efflux pumps from various superfamilies with broad substrate preferences sequester and pump drugs across the inner membrane to supply the highly polyspecific and powerful tripartite resistance–nodulation–cell division (RND) efflux pumps with compounds to be extruded across the outer membrane barrier. In Escherichia coli, the tripartite efflux system AcrAB–TolC is the archetype RND multiple drug efflux pump complex. The homotrimeric inner membrane component acriflavine resistance B (AcrB) is the drug specificity and energy transduction center for the drug/proton antiport process. Drugs are bound and expelled via a cycle of mainly three consecutive states in every protomer, constituting a flexible alternating access channel system. This review recapitulates the molecular basis of drug and inhibitor binding, including mechanistic insights into drug efflux by AcrB. It also summarizes 17 years of mutational analysis of the gene acrB, reporting the effect of every substitution on the ability of E. coli to confer resistance toward antibiotics (http://goethe.link/AcrBsubstitutions). We emphasize the functional robustness of AcrB toward single‐site substitutions and highlight regions that are more sensitive to perturbation.