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The molecule of the title compound, C14H16N2O2, is located on a crystallographic twofold rotation axis. The central O-C-C-O bridge adopts a gauche conformation. One of the amine H atoms is disordered over two equally occupied positions. The crystal structure is stabilized by N-H...O and N-H...N hydrogen bonds. Key indicators: single-crystal X-ray study; T = 173 K; mean σ(C–C) = 0.002 Å; disorder in main residue; R factor = 0.049; wR factor = 0.119; data-to-parameter ratio = 17.9.
In the title compound, C27H16F6N2O6, the nitro groups are almost coplanar with the aromatic rings to which they are attached [dihedral angles = 3.5 (5) and 6.2 (3)°]. The dihedral angles between adjacent aromatic rings are 78.07 (8) and 71.11 (8)° for nitrophenyl/phenyl and 69.50 (8)° for phenyl/phenyl. An intermolecular C-H...[pi] interaction seems to be effective in the stabilization of the structure. Key indicators: single-crystal X-ray study; T = 173 K; mean σ(C–C) = 0.004 Å; R factor = 0.045; wR factor = 0.092; data-to-parameter ratio = 12.5.
The research presented in this thesis characterizes U2AF homology motifs (UHM) and their interactions with UHM ligand motifs (ULM) in the context of splicing regulation. UHM domains are a subgroup of RNA recognition motifs (RRM) originally discovered in the proteins U2AF65 and U2AF35. Whereas canonical RRMs are usually involved in binding of RNA, UHM domains bind tryptophan containing linear protein motifs (ULM) instead. In the first article, we analyze the complex network of interactions between splicing factors and RNA that initiate the assembly of the spliceosome at the 3´ splice site of an intron. The protein U2AF65 binds a pyrimidine-rich element in introns and recruits U2snRNP by binding its protein component SF3b155. My contribution was to define the binding site of the protein U2AF65 to the intrinsically unstructured N-terminus of the scaffolding protein SF3b155. I could show that the UHM domain of U2AF65 recognizes a ULM in SF3b155, and that this binding site is not overlapping with the binding sites of other splicing factors, like p14, to SF3b155. As the U2AF65-UHM:SF3b155-ULM interaction is mutually exclusive with an interaction between U2AF65-UHM and a ULM in the splicing factor SF1, which was reported to initially recognize the branch point sequence, my results provide the molecular details on how SF3b155 replaces SF1 during spliceosomal reorganizations. In the second article, we show that overexpression of the UHM domain of the splicing factor SPF45 induces exon 6 skipping in the pre-mRNA of Fas (CD95/APO-1). I provide evidence for in vitro binding of SPF45-UHM to ULM sequences in the splicing factors U2AF65, SF1, and SF3b155. I crystallized free and SF3b155-bound SPF45 UHM and solved both structures by X-ray crystallography. The analysis of the complex interface and sequence differences in the ULMs allowed me to design mutations of SPF45-UHM, which selectively inhibit binding to distinct ULMs. After assessing the ULM binding properties in vitro, we could show that the activity of SPF45-UHM in influencing the splicing pattern of Fas relies on interactions with SF3b155 and/or SF1, but that an interaction with U2AF65 is dispensable. A mechanism for the activity of SPF45-UHM could thus be engaging in ULM interactions and thus interfering with the network of interactions that initiate the assembly of the spliceosome at the 3´splice site, as described above. In the third article, we describe an unusual flexible homodimerization mode of the UHM in the splicing factor Puf60, which enables simultaneous interactions with ULM sequences on other splicing factors. I could show that the NMR relaxation properties of Puf60-UHM are inconsistent with a model of a rigid dimer, but rather indicate a dimerization via a flexible linker. I identified a flexible loop in the peptide backbone of Puf60-UHM, and showed that mutiation of acidic residues in this loop impairs the dimerization. To analyze the dimerization interface in further detail, I solved the structure of Puf60-UHM by X-ray crystallography. The acidic residues in the flexible loop of one UHM dimer subunit mediate the dimerization by contacting basic residues on the β-sheet surface of the other dimer subunit. Differences in the four dimer interfaces observed for the eight molecules in the asymmetric unit of the crystal support the model of an undescribed, flexible mode of dimerization, and thus complement the NMR relaxation data. Furthermore, I could show that the Puf60-UHM dimer and U2AF65-UHM contact different ULM sequences on the SF3b155 N-terminus in vitro, thus providing a possible explanation for the mutual cooperative activation of Puf60 and U2AF65 in splicing assays described in the literature. The fourth article is a review about recent research on the recognition of DNA double strand breaks (DSB) by covalent histone modifications. The p53 binding protein 1 (53BP1) is a DSB sensor and a checkpoint protein for mitosis. Recent crystallographic evidence indicates that 53BP1 recognizes DSB sites by binding histone H4 dimetylated at lysine 20 (H4-K20). We provide a comprehensive overview of the atomic resolution structures that revealed how proteins can specifically recognize histone tail modifications, especially methylated lysines, to read the information stored in what is called the histone code.
Determination of the distribution of halocarbons in the tropical upper troposphere and stratosphere
(2008)
The aim of this thesis was to investigate distributions of 32 volatile chlorinated and/or brominated halocarbons that are currently believed to be present in the tropical upper troposphere and stratosphere and to contribute to stratospheric ozone depletion and also to global warming. For this purpose an analytical system was established, which is capable to measure ultra-low concentrated atmospheric trace gases. A quadrupole Mass Spectrometric (MS) Detector was attached to an existing Gas Chromatograph with pre-concentration system and Electron Capture Detector (ECD). The characterisation of the chromatographic system was significantly enhanced by the subsequent identification of 48 additional volatile organic compounds. Furthermore a Gaussian fit algorithm, which was developed in the workgroup, was applied to the chromatographic signals. This algorithm was proven to reflect peaks quantitatively and to enhance the performance of the integration process – especially the reproducibilities for peaks with a low signal to noise ratio. As it is known that the Electron Capture Detector responds nonlinear the new MS detector was checked for such behaviour and found to respond linear. In logical consistency the complete quantification process including e.g. pre-concentration of trace gases and signal integration can be considered as linear responding within the investigated parameter ranges. Moreover, the long term stability of the targeted halocarbons was proven inside the calibration standard containers over a period of 25 months. Many substances were also found to be stable inside the containers used for storage of air samples but a number of substances showed significant concentration changes. These were mainly CH3Cl (methyl chloride), CH3Br (methyl bromide), CH2Cl2 (dichloromethane), CHCl3 (chloroform), CCl4 (tetrachloromethane), C2Cl4 (tetrachloroethene), CH3CCl3 (methyl chloroform), CH2ClCH2Cl (1,2-dichloroethane) und C2H5Cl (chloroethane). But the number of affected substances and also the corresponding concentration changes varied between the individual containers. A systematic investigation of the influence of possible causes (e.g. air sampling methods, container materials) is recommended. Results from both internal detectors were compared and revealed biases and disadvantages of the ECD caused by its lower selectivity and its nonlinear response behaviour. Consequently the MS detector was chosen for the quantification of atmospheric trace gases. The quantification process was performed relative to externally calibrated air standards. To assess the uncertainties connected with different absolute calibration scales cross-comparisons between calibration standards of three different laboratories were carried out. Most substances’ calibrations agreed within the measurement uncertainties but significant differences were observed for CF2ClBr (H1211), CH3Cl (methyl chloride), CH2Cl2 (dichloromethane), CHCl3 (chloroform), CCl4 (tetrachloromethane) and CH3CCl3 (methyl chloroform). As five of these substances were also observed to show concentration changes inside sample containers it is likely, that such changes are responsible for calibration differences. In addition to the detailed assessment of uncertainties connected with the analytical quantification process a set of air samples was available for measurements. These samples mainly originated from the upper troposphere and lower and middle stratosphere in the tropics and the determined halocarbon quantities were used to investigate their distributions in the respective atmospheric regions. In detail, the altitudinal distributions and interrelations of 17 long-lived halocarbons in the tropical stratosphere were determined and compared with those of other stratospheric regions. Tracer-tracer-correlations of these substances in the tropical stratosphere were found to differ from those in mid- and high-latitudes. Characteristic fit functions relative to CF2Cl2 (F12) which are valid for the tropical stratosphere in 2005 were derived as well as time-independent fit functions of fractional release factors (FRFs) relative to the mean age of air. Both sets of correlations could be used for the parameterisation and evaluation of models and also to reassess the Global Warming Potentials (GWPs) of the corresponding halocarbons which might affect future climate predictions. However, the data set on halocarbons in the tropical stratosphere is still insufficient to investigate the variability of tracer-tracer-correlations and FRFs caused by dynamical and photochemical processes. Therefore it is important for future research to perform additional measurements there and – if possible – to extend the measurements to the upper tropical stratosphere in order to characterise the sink of those halocarbons that are still present in these altitudes. In addition, the amount of chlorine and bromine present in the form of organic compounds inside and above the main stratospheric entrance region (the Tropical Tropopause Layer, TTL) was quantified in the frame of a case study. This was possible because of a cooperation with scientists from the University of East Anglia which carried out measurements of six additional halocarbons leading to a total of 28 quantified target substances. Ten of these substances have short atmospheric lifetimes compared with the mean transport times of tropospheric air to the stratosphere (i.e. lifetimes below 0.5 years) and show non-uniform distributions in the upper troposphere. The contribution of these substances to stratospheric ozone depletion is subject of an ongoing scientific debate. In the performed case study a fraction range of short-lived halocarbons of 6 – 8 % (0.98 – 1.25 ppt) relative to the sum of bromine from organic substances and of 1.1 – 1.4 % (36.6 – 47.1 ppt) for the corresponding sum of chlorine was calculated to enter the stratosphere above Brazil in June 2005. Moreover by combining the data with tropospheric reference data and age of air observations the abundances of inorganic chlorine and bromine (Cly and Bry) were derived. At an altitude of 34 km an amount of 3062 ppt of Cly and 17.5 ppt of Bry from organic source gases was calculated. The latter is significantly lower than Bry mixing ratios inferred from quasisimultaneous BrO measurements at 33 km altitude above Brazil (Dorf, 2005, Dorf et al., 2008). But at the University of East Anglia indications for the presence of unknown brominated organic substances in the TTL were found which might cause this difference. Finally, a major result of this thesis adds to the knowledge of the composition of the troposphere as three Chlorofluorocarbons (CFCs) were first observed. Trifluorochloroethene, 3-chloropentafluoropropene and 4,4-dichlorohexafluoro-1-butene were found in air samples collected at the Taunus Observatory near Frankfurt (Main) and the Jungfraujoch High Altitude Research Station in Switzerland (Laube and Engel, 2008). Identification was possible because of an air plume containing high concentrations of these substances. It is suggested that the abundances found on this occasion originated from a local source. The atmospheric lifetimes of these substances are expected to be rather short as they contain a double bond. A quantitative calibration could only be derived for trifluorochloroethene but not for the other species by now. Thus, a relative sensitivity method was derived to get a first indication of the observed atmospheric abundances. All three CFCs could also be detected in air masses representative of background conditions, though with much lower concentrations. These species and some of their degradation products are toxic and could also be relevant for stratospheric and tropospheric ozone depletion. It is important to find out more about their atmospheric distributions, lifetimes, sinks and sources and their ability to reach the stratosphere to assess their possible influence on the global atmosphere. This will be done in the frame of the project "CLEARFOGG – Checking Layers of the Earths AtmospheRe For halogenated Ozone-depleting and Greenhouse Gases". This research project aims to perform a systematic scan of the atmosphere because there are indications for the presence of a number of halogenated organic compounds which are unknown by now. It was recently decided to be funded by the British National Environmental Research Council and will be carried out at the University of East Anglia mainly by the author of this thesis.
Mitochondrial complex I (NADH:ubiquinone oxidoreductase) undergoes reversible deactivation upon incubation at 30–37 °C. The active/deactive transition could play an important role in the regulation of complex I activity. It has been suggested recently that complex I may become modified by S-nitrosation under pathological conditions during hypoxia or when the nitric oxide:oxygen ratio increases. Apparently, a specific cysteine becomes accessible to chemical modification only in the deactive form of the enzyme. By selective fluorescence labeling and proteomic analysis, we have identified this residue as cysteine-39 of the mitochondrially encoded ND3 subunit of bovine heart mitochondria. Cysteine-39 is located in a loop connecting the first and second transmembrane helix of this highly hydrophobic subunit. We propose that this loop connects the ND3 subunit of the membrane arm with the PSST subunit of the peripheral arm of complex I, placing it in a region that is known to be critical for the catalytic mechanism of complex I. In fact, mutations in three positions of the loop were previously reported to cause Leigh syndrome with and without dystonia or progressive mitochondrial disease.
Location and orientation of serotonin receptor 1a agonists in model and complex lipid membranes
(2008)
Magic angle spinning (MAS) NMR has been used to investigate the location and orientation of five serotonin receptor 1a agonists (serotonin, buspirone, quipazine, 8-OH-DPAT, and LY-163,165) in single component model lipid and brain lipid membranes. The agonist locations are probed by monitoring changes in the lipid proton chemical shifts and by MAS-assisted nuclear Overhauser enhancement spectroscopy, which indicates the orientation of the agonists with respect to the 1,2-dioleoyl-sn-glycero-3-phosphocholine lipids. In the single component bilayer, the membrane agonists are found predominantly in the top of the hydrophobic chain or in the glycerol region of the membrane. Most of the agonists orient approximately parallel to the membrane plane, with the exception of quipazine, whose piperazine ring is found in the glycerol region, whereas its benzene ring is located within the lipid hydrophobic chain. The location of the agonist in brain lipid membranes is similar to the 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid bilayers; however, many of the agonists appear to locate close to the cholesterol in the membrane in preference to the phospholipids.
Cytotoxic T lymphocytes eliminate infected cells upon surface display of antigenic peptides on major histocompatibility complex I molecules. To promote immune evasion, UL49.5 of several varicelloviruses interferes with the pathway of major histocompatibility complex I antigen processing. However, the inhibition mechanism has not been elucidated yet. Within the macromolecular peptide-loading complex we identified the transporter associated with antigen processing (TAP1 and TAP2) as the prime target of UL49.5. Moreover, we determined the active oligomeric state and crucial elements of the viral factor. Remarkably, the last two residues of the cytosolic tail of UL49.5 are essential for endoplasmic reticulum (ER)-associated proteasomal degradation of TAP. However, this process strictly requires additional signaling of an upstream regulatory element in the ER lumenal domain of UL49.5. Within this new immune evasion mechanism, we show for the first time that additive elements of a small viral factor and their signaling across the ER membrane are essential for targeted degradation of a multi-subunit membrane complex.
By translocating proteasomal degradation products into the endoplasmic reticulum (ER) for loading of major histocompatibility complex (MHC) class I molecules, the ATP binding cassette (ABC) transporter associated with antigen processing (TAP) plays a pivotal role in the adaptive immunity against infected or malignantly transformed cells. A key question regarding the transport mechanism is how the inter-domain communication and conformational dynamics of the TAP complex are connected during the peptide transport. To identify residues involved in this processes, we evolved a Trojan horse strategy in which a small artificial protease is inserted into antigenic epitopes. After binding, the TAP backbone in contact is cleaved, allowing the peptide sensor site to be mapped by mass spectrometry. Within this study, the peptide sensor and transmission interface have been identified. This region aligns with the cytosolic loop 1 (CL1) of Sav1866 and MsbA. Based on a number of experimental data and the homology to the bacterial ABC exporter Sav1866, we constructed a 3D structural model of the core TAP complex. According to this model, the CL1 and CL2 of TAP1 are extended cytosolic loops connecting the transmembrane helices (TMH) 2 and 3, and TMH4 and 5 respectively, and contact both nucleotide binding domains (NBDs) of the opposite subunit. In contrast to exporters, the cytosolic loop (named L-loop) of BtuCD importer is much shorter, and contacts only one NBD. The data confirm that the CL1 of TAP1 functions as signal transducer in ABC exporters, because it does not interfere with substrate binding but with substrate transport. The peptide contact site identified herein is restructured during the ATP hydrolysis cycle. Importantly, TAP showed a structural change trapped in the ATP hydrolysis transition state, because direct contact between peptide and CL1 is abolished. By cysteine scanning, the most conserved residues within CL1 were identified, which disrupted the tight coupling between peptide binding and transport. Together with Val-288, these residues are essential in sensing the bound peptide and inter-domain signal transmission. To characterize the molecular architecture of CL1, a convenient and minimally perturbing approach was used, which combined cysteine substitution in the CL1 region and determination of accessibility to thiol specific compounds with different properties. These studies revealed that the N-terminal region of CL1 has a good accessibility for hydrophilic (iodoacetamidofluorescein, IAF) and amphiphilic probes (BODIPY maleimide, BM), whereas the C-terminal region is accessible for hydrophobic probe (coumarin maleimide, CM). Kinetic studies of fluorescence labeling suggest that this region displayed a different accessibility to probes when the protein undergoes distinct conformations (e. g. nucleotide free state), thereby reflecting conformational transitions. Fluorescence labeling with BM induces a lost of peptide transport, whereas the peptide binding remains unaffected. These results indicate that covalent modifications of the CL1 residues influenced the inter-domain communication between transmembrane domain (TMD) and NBD. The X-loop is a recently discovered motif in the NBD of ABC exporters, which stays in close contact to the CLs. Moreover, because the X-loop precedes the ABC signature motif, it probably responds to ATP binding and hydrolysis and may transmit conformational changes to the CLs. By substitution of the highly conserved Glu-602 of TAP2 with residues that have different chemical properties, it was shown for the first time that the X-loop is a functional important element, which plays an key role in coupling substrate binding to downstream events in the transport cycle. We further verified domain swapping in the TAP complex by cysteine cross-linking. The TAP complex can be reversibly arrested either in a binding or translocation incompetent state by cross-linking of the X-loop to CL1 or CL2, respectively. These results resolve the structural arrangement of the transmission interface and point to different functions of the cytosolic loops in substrate recognition, signaling and transport.
Life-threatening fungal infections are becoming increasingly common for immunocompromised patients such as those with AIDS, or those undergoing organ transplantation or chemotheraphy, as well as for other health-vulnerable patients. Excellent targets for antifungal drugs are chitin synthases, which are essential for survival of the fungus and lacking in humans. To design new antifungal drugs, knowledge of the three-dimensional structure and mechanism of action of chitin synthases are crucial. Chitin synthases are members of an important family of enzymes that synthesize structural polysaccharides, such as cellulose, β(1,3)-glucan, β(1,4)-mannan and hyaluronan. Therefore, chitin synthases could be used as a model system to understand these more complex enzymes, which are also of major medical and commercial importance. Chitin synthase 2 from Saccharomyces cerevisiae (ScChS2), the protein under study, is an integral membrane protein that synthesizes the primary septum between mother and daughter cells in budding yeast. It is essential for proper cell separation and expected to be highly regulated. An important aspect is that ScChS2 shows 55% sequence identity and is functionally analogous to chitin synthase 1 from the human opportunistic pathogen Candida albicans, this enzyme is also essential for cell survival (Munro, Winter et al. 2001). ...
Chlamydia are obligate intracellular bacteria that cause variety of human diseases. Host cells infected with Chlamydia are protected against many different apoptotic stimuli. The induction of apoptosis resistance is thought to be an important immune escape mechanism allowing Chlamydia to replicate inside the host cell. Infection with C. trachomatis activates the Raf/MEK/ERK pathway and the PI3K/AKT pathway. Here we show that inhibition of these two pathways by chemical inhibitors sensitized C. trachomatis infected cells to granzyme B-mediated cell death. Infection leads to the Raf/MEK/ERK-mediated up-regulation and PI3K-dependent stabilization of the anti-apoptotic Bcl-2 family member Mcl-1. Consistently, interfering with Mcl-1 up-regulation sensitized infected cells for apoptosis induced via the TNF receptor, DNA damage, granzyme B and stress. Our data suggest that Mcl-1 up-regulation is primarily required to maintain apoptosis resistance in C. trachomatis-infected cells.