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"Ästhetisch ist, was hilft"
(2017)
The asymmetric unit of the title compound, C21H28N4O, consists of two unique molecules linked by an O—H⋯N hydrogen bond. The conformation of both C=N bonds is E and the azomethine functional groups lie close to the plane of their associated benzene rings in each of the independent molecules. The dihedral angles between the two benzene rings are 83.14 (4) and 75.45 (4)°. The plane of the one of the N(CH3)2 units is twisted away from the benzene ring by 18.8 (2)°, indicating loss of conjugation between the lone electron pair and the benzene ring. In the crystal structure, O—H⋯N hydrogen bonds together with C—H⋯O hydrogen bonds link neighbouring supramolecular dimers into a three-dimensional network.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition of abnormal heart rhythm (arrhythmia), induced by physical activity or stress. Mutations in ryanodine receptor 2 (RyR2), a Ca2+ release channel located in the sarcoplasmic reticulum (SR), or calsequestrin 2 (CASQ2), a SR Ca2+ binding protein, are linked to CPVT. For specific drug development and to study distinct arrhythmias, simple models are required to implement and analyze such mutations. Here, we introduced CPVT inducing mutations into the pharynx of Caenorhabditis elegans, which we previously established as an optogenetically paced heart model. By electrophysiology and video-microscopy, we characterized mutations in csq-1 (CASQ2 homologue) and unc-68 (RyR2 homologue). csq-1 deletion impaired pharynx function and caused missed pumps during 3.7 Hz pacing. Deletion mutants of unc-68, and in particular the point mutant UNC-68(R4743C), analogous to the established human CPVT mutant RyR2(R4497C), were unable to follow 3.7 Hz pacing, with progressive defects during long stimulus trains. The pharynx either locked in pumping at half the pacing frequency or stopped pumping altogether, possibly due to UNC-68 leakiness and/or malfunctional SR Ca2+ homeostasis. Last, we could reverse this ‘worm arrhythmia’ by the benzothiazepine S107, establishing the nematode pharynx for studying specific CPVT mutations and for drug screening.
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.
Biophysical studies of the translation-regulating add adenine riboswitch from Vibrio vulnificus
(2017)
Bacterial gene expression can be regulated at mRNA level by cis-acting mRNA elements termed riboswitches. Riboswitches operate by conformational switching between a ligand-free and a ligand-bound state with different structures that either activate or inhibit gene expression. This PhD thesis contributes to the molecular level understanding of full-length purine riboswitches. It presents biophysical investigations on the ligand-dependent folding of the full-length translation-regulating add adenine riboswitch from the gram-negative human pathogenic marine bacterium Vibrio vulnificus (Asw). Asw has the typical bipartite riboswitch architecture with a 5’ ligand-sensing aptamer domain and a 3’ regulatory domain termed expression platform. According to the working hypothesis, Asw employs a unique thermodynamically-controlled 3-state conformational switching mechanism between an apoB, an apoA and a holo conformation to regulate translation initiation in a temperature-compensated manner. The two apo conformations are the putative translation-OFF states and the holo conformation is the putative translation-ON state of Asw. In the main project of this PhD thesis, an integrated nuclear magnetic resonance (NMR) and smFRET spectroscopic study of the full-length 112-nucleotide Asw (112Asw) was performed. The adenine-dependent folding of 112Asw was monitored at the level of base pairing interactions by NMR of the RNA imino protons, and at the level of three long-range intramolecular distances by smFRET of immobilized molecules. The integrated NMR and smFRET spectroscopic study of 112Asw yielded two major findings. First, NMR and smFRET both revealed that adenine binding to 112Asw impedes apoB formation by stabilizing the apoA secondary structure in the holo conformation without modulating tertiary structural interactions between the two riboswitch domains. This highlights the central role of competitive P1 and P4 helix formation at the interface of the aptamer and the expression platform for switching the accessibility of the ribosome binding site of 112Asw. Moreover, it strongly corroborates the hypothesis that purine riboswitches in general operate according to the key principle of a spatially decoupled secondary structural allosteric switch that proceeds without ligand-induced tertiary structural interactions between the aptamer domain and the expression platform. Second, it was uncovered by smFRET that the apoA and the holo conformation of 112Asw do not adopt a single folding state at near-physiological Mg2+ concentration. Instead, apoA and holo exhibit a persistent dynamic equilibrium between substates with an undocked (U), a short-lived docked (D1; ~s) and a Mg2+-bound long-lived docked (D2; ~10 s) aptamer kissing loop motif. In the holo conformation, the fractional population of the long-lived docked substate is ~2-fold increased compared to the apoA conformation, but undocked and docked substates are still comparably stable. The here described multiple folding states of the apoA and the holo conformation might have regulatory properties that are in between the apoB translation-OFF state and the holo-D2 translation-ON state. Additonally, an integrated NMR and smFRET analysis of 127-nucleotide Asw (127Asw) is presented. Compared to 112Asw, 127Asw is 3’-elongated by 15 nucleotides of the adenosine deaminase encoding sequence of the add gene from Vibrio vulnificus. 127Asw was chosen as mRNA template for future investigations of the interaction between Asw and the 30S ribosomal subunit. The NMR spectra of 127Asw demonstrated that 127Asw has the same overall secondary structure as 112Asw. Like for 112Asw, the combined NMR and smFRET analysis of 127Asw showed that adenine binding impedes apoB formation and stabilizes a long-lived docked aptamer kissing loop fold. However, compared to 112Asw, 127Asw has a destabilized aptamer kissing loop motif and a stabilized P4 helix in the expression platform. Finally, ligand-observed studies of the transient encounter complex between Asw and the near-cognate ligand hypoxanthine are described. By competition binding WaterLOGSY NMR experiments with hypoxanthine and the adenine analogue 2,6-diaminopurine, it could be shown that hypoxanthine binds to the same binding site of 112Asw as the cognate ligand adenine. The hypoxanthine binding constant measured with the WaterLOGSY method is in the low mM range (1.8 mM) and substantially exceeds the physiological hypoxanthine concentration in E. coli (~0.3 mM), thus ruling out that hypoxanthine binding can significantly impact the translational regulation of Asw in vivo. Also, preliminary FTIR difference spectra of 13C,15N-labelled and unlabelled hypoxanthine in complex with the pbuE adenine riboswitch aptamer and the xpt guanine riboswitch aptamer are discussed. These spectra showed a pattern of multiple IR bands that appeared to be characteristic for the respective complex.
Cells respond to protein misfolding and aggregation in the cytosol by adjusting gene transcription and a number of post-transcriptional processes. In parallel to functional reactions, cellular structure changes as well; however, the mechanisms underlying the early adaptation of cellular compartments to cytosolic protein misfolding are less clear. Here we show that the mammalian ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP), if freed from chaperones during acute stress, can dock on cellular membranes thus performing a proteostasis sensor function. We reconstituted this process in vitro and found that mainly phosphatidic acid and phosphatidylinositol-4-phosphate enhance association of chaperone-free CHIP with liposomes. HSP70 and membranes compete for mutually exclusive binding to the tetratricopeptide repeat domain of CHIP. At new cellular locations, access to compartment-specific substrates would enable CHIP to participate in the reorganization of the respective organelles, as exemplified by the fragmentation of the Golgi apparatus (effector function).
N-Allyltetramethylpiperidine is readily isomerized to the corresponding enamine by treatment with catalytic amounts of B(C6F5)3. It adds HB(C6F5)2 at the nucleophilic enamine carbon atom to form a C/B Lewis adduct. This reacts with two molar equivalents of carbon monoxide by selective head to tail coupling to give a five-membered C2O2B heterocycle. In contrast the enamine/HB(C6F5)2 Lewis pair reacts with two molar equiv. of nitric oxide by head to head coupling. This reaction probably proceeds via equilibrium with the corresponding vicinal N/B Lewis pair. Most products were characterized by X-ray diffraction.
Na+/H+ exchange is essential for survival of all organisms, having a role in the regulation of the intracellular Na+ concentration, pH and cell volume. Furthermore, Na+/H+ exchangers were shown to be involved in the virulence of the bacterium Yersinia pestis, indicating they might be potential targets for novel antibiotic treatments. The model system for Na+/H+ exchangers is the NhaA transporter from Escherichia coli, EcNhaA. Therefore, the general transport mechanism of NhaA exchangers is currently well characterized. However, much less is known about NhaB exchangers, with only a limited number of studies available. The pathogen Klebsiella pneumoniae, which is a major source of nosocomial infection, possesses three electrogenic Na+/H+ exchangers, KpNhaA1, KpNhaA2 and KpNhaB, none of which have been previously investigated. Our aim in this study was to functionally characterize KpNhaB using solid supported membrane-based electrophysiology as the main investigation technique, and thus provide the first electrophysiological investigation of an NhaB Na+/H+ exchanger. We found that NhaB can be described by the same competition-based mechanism that was shown to be valid for electrogenic NhaA and NapA, and for electroneutral NhaP Na+/H+ exchangers. For comparison we also characterized the activity of KpNhaA1 and KpNhaA2 and found that the three exchangers have complementary activity profiles, which is likely a survival advantage for K. pneumoniae when faced with environments of different salinity and pH. This underlines their importance as potential antibiotic drug targets.
Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3′-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level.
Background: How a dentist works, such as the patterns of movements performed daily, is also largely affected by the workstation Dental tasks are often executed in awkward body positions, thereby causing a very high degree of strain on the corresponding muscles. The objective of this study is to detect those dental tasks, during which awkward postures occur most frequently. The isolated analysis of static postures will examine the duration for which these postures are maintained during the corresponding dental, respectively non-dental, activities.
Methods: 21 (11f/10 m) dentists (age: 40.1 ± 10.4 years) participated in this study. An average dental workday was collected for every subject. To collect kinematic data of all activities, the CUELA system was used. Parallel to the kinematic examination, a detailed computer-based task analysis was conducted. Afterwards, both data sets were synchronized based on the chronological order of the postures assumed in the trunk and the head region. All tasks performed were assigned to the categories "treatment" (I), "office" (II) and "other activities" (III). The angle values of each body region (evaluation parameter) were examined and assessed corresponding to ergonomic standards. Moreover, this study placed a particular focus on static positions, which are held statically for 4 s and longer.
Results: For "treatment" (I), the entire head and trunk area is anteriorly tilted while the back is twisted to the right, in (II) and (III) the back is anteriorly tilted and twisted to the right (non-neutral position). Static positions in (I) last for 4–10s, static postures (approx. 60%) can be observed while in (II) and (III) in the back area static positions for more than 30 s are most common. Moreover, in (II) the back is twisted to the right for more than 60 s in 26.8%.
Conclusion: Awkward positions are a major part of a dentists’ work. This mainly pertains to static positions of the trunk and head in contrast to "office work." These insights facilitate the quantitative description of the dentist profession with regard to the related physical load along with the health hazards to the musculoskeletal system. Moreover, the results allow for a selective extraction of the most unfavorable static body positions that dentists assume for each of the activities performed.
CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin
(2017)
Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain 3 of the soluble toxin refolds into two ~85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel. The apolar outer barrel surface with large sidechains is immersed in the lipid bilayer, while the inner barrel surface is highly charged. Comparison of the cryoEM pore complex to the prepore structure obtained by electron cryo-tomography and the x-ray structure of the soluble form reveals the detailed mechanisms by which the toxin monomers insert into the lipid bilayer to perforate the target membrane.
In the title compound, C17H18N2O, the central carbon atom with the OH substituent and one of the (E)-benzylideneamino substituents are disordered over two sets of sites with occupancies of 0.851 (4) and 0.149 (4). The relative positions of the two disorder components is equivalent to a rotation of approximately 60° about the C—N single bond. In the crystal, the molecules are held together by O—H...N hydrogen bonds, forming simple C(5) chains along the b-axis direction. In addition, pairs of the chains are further aggregated by weak C—H...π interactions.
In the title compound, C26H24N2O2, the oxazine moiety is fused to a naphthalene ring system. The asymmetric unit consists of one half of the molecule, which lies about an inversion centre. The C atoms of the ethylene spacer group adopt an antiperiplanar arrangement. The oxazine ring adopts a half-chair conformation. In the crystal, supramolecular chains running along the b axis are formed via short C—H⋯π contacts. The crystal studied was a non-merohedral twin with a fractional contribution of 0.168 (2) of the minor twin component.
The asymmetric unit of the title co-crystalline adduct, 1,3,6,8-tetraazatricyclo[4.4.1.13,8]dodecane (TATD)–4-iodophenol (1/2), C8H16N4·2C6H5IO, comprises a half molecule of the aminal cage polyamine plus a 4-iodophenol molecule. A twofold rotation axis generates the other half of the adduct. The components are linked by two intermolecular O—H⋯N hydrogen bonds. The adducts are further linked into a three-dimensional framework structure by a combination of N⋯I halogen bonds and weak non-conventional C—H⋯O and C—H⋯I hydrogen bonds.
Biogenesis of mitochondrial cytochrome c oxidase (COX) is a complex process involving the coordinate expression and assembly of numerous subunits (SU) of dual genetic origin. Moreover, several auxiliary factors are required to recruit and insert the redox-active metal compounds, which in most cases are buried in their protein scaffold deep inside the membrane. Here we used a combination of gel electrophoresis and pull-down assay techniques in conjunction with immunostaining as well as complexome profiling to identify and analyze the composition of assembly intermediates in solubilized membranes of the bacterium Paracoccus denitrificans. Our results show that the central SUI passes through at least three intermediate complexes with distinct subunit and cofactor composition before formation of the holoenzyme and its subsequent integration into supercomplexes. We propose a model for COX biogenesis in which maturation of newly translated COX SUI is initially assisted by CtaG, a chaperone implicated in CuB site metallation, followed by the interaction with the heme chaperone Surf1c to populate the redox-active metal-heme centers in SUI. Only then the remaining smaller subunits are recruited to form the mature enzyme which ultimately associates with respiratory complexes I and III into supercomplexes.
The asymmetric unit of the title compound, C18H18I2N2O2, consists of one half-molecule, completed by the application of inversion symmetry. The molecule adopts the typical structure for this class of bis-benxozazines, characterized by an anti orientation of the two benzoxazine rings around the central C—C bond. The oxazinic ring adopts a half-chair conformation. In the crystal, molecules are linked by C—I⋯N short contacts [I⋯N = 3.378 (2) Å], generating layers lying parallel to the bc plane.
Der viersemestrige Master-Studiengang Biochemie leitet sich aus der langjährigen Tradition in biomolekularer Forschung und Lehre in der Frankfurter Forschungslandschaft her und ist stark forschungsorientiert.
Ziel des Studienganges ist es, fachliche Kenntnisse, Fähigkeiten und Methodenkompetenzen zu vermitteln, mit denen die Absolventen in die Lage versetzt werden, in einem forschungsbezogenem Kontext selbstständig zu arbeiten.
Inhaltlich erstreckt sich der Studiengang von zellulärer Biochemie über Strukturbiologie bis hin zur Biophysik/Biophysikalischen Chemie und ermöglicht den Studierenden die Setzung individueller Schwerpunkte. Für eine forschungsnahe Ausbildung auf hohem Niveau ist der Studiengang integral mit dem lokalen Forschungsumfeld verknüpft, was neben dem Heimatfachbereich Biochemie / Chemie / Pharmazie auch die Fachbereiche Physik, Biowissenschaften, Medizin als auch außeruniversitäre Institutionen wie das Max-Planck-Institut für Biophysik und das Paul-Ehrlich-Institut involviert. Den besonderen Frankfurter Schwerpunkten Strukturbiologie und Membranproteinforschung wird im Studiengang Rechnung getragen.
Das Masterstudium am hochmodernen Life-Science Campus der Goethe-Universität bietet in einer einzigartigen Mischung aus Theorie und Praxis in Biochemie und Biophysikalischer Chemie das Rüstzeug für eine Karriere in der universitären sowie industriellen Umgebung. Die Beteiligung diverser Forschungsinstitute am Masterprogramm ermöglicht eine Spezialisierung in Immunologie, Tumorbiologie oder in Membranbiochemie. Die starke Vernetzung der Universität in nationale sowie internationale Forschungsverbünde gewährleistet den Studierenden einen engen Kontakt mit international führenden Wissenschaftlern und die Möglichkeit zu weltweiten Forschungsaufenthalten. ...
We have determined the crystal structures of two decachlorocyclopentasilanes, namely bis(tetra-n-butylammonium) dichloride decachlorocyclopentasilane dichloromethane disolvate, 2C16H36N+·2Cl−·Si5Cl10·2CH2Cl2, (I), and bis(tetraethylammonium) dichloride decachlorocyclopentasilane dichloromethane disolvate, 2C8H20N+·2Cl−·Si5Cl10·2CH2Cl2, (II), both of which crystallize with discrete cations, anions, and solvent molecules. In (I), the complete decachlorocyclopentasilane ring is generated by a crystallographic twofold rotation axis. In (II), one cation is located on a general position and the other two are disordered about centres of inversion. These are the first structures featuring the structural motif of a five-membered cyclopentasilane ring coordinated from both sides by a chloride ion. The extended structures of (I) and (II) feature numerous C—H⋯Cl interactions. In (II), the N atoms are located on centres of inversion and as a result, the ethylene chains are disordered over equally occupied orientations.
The transcriptional regulator far upstream binding protein 1 (FUBP1) is essential for fetal and adult hematopoietic stem cell (HSC) self-renewal, and the constitutive absence of FUBP1 activity during early development leads to embryonic lethality in homozygous mutant mice. To investigate the role of FUBP1 in murine embryonic stem cells (ESCs) and in particular during differentiation into hematopoietic lineages, we generated Fubp1 knockout (KO) ESC clones using CRISPR/Cas9 technology. Although FUBP1 is expressed in undifferentiated ESCs and during spontaneous differentiation following aggregation into embryoid bodies (EBs), absence of FUBP1 did not affect ESC maintenance. Interestingly, we observed a delayed differentiation of FUBP1-deficient ESCs into the mesoderm germ layer, as indicated by impaired expression of several mesoderm markers including Brachyury at an early time point of ESC differentiation upon aggregation to EBs. Coculture experiments with OP9 cells in the presence of erythropoietin revealed a diminished differentiation capacity of Fubp1 KO ESCs into the erythroid lineage. Our data showed that FUBP1 is important for the onset of mesoderm differentiation and maturation of hematopoietic progenitor cells into the erythroid lineage, a finding that is supported by the phenotype of FUBP1-deficient mice.