Biochemie und Chemie
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Die Aufklärung der dreidimensionalen Helix-Struktur der DNA, des Trägermoleküls der genetischen Information aller Lebewesen, durch Watson und Crick im Jahre 1953 ermöglichte eine ganz neue Sichtweise auf ihre Eigenschaften und viele zelluläre Prozesse. Von besonderem Interesse sind hier u.a. Mechanismen, bei denen die DNA an den Phosphaten nucleophil substituiert wird, wie dies beispielsweise bei der Rekombination oder der Transkription geschieht. Dies ist daher interessant, weil sich die DNA gegenüber nucleophilen Angriffen in verschiedenen Experimenten als überaus stabil und reaktionsträge gezeigt hat. Spezialisierte Enzyme wie die Staphylokokkennuklease oder Restriktionsendonukleasen nutzen u.a. Metall-Ionen, um Phosphoryltransfer-Reaktionen zu katalysieren und in eine akzeptable Zeitskala zu verschieben. Die Topoisomerase vom Typ I zeigt eindrucksvoll, dass Katalyse solcher Reaktionen auch ohne Metall-Ion möglich ist, womit auch gleichzeitig die Quelle für eine potentielle oxidative Schädigung der DNA entfernt ist. Leider ist die Palette der natürlich vorkommenden Enzyme begrenzt. Die Erforschung und Entwicklung von künstlichen Nukleasen ermöglicht daher potentiell den zukünftigen Einsatz neuer, maßgeschneiderter Werkzeuge für die Biochemie und die Biotechnologie, sowie langfristig die Bereitstellung neuartiger Chemotherapeutika. Vom aktiven Zentrum der Staphylokokkennuklease abgeleitete Moleküle auf Bisguanidinium-Naphthol-Basis bzw. deren Derivate zeigten in der Vergangenheit deutliche Aktivität als metallfreie, unspezifische Spalter von Plasmid-DNA. Die vorliegende Arbeit beschreibt die weitere Entwicklung und Charakterisierung neuer unspezifischer und potentiell sequenzselektiver Bisguanidinium-Naphthol-Derivate. Hierbei wurde eine neue, zuverlässige Synthesestrategie für Bisguanidinium-Naphthole und parallel dazu ein neuer und flexibler Weg der Flüssigphasen-Synthese von DNA-bindenden Polyamiden ausgearbeitet, um daraus DNA-bindende Konjugate herzustellen. Vier unspezifische Moleküle (45, 94, 95, 97) und zwei Konjugate (46 und 140) wurden dann bei physiologischen Bedingungen auf ihre Spaltaktivität gegenüber Plasmid-DNA und linearer Duplex-DNA untersucht. Bei allen oben genannten Verbindungen konnte - verglichen mit der Stamm-Verbindung 36 aus Vorgängerarbeiten - eine erhöhte Aktivität gegenüber Plasmid-DNA bestimmt werden, die im Falle der Konjugate zwischen 4000- und 8000-fach liegt. Zur weiteren Charakterisierung wurden Experimente in Anwesenheit von EDTA oder Mg2+, zur pH-Abhängigkeit und zur Kinetik der Spalt-Reaktion durchgeführt. Erste Testreihen zum Nachweis sequenzselektiver DNA-Spaltung lieferten kein abschließendes Ergebnis, gaben jedoch erste Hinweise auf Selektivität, welche zur Zeit näher untersucht und überprüft werden.
In this thesis the integral membrane protein diacylglycerol kinase (DAGK) from E.coli is investigated with solid-state NMR. The aim is to gain an insight into the enzyme’s mechanism through integration of kinetic, structural and dynamic data. The biological function of DAGK is the transfer of the γ-phosphate group from Mg*ATP to diacylglycerol (DAG) building phosphatidic acid (PA)[6] as port of the membrane-derived oligosaccharide cycle[31,34]. Surprisingly, DAGK does not share structural or sequential similarities with other kinases[12]. Typical sequence motives found in other kinases, which catalyze phosphoryl transfer reactions, are not found[13]. In its physiological form DAGK is a homo-trimer with nine transmembrane helices, three catalytic centers and a size of 39.6 kDa.
First, the set-up of a real-time 31P MAS NMR experiment is shown. This experiment allows measuring in real-time the simultaneous ATP hydrolysis in the aqueous phase and lipid substrate phos-phorylation in the membrane phase with atomic resolution under magic angle spinning[56]. After fast transfer of the sample into the NMR spectrometer the enzymatic reaction is started with a temperature jump. This approach of real-time MAS NMR in a dual-phase system was demonstrated for the lipid substrate analogs dioleoyl- (DOG) and dibutyrylglycerol (DBG), with a C8 and C4 aliphatic chain, respectively. The combination of 31P direct and cross polarization functions as a dynamic filter. In the 31P direct polarized experiment nuclei in both phases are detected, while in the 31P cross polar-ized experiment, only nuclei in the membrane phase are detected. Rates for substrate turnover, i.e. degradation of γP-, βP, αP-ATP and build-up of βP-, αP-ADP, free phosphate as side reaction, and PA are obtained, which reveal a Michaelis-Menten behavior with regard to Mg*ATP and DBG. Here Mg*ATP and DBG follow a random-equilibrium model, where every substrate can bind indepen-dently from the other substrate. Analyses of the peak integrals from educts and products of the enzymatic reaction, revealed the stoichiometry of the reaction: 1.5 ATP molecules are used to phos-phorylate one DBG molecule. The excess of ATP is attributed to the basal ATPase activity. Further-more, experiments with ATPγS, usually regarded as a non-hydrolysable ATP-analog, where carried out. Surprisingly, DAGK hydrolyzes ATPγS and also transfers the thio-phosphate group to the lipid acceptor DBG, which points to a certain degree of plasticity in the active center. A phosphorylated enzyme intermediate was not detected. These results suggest the building of a ternary complex of Mg*ATP, DBG and DAGK performing a direct-phosphoryl transfer reaction, without passing through a phosphorylated enzyme intermediate. Experiments with the transition state analog ortho-vanadate (Vi) showed a decoupling of the ATP hydrolysis activity from lipid substrate phosphorylation. This indicates a specific transfer site for the γ-phosphate group from ATP to DAG, which can be blocked by Vi.
A general disadvantage of NMR spectroscopy compared to other spectroscopic methods is its inherent low sensitivity. One possible starting point for the improvement of signal-to-noise per unit time is the reduction of the spin-lattice relaxation time of protons[209]. Usually 95 % of the experi-mental time is required for the relaxation of the 1H to equilibrium. The addition of paramagnetic species can be used to reduce the 1H T1[233]. In a comprehensive study four different paramagnetic agents were tested: Cu2+-EDTA, Cu2+-EDTA-tag, Gd3+-TTAHA and Gd3+-DOTA. The titration of these paramagnetic complexes showed the principle feasibility of this approach, but differences between the tested species exist. The most promising complex is Gd3+-DOTA which, at a concentration of 2 mM, causes a 10-time improvement of signal-to-noise ratio per unit time. This allowed measuring 2D 13C-13C correlation spectra of proteoliposomes in one tenth of the usual required experimental time (i.e. 10 hours vs. 4 days) with good signal-to-noise.
For the investigation of structural or dynamic changes in the protein upon substrate interaction with MAS NMR, the spectral properties CP efficiency and resolution of the DAGK in liposomes needed to be improved. The most critical step during sample preparation is the reconstitution of the membrane protein from detergent micelles into a membrane of synthetic lipids under detergent removal. For this procedure the important criteria are enzymatic activity, measured in a coupled ATPase assay[55], and homogeneity of the proteoliposomes, which was tested e.g. on a discontinuous sucrose step gradient. Therefore an extensive study was carried out, in which different detergents, lipids and lipid mixtures, techniques for detergent removal and different protein-to-lipid ratios were tested. A direct correlation between high ATPase activity and good resolution was not found. Moreover, active DAGK in a mixture of DMPC and cholesterol, which emulates the membrane features of a membrane containing DAG, showed the best CP efficiency and resolution.
The assignment of the protein backbone and amino acid side chains the first mandatory step towards the investigation of structural and dynamical features influencing and defining the enzymatic mechanism by MAS NMR. As the assignment procedure is very time consuming for a total protein, a special labeling scheme for DAGK was developed, which allows assigning most of the protein areas presumably involved in enzyme catalysis. The assignment of DAGK with solution NMR[132] was not transferable to the MAS NMR spectra. Most important for the assignment process were the unique pairs[335], two consecutive amino acids which only appear once in the amino acid sequence. These unique pairs served as anchor points. Five different multinuclear MAS NMR experiments (DARR, NCO, NCA, NCACX, NCOCX) were required for the sequential assignment. It was possible to assign 35 % of the total amino acid sequence with one sample and 8 experiments acquired at 850 MHz. The secondary structure analysis showed subtle differences to the DAGK assignment with solution NMR[132], which can be attributed to the different environment in lipid bilayers and detergent micelles.
Data about structural and dynamical changes under substrate interaction can reveal details about the enzymatic mechanism. Therefore changes in chemical shift in 2D heteronuclear correlation experiments in the apo-state and under substrate saturated conditions with the substrates Mg*AMP-PNP, a non-hydrolysable ATP-analog, DOG, a mixture of Mg*AMP-PNP and DOG as well as inhibited by Vi were recorded. The most significant peak changes were observed at the interface membrane-cytoplasm as well as the the N-terminal amphipathic helix. The residues revealing chemical shift perturbations correlate with conserved residues or such residues, for which importance for catalysis and/or folding could be shown in mutation studies[8]. Especially noticeable were the changes at the amino acids Asn 72, Lys 64, His 87, Tyr 86 and Asp 95.
Beside changes of the chemical shift, changes of line width or signal doubling were observable. These changes can point to a correlation with dynamic reorientations in the μs-ms time regime, which are most relevant for enzymatic processes. The protein backbone dynamics in the apo-state as well as saturated with the substrates or inhibited with Vi were investigated with a 15N-CODEX experiment, which is based on the reorientation of the CSA tensor upon dynamical changes[350]. Specific effects of the different substrates or analogs on the protein backbone dynamic were revealed complementing the structural data and the chemical shift perturbation experiments.
Infections with multidrug resistant bacterial strains like Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa or Acinetobacter baumanii that can accumulate resistance mechanisms against different groups of drugs cause increasing problems for the health care system. Multidrug efflux pumps are able to transport different classes of substances, providing a basic resistance to different antibiotics. Especially when they are overexpressed they can keep bacterial cells alive under antibiotic pressure unless other high level resistance mechanisms like expression of β-lactamases are established. One example for a clinically relevant multidrug efflux pump is the AcrAB/TolC tripartite system of E. coli, that transports a variety of different substrates, including besides antibiotics dyes, detergents, bile salts and organic compounds from the periplasm or the inner membrane out of the cell. AcrB is the inner membrane component of the protein complex that determines not only the substrate specificity of the tripartite system but energises the transport through the whole system process via proton transduction as well. TolC is the outer membrane spanning protein that forms a pore in the outer membrane enabling the system to transport drugs over the latter out of the cell. The periplasmic membrane fusion protein AcrA connects AcrB and TolC in the periplasm completing the channel from the periplasm, respective the inner membrane to the extracellular space. AcrB assembles as trimers, in asymmetric crystal structures each of the protomers adapts a different conformation designated L(oose), T(ight) and O(pen). In the protomers tunnels open up and collaps in different conformations. In the L protomer a periplasmic cleft opens up that can initially bind substrates to the periplasmic part of AcrB. In the T conformation the deep binding pocket opens that is assumed to bind substrates tightly that were bound to the access pocket before. As well in the T conformation a second pathway leading to the deep binding pocket opens that can guide substrates from a groove between transmembrane helices TM7, TM8 and TM9, the TM8 groove, that is connected with socalled tunnel 1 that ends in the deep binding pocket. In the O conformation a new tunnel opens that connects the collapsing deep binding pocket with the periplasmic space, respective the channel through the periplasmic space formed from AcrA and TolC. Substrates were cocrystallised in access and deep binding pocket verifying their role in substrate transport. In the TM8 groove in high resolution crystal structures DDM molecules were cocrystallised in L and T conformation, indicating that the AcrB substrate DDM may utilise this entrance to the deep binding pocket. The asymmetry observed in the AcrB trimers trongly suggests a peristaltic pump mechanism. The functional rotation cycle demands communication between the subunits and tight control of substrate load of protomers during the transport to optimise the ration between protons that are transduced and substrates transported. Indeed it was shown that AcrB transport mechanism is positively cooperative for some β-lactam substrates. For the communication between the subunits it was assumed that ionic interaction between ion pairs established between charged amino acids at the interfaces of protomers in different conformations are of special importance. Thus the amino acids engaged in ionic interactions, respective ion pairs D73-K131, E130-K110, D174-K110, R168, R259-E734 were substituted with non-charged amino acids pairwise and phenotypes were determined in plate dilution assays and MIC experiments. No evidence for a general, substrate independent, reduction of AcrB activity, that would be expected when the ionic residues are of special importance for AcrB function, could be found with the methods applied. Substitutions were not only combined pairwise according to the putative ion pairs but as well in combinations of R168A with D174N, E130Q and K131M. AcrB activity is reduced for the variant R168A_D174N significantly, activity decreases further for quadruple variant E130Q_K131M_ R168A_D174N. Because the reduced activity is only observed in this combination of substitutions the phenotype must result from accumulation of small effects of the single substitutions. R168A may destabilise the protomer interfaces, as its side chain is oriented in direction to the neighbouring protomer at all interfaces, enhancing substratespecific effects of substitutions E130Q, K131M, D174N that are not in all conformations oriented towards the neighbouring protomer but as well along the substrate transport pathway. Further investigations to figure out the details of the effects observed were not conducted because fluctuating expression of the variants hindered experimental procedures.
In another approach TM8 was in focus of the interest. As mentioned above it is a possible substrate entrance in the inner membrane. The linker between TM8 and the periplasmic PC2 subdomain undergoes a coil-to-helix transition when AcrB cycles through L, T and O conformations. Linking the transmembrane part of AcrB that provides the energy for the transport process via proton transduction with the periplasmic part harbouring the major part of the substrate pathway assignes TM8 and the periplasmic linker (859-876) an important role in the function of AcrB. Thus it was investigated with an alanine-scan of residues 859 to 884 and G/P respective P/G exchange followed by phenotype characterisation in growth curve and plate dilution assays of selected variants. In the phenotype determinations none of the variants, except G861P that seems to cause massive sterical restriction in an α-helical region, displayed a general, substrate independent decrease of AcrB activity. Thus it is concluded that the individual properties of amino acids in TM8 and the periplasmic linker are not of general importance for the mechanism of AcrB. The substitution of individual amino acids had impact on uptake of different substrates in plate dilution assays in a substrate dependent manner. The uptake of some substrates, like erythromycin or chloramphenicol is more affected than that of others with rhodamine 6G resistance being only reduced for the G861P variant. A relation between the PSA of substrates and reduced activity of AcrB was observed. in Substrates with higher PSA values are more affected by substitutions in TM8 or periplasmic linker, resulting in the conclusion that substrates with higher PSA are more likely to be taken up via the TM8 groove/tunnel 1 pathway than those with lower PSA values.
The miRNA biogenesis is tightly regulated to avoid dysfunction and consequent disease development. Here, we describe modulation of miRNA processing as a novel noncanonical function of the 5-lipoxygenase (5-LO) enzyme in monocytic cells. In differentiated Mono Mac 6 (MM6) cells, we found an in situ interaction of 5-LO with Dicer, a key enzyme in miRNA biogenesis. RNA sequencing of small noncoding RNAs revealed a functional impact, knockout of 5-LO altered the expression profile of several miRNAs. Effects of 5-LO could be observed at two levels. qPCR analyses thus indicated that (a) 5-LO promotes the transcription of the evolutionarily conserved miR-99b/let-7e/miR-125a cluster and (b) the 5-LO-Dicer interaction downregulates the processing of pre-let-7e, resulting in an increase in miR-125a and miR-99b levels by 5-LO without concomitant changes in let-7e levels in differentiated MM6 cells. Our observations suggest that 5-LO regulates the miRNA profile by modulating the Dicer-mediated processing of distinct pre-miRNAs. 5-LO inhibits the formation of let-7e which is a well-known inducer of cell differentiation, but promotes the generation of miR-99b and miR-125a known to induce cell proliferation and the maintenance of leukemic stem cell functions.
Im ersten Teil dieser Studie wurde zunächst untersucht, welche Mechanismen für die akute und chronische Desensitivierung der glattmuskulären löslichen Guanylylcyclase (GC) gegenüber NO bei intakter Gefäßfunktion verantwortlich sind. Das Kontraktions- und Relaxationsverhalten von isolierten Rattenaorten im Organbad diente als Maß für die GC-Aktivität. Es wurden sowohl der Einfluß des endogen gebildeten NO als auch der Einfluß von exogen zugeführtem NO untersucht. Hierbei wurde ermittelt, ob die Sensitivitätsänderung der GC gegenüber NO direkt durch NO vermittelt wird oder ob eine gesteigerte cGMP-Bildung die indirekte Ursache für die Desensitivierung des Rezeptorproteins ist. Es konnte gezeigt werden, daß die Desensitivierung aufgrund der basalen NO-Freisetzung erfolgte. Wir konnten weiterhin zeigen, daß exogen zugeführtes NO die glatte Gefäßmuskulatur der Ratte gegenüber NO in gleichem Maße desensitiviert wie endogen gebildetes NO. Durch Untersuchungen mit dem Atrialen Natriuretischen Faktor konnten wir den Signaltransduktionsweg näher charakterisieren und zeigen, daß sehr wahrscheinlich eine gesteigerte cGMP-Bildung für die akute Desensitivierung der GC verantwortlich ist. Ergänzend führten wir Analysen über das Redoxgleichgewicht der GC durch. Hierzu wurden neu entwickelte selektive Aktivatoren der Eisen (III)-Form der GC - S973448 und HMR1766 (schwefelsubstituierte Sulfonylamino-carbonsäure-N-arylamide) - verwendet. Wir konnten in unseren Befunden zeigen, daß die akute Desensitivierung der GC durch physiologische Konzentrationen von NO nicht auf einer Oxidation des Hämeisens des Enzyms beruht. In Tiermodellen wurden die Auswirkungen einer chronischen Aktivierung der Eisen (II)-Form und der Eisen (III)-Form der GC auf die NO-Sensitivität, den Redoxstatus und die Proteinexpression der GC analysiert. Außerdem wurde untersucht, ob Aktivatoren der Häm- oxidierten GC bei chronischer in vivo Anwendung eine Toleranz induzieren. Hierbei wurde insbesondere die Frage berücksichtigt, inwieweit diese Substanzen langfristig in der Lage wären, organische Nitrate bei der Behandlung von Krankheitsbildern mit endothelialer Dysfunktion, wie z. B. Atherosklerose und Bluthochdruck, zu ersetzen. Wir konnten erstmals zeigen, daß die Aktivatoren der Eisen (III)-Form der GC wesentliche Vorteile gegenüber den organischen Nitraten aufweisen, da dieBehandlung der Ratten mit S973448 und HMR1766 weder über einen Zeitraum von drei Tagen noch über vier Wochen zu einer Wirkungsabschwächung dieser Substanzen (Eigentoleranz) führt. Auch gegenüber organischen Nitraten sowie endogenem NO (Kreuztoleranz) konnten wir trotz der chronischen Aktivierung der Häm-oxidierten GC keinen Wirkungsverlust dieser Pharmaka beobachten. Expressionsänderungen der GC konnten wir ebenfalls nicht feststellen. Es ist daher anzunehmen, daß die neuen, selektiven Aktivatoren günstigere Eigenschaften als die bisher verwendeten organischen Nitrate aufweisen und therapeutisch zur Behandlung der genannten Herz- Kreislauf- Erkrankungen geeignet sind. Im zweiten Teil der Arbeit untersuchten wir in Tiermodellen mit einer experimentell induzierten Störung der Funktion des glatten Gefäßmuskels, ob die chronische Aktivierung der Eisen (II)-Form der GC zu einer Veränderung der NO-Empfindlichkeit, des Oxidationsstatus des Enzyms und der Expression der GC-Untereinheiten a (alpha) 1 und ß(beta) 1 führt. Wir konnten im Tiermodell der nitrattoleranten Ratte sowohl eine Toleranz gegenüber Nitroglycerol als auch gegenüber endothelabhänigen Dilatatoren und - wenn auch nicht so ausgeprägt - gegenüber Natriumnitroprussid (Kreuztoleranz) beobachten. Einen Wirkungsverlust gegenüber den Aktivatoren der Eisen (III)-Form der GC konnten wir nicht feststellen. Weiterhin zeigten wir, daß die chronische Aktivierung des Renin-Angiotensin-Systems zu einer gesteigerten Superoxidbildung führt, die wiederum eine verminderte Bioverfügbarkeit von NO bewirkt. Dies führt zu einer kompensatorisch gesteigerten Expression der GC-Untereinheiten ( a(alpha) 1 / ß (beta) 1 ) in nitrattoleranten Rattten. Im Tiermodell der genetisch salzsensitiven Dahl-S-Ratte konnten wir nachweisen, daß die durch den renalen Bluthochdruck hervorgerufene Gefäßdysfunktion in den Rattenaorten zu einer Abnahme der Sensitivität gegenüber Acetylcholin und Natriumnitroprussid sowie dem NO- unabhängigen, direkten Aktivator der GC, YC-1, führt. Auch war die Expression beider GC-Untereinheiten in den Aorten der hypertensiven Ratten vermindert.
The title compound, C8H11FN5 +·Cl-, crystallized with a monoprotonated 1-(4-fluorophenyl)biguanidinium cation and a chloride anion in the asymmetric unit. The biguanidium group is not planar [dihedral angle between the two CN3 groups = 52.0 (1)°] and is rotated with respect to the phenyl group [tau = 54.3 (3)°]. In the crystal, N—H ... N hydrogen-bonded centrosymmetric dimers are connected into ribbons, which are further stabilized by N—H ... Cl interactions, forming a three-dimensional hydrogen-bonded network.
In the title compound, C4H7N3O·C2H6OS, creatinine [2-amino-1-methyl-1H-imidazol-4(5H)one] exists in the amine form. The ring is planar (r.m.s. deviation for all non-H atoms = 0.017 Å). In the crystal, two creatinine molecules form centrosymmetric hydrogen-bonded dimers linked by pairs of N—H[cdots, three dots, centered]N hydrogen bonds. In addition, creatinine is linked to a dimethyl sulfoxide molecule by an N—H[cdots, three dots, centered]O interaction. The packing shows layers parallel to (120).
Diese Arbeit beschäftigt sich mit den Strukturen supramolekularer Komplexe, die aus einem Wirkstoff und einem Modellrezeptor bestehen. Um die spezifische Bindung durch H-Brückenbildung nachzuahmen, wurden Co-Kristallkomponenten ausgesucht, die komplementäre Bindungsstellen besitzen. Die Strukturen der erhaltenen Komplexe sowie einiger (pseudo)polymorpher Formen wurden mit Hilfe der Einkristallstrukturanalyse bestimmt. Ein Vergleich mit Kristallstrukturen ähnlicher Verbindungen ergab Hinweise auf die bevorzugten Konformationen sowie die am häufigsten gebildeten H-Brückenmotive. Theoretische Berechnungen mit den Programmen MOMO und GAUSSIAN wurden bei der Einstufung der Stabilität der Konformere und Tautomere sowie bei der Abschätzung der Komplexbildungsenergien eingesetzt.
Zunächst wurden Co-Kristalle synthetisiert, deren Komponenten ausschließlich fixierte H-Brücken-bindungsstellen besitzen. Die Co-Kristallisationsversuche des Antimalariamittels Pyrimethamin mit Orotsäure führten zur Bildung einer neuen polymorphen Form, zwei Solvaten sowie dem gewünschten Co-Kristall.
In dem ADA/DAD-Komplex zwischen dem Antibiotikum Nitrofurantoin und 2,6-Diacetamidopyridin werden die Co-Kristallkomponenten durch drei H-Brücken verbunden. In den Kristallstrukturen wird die energetisch ungünstigere sp-Konformation von Nitrofurantoin bevorzugt. In dieser Konfomation besitzt das Molekül eine positive und eine negative Seite; dies ermöglicht eine dichtere Kristallpackung.
Aufgrund der Elektronegativitäten der O- und S-Atome sollte das Watson-Crick-Basenpaar zwischen den Nucleosiden 2-Thiouridin und Adenin, das durch eine N-H•••O-Brücke verbunden ist, stabiler sein als das entsprechende Wobble-Basenpaar mit einer N-H•••S-Brücke. Um die Stabilitäten der beiden H-Brücken zu untersuchen, wurden Co-Kristallisationsversuche mit dem Thyreostatikum 6-Propyl-2-thiouracil durchgeführt. Im Co-Kristall mit 2-Aminopyrimidin wird das R_2^2(8)-Heterodimer durch eine N-H•••N- und eine N-H•••S-Brücke verbunden, während N-H•••O-Brücken die 6-Propyl-2-thiouracilmoleküle zu Ketten verknüpfen. Aufgrund der ungünstigen intramolekularen Donor/Akzeptor-Abstände wird im Co-Kristall mit 2,6-Diacetamidopyridin der gewünschte ADA/DAD-Komplex nicht beobachtet. Stattdessen bildet 6-Propyl-2-thiouracil mit Hilfe zweier N-H•••S-Brücken R_2^2(8)-Homodimere, mit denen 2,6-Diacetamidopyridin nur durch eine N-H•••O-Brücke verbunden ist. Die Mitwirkung der N-H•••S-Brücke bei der „Basenpaarung“ kann dadurch erklärt werden, dass bei der Beteiligung der N-H•••O-Brücken an dem R_2^2(8)-Motiv N-H•••S-Brücken für die Kettenbildung zuständig wären; dieses Strukturmotiv wird jedoch in Kristallstrukturen selten beobachtet. Insgesamt zeigen diese Untersuchungen, dass C-O- und C-S-Gruppen konkurrenzfähige H-Brückenakzeptoren sind.
Anschließend wurden mehrere Co-Kristalle des Antimykotikums 5-Fluorcytosin synthetisiert. Im Co-Kristall mit 2-Aminopyrimidin wird das gewünschte AD/DA-Heterodimer beobachtet. Ein ähnliches R_2^2(8)-Heterodimer könnte zwischen 5-Fluorcytosin und N-Acetylkreatinin gebildet werden, jedoch werden die Komponenten lediglich durch eine H-Brücke miteinander verknüpft. Energieberechnungen machen dies plausibel. Trotz der komplementären AAD/DDA-Bindungsstellen wird im Co-Kristall mit 6-Aminouracil das Heterodimer nur durch zwei H-Brücken verbunden. Die dadurch gewonnene Energie reicht offenbar aus, um den Energieunterschied zum AAD/DDA-Heterodimer zu kompensieren. Die Co-Kristalle des 5-Fluorcytosins mit 6-Aminoisocytosin sowie der Co-Kristall mit dem antiviralen Wirkstoff Aciclovir bestätigen die Stabilität des AAD/DDA-H-Brückenmusters, welches dem Watson-Crick-Basenpaar C-G ähnelt.
Es gelang auch, das Konformations- und das Tautomerengleichgewicht durch eine spezifische Bindung zu beeinflussen. In den Co-Kristallen von 5-Fluorcytosin mit den beiden konformationell flexiblen Molekülen Biuret und 6-Acetamidouracil wird nur diejenige Konformation gefunden, die zur Bildung des gewünschten AAD/DDA-Heterodimers führt. Dabei liegt Biuret in der energetisch günstigeren trans-Form, 6-Acetamidouracil jedoch in der ungünstigeren cis-Form vor. Die drei AAD/DDA-Komplexe von 6-Methylisocytosin zeigen, dass durch die Bildung komplementärer H-Brückeninteraktionen Tautomere getrennt kristallisiert werden können: in den Co-Kristallen mit 5-Fluorcytosin findet man ausschließlich die 3H-Form, während in dem Komplex mit 6-Aminoisocytosin lediglich die 1H-Form vorliegt.
In dieser Studie werden somit neue Einblicke in die Anwendung von Co-Kristallen als Modellsysteme für die Untersuchung von Wirkstoff/Rezeptor-Wechselwirkungen gewonnen. Um Wirkstoff/Rezeptor-Komplexe noch besser nachzuahmen, sollten zukünftig Co-Kristallisationsversuche mit größeren und flexibleren Modellrezeptoren vorgenommen werden. Weiterhin wäre die Berücksichtigung schwacher Wechselwirkungen bei der Synthese von Co-Kristallen von Interesse.
Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyse biomolecules in situ by subtomogram averaging (STA). Specimen thickness is a key factor affecting cryo-ET data quality. Cells that are too thick for transmission imaging can be thinned by cryo-focused-ion-beam (cryo-FIB) milling. However, optimal specimen thickness for cryo-ET on lamellae has not been systematically investigated. Furthermore, the ions used to ablate material can cause damage in the lamellae, thereby reducing STA resolution. Here, we systematically benchmark the resolution depending on lamella thickness and the depth of the particles within the sample. Up to ca. 180 nm, lamella thickness does not negatively impact resolution. This shows that there is no need to generate very thin lamellae and thickness can be chosen such that it captures major cellular features. Furthermore, we show that gallium-ion-induced damage extends to depths of up to 30 nm from either lamella surface.
Nichtribosomale Peptid Synthetasen sind Quelle für eine Vielzahl an Sekundärmetaboliten mit antibiotischer Wirkung. Jede Synthetase besteht aus einer Abfolge von Modulen, wobei jedes Modul die nötigen Domänen für den Einbau eines Bausteins in das gebildeten Peptids enthält. Ein Ansatz zur Gewinnung neuer Peptidantibiotika, die angesichts der steigenden Zahl multiresistenter Keime dringend benötigt werden, ist der Austausch von Domänen oder Modulen. Aufgrund bisher noch nicht verstandener Selektivitäten, entweder zwischen den Domänen oder zwischen einzelnen Domänen und Zwischenstufen des gebildeten Peptids, führt dieser Ansatz jedoch in der Praxis oft zu keiner oder nur geringer Ausbeute.
Ziel der vorgelegten Arbeit war es, einige dieser Selektivitäten zu untersuchen, wobei der Fokus auf Peptidyl Carrier Proteinen Domänen (PCPs) lag. An diese Domänen sind alle Intermediate während der Reifung des Peptids kovalent über einen Phosphopantethein-Kofaktor (Ppan-Arm) gebunden.
Im ersten Teil der Arbeit sollte die Struktur einer mit einem Heptapeptid beladenen PCP mittels Lösungs-Kernspinresonanzspektroskopie (NMR) bestimmt werden. Hierbei konnte die natürliche Verknüpfung zwischen Ppan-Arm und Peptid über einen Thioester nicht verwendet werden, da diese Bindung zu Hydrolyse-anfällig war. Es konnte jedoch gezeigt werden, dass die Substitution des Thioesters durch eine nicht hydrolysierbare Amidbindung keinen Einfluss auf die Struktur hat, wodurch die Strukturbestimmung möglich war. Hierbei zeigte sich, dass die Peptid-beladene PCP in der sogenannten A/H state Konformation vorliegt, wobei das an sie gebundene Peptid frei beweglich ist. Somit scheint es wahrscheinlich, dass die PCP keine Selektivität für das an sie gebundene Peptid aufweist. Dies ist ein Unterschied zu den strukturell ähnlichen Acyl Carrier Proteinen (ACPs) aus der bakteriellen Fettsäurebiosynthese, da diese eine Bindungstasche für die an sie gebundenen Fettsäuren ausbilden.
Untersuchungen der Selektivität der Kondensationsdomäne (C Domäne) für das PCP gebundene Peptid mittels NMR-Titrationen und biochemischer Analysen konnten nicht durchgeführt werden, da sich im Laufe des Projekts zeigte, dass die aus der Synthetase herausgetrennte C Domäne katalytisch nicht aktiv war. Stattdessen sollte die Kristallstruktur einer Peptid-beladenen PCP-C Bidomäne, für welche eine katalytische Aktivität bereits gezeigt worden war, gelöst werden. Da aber bereits ein signifikanter Anteil der Bidomäne während der Expression mit dem Ppan-Arm beladen wurde, war die nötige quantitative Beladung mit dem Peptid gekoppelten Ppan-Arm in vitro nicht möglich. Eine quantitative Modifizierung mit dem Ppan-Arm in vitro war hingegen erfolgreich, und die Struktur der Ppan-beladenen Bidomäne konnte gelöst werden. Aufgrund des großen Abstands zwischen den aktiven Zentren der beiden Domänen kann es sich bei der beobachteten Orientierung nicht um jene handeln, die die beiden Domänen zueinander annehmen, wenn die C Domäne das PCP-gebundene Peptid bindet.
Im zweiten Teil der Arbeit wurde die Modifizierung einer PCP durch eine Gruppe II Phosphopantetheintransferase (PPT) untersucht. PPTs katalysieren die Übertragung des Ppan Arms auf die Seitenkette eines in PCPs konservierten Serins. In dieser Magnesium-abhängigen Reaktion dient Coenzym A (CoA) als Quelle für den Ppan-Arm. Durch Mutation des konservierten Serins in der PCP zu Alanin konnte ein stabiler Komplex aus PCP und PPT in Anwesenheit von CoA und Magnesium kristallisiert und seine Struktur bestimmt werden.
In einem Strukturmodell für den PCP/PPT Komplex war eine andere Konformation für die PCP postuliert worden, als sie in der Kristallstruktur des Komplexes zu beobachten ist. Durch Strukturbestimmung der PCP mittels Lösungs-NMR und anschließender Titrationsexperimente konnte jedoch gezeigt werden, dass sowohl die freie als auch die komplexierte PCP in Lösung ebenfalls die in der Kristallstruktur beobachtete Konformation einnehmen.
Aufgrund der gelösten Kristallstruktur konnten zwei Bereiche identifiziert werden, in denen die beiden Proteine im Komplex in direktem Kontakt zueinander stehen. Der eine Bereich ist durch eine intermolekulare Wasserstoffbrücke, der andere durch hydrophobe Wechselwirkungen zwischen den Proteinen gekennzeichnet. Durch ortsspezifische Mutagenese konnten beide Wechselwirkungen gestört werden, was sich in einer Abnahme der Komplexstabilität und einer veränderten Geschwindigkeit der Übertragung des Ppan-Arms äußerte.
Die große strukturelle Ähnlichkeit zwischen dem in dieser Arbeit untersuchten Komplex aus zwei in Bacillus vorkommenden Proteinen und einem humanen ACP/PPT Komplex legt die Vermutung nahe, dass die beobachteten Wechselwirkungen in vielen Organismen konserviert sind.
Human Transformer2-beta (hTra2-beta) is an important member of the serine/arginine-rich protein family, and contains one RNA recognition motif (RRM). It controls the alternative splicing of several pre-mRNAs, including those of the calcitonin/calcitonin gene-related peptide (CGRP), the survival motor neuron 1 (SMN1) protein and the tau protein. Accordingly, the RRM of hTra2-beta specifically binds to two types of RNA sequences [the CAA and (GAA)2 sequences]. We determined the solution structure of the hTra2-beta RRM (spanning residues Asn110–Thr201), which not only has a canonical RRM fold, but also an unusual alignment of the aromatic amino acids on the beta-sheet surface. We then solved the complex structure of the hTra2-beta RRM with the (GAA)2 sequence, and found that the AGAA tetra-nucleotide was specifically recognized through hydrogen-bond formation with several amino acids on the N- and C-terminal extensions, as well as stacking interactions mediated by the unusually aligned aromatic rings on the beta-sheet surface. Further NMR experiments revealed that the hTra2-beta RRM recognizes the CAA sequence when it is integrated in the stem-loop structure. This study indicates that the hTra2-beta RRM recognizes two types of RNA sequences in different RNA binding modes.
The CUG-binding protein 1 (CUG-BP1) is a member of the CUG-BP1 and ETR-like factors (CELF) family or the Bruno-like family and is involved in the control of splicing, translation and mRNA degradation. Several target RNA sequences of CUG-BP1 have been predicted, such as the CUG triplet repeat, the GU-rich sequences and the AU-rich element of nuclear pre-mRNAs and/or cytoplasmic mRNA. CUG-BP1 has three RNA-recognition motifs (RRMs), among which the third RRM (RRM3) can bind to the target RNAs on its own. In this study, we solved the solution structure of the CUG-BP1 RRM3 by hetero-nuclear NMR spectroscopy. The CUG-BP1 RRM3 exhibited a noncanonical RRM fold, with the four-stranded b-sheet surface tightly associated with the N-terminal extension. Furthermore, we determined the solution structure of the CUG-BP1 RRM3 in the complex with (UG)3 RNA, and discovered that the UGU trinucleotide is specifically recognized through extensive stacking interactions and hydrogen bonds within the pocket formed by the b-sheet surface and the N-terminal extension. This study revealed the unique mechanism that enables the CUG-BP1 RRM3 to discriminate the short RNA segment from other sequences, thus providing the molecular basis for the comprehension of the role of the RRM3s in the CELF/Bruno-like family.
The well-resolved helium(I) photoelectron spectrum of H3C-Se—Se-CH3 exhibits distinct bands corresponding to 11 of the total 13 valence electron ionizations. The unequivocal assignment is supported by EHMO calculations including spin/orbit coupling. The two selenium lone pair ionizations differ by 0.23 eV; a split observed also for dimethyl disulfide and discussed within a general model for interactions between adjacent lone pairs.
The photoelectron spectrum of H5C6-Te-CH3 displays in its low energy region overlapping bands of gas-phase conformers. Depending on the dihedral angle between the plane of the phenyl ring and the tellurium lone pair, the π conjugation amounts to only 0.1 eV and 0.3 eV, respectively. These values are compared to the considerably larger ones found for the analogous phenyl derivatives H5C6-X-CH3 with X = O, S and Se.
Three-dimensional structure of the glycine-betaine transporter BetP by cryo electron crystallography
(2008)
The soil bacterium Corynebacterium glutamicum has five secondary transporters for compatible solutes allowing it to cope with osmotic stress. The most abundant of them, the transporter BetP, performs a high affinity uptake of glycine-betain when encountering hyperosmotic stress. BetP belongs to the betaine/carnitine/choline/transporter (BCCT) family, and is predicted to have twelve transmembrane helices with both termini facing the cytoplasm. The goal of this thesis is to facilitate understanding of BetP function by determining a three dimensional (3D) model of its structure. Two-dimensional (2D) crystallization of wild-type (WT) BetP has been successfully performed by reconstitution into a mixture of E. coli lipids and bovine cardiolipin, which resulted in vesicular crystals diffracting to 7.5 Å resolution (Ziegler, Morbach et al. 2004). Diffraction patterns of these crystals however showed unfocused spots, generally due to high mosaicity. Better results were obtained by using the constitutively active mutant BetPdeltaC45 in which the first 45 amino acids of the positively charged C-terminus were removed. BetPdeltaC45 crystals obtained under the same conditions for BetP WT were concluded to be pseudo crystals, based on the inconsistence of symmetry. These crystals had BetPdeltaC45 molecules randomly up/downwards inserted into membrane crystals, and cannot be used for structure determination, even though they diffracted up to 7 Å. The problem of pseudo crystal formation could be solved by changing the lipids used for 2D crystallization to a native lipid extract from C. glutamicum cells. This change of lipids improved the crystals to well-ordered packing with exclusive p121_b symmetry. To understand the role of lipids in crystal packing and order, lipids were extracted at different stages during crystallization, and identified by using multiple precursor ion scanning mass spectrometry. The results show that phosphatidyl glycerol (PG) 16:0-18:1 is the most dominant lipid species in C. glutamicum membranes, and that BetP has a preference for the fatty acid moieties 16:0-18:1. Crystallization with synthetic PG 16:0-18:1 proved that an excess of this lipid prevents pseudo crystal formation, but these crystals did not reach the quality as previously achieved by using the C. glutamicum lipids. Apart from the effect of lipids in crystallinity, the concentration and type of salts influenced crystal growth and morphology. High salt conditions (>400 mM LiCl or KCl) yielded tubular crystals, whereas low salt conditions (<300 mM LiCl, NaCl or KCl) led to formation of up to 10 µm large sheet-like crystals. The intermediate concentration gave a mixture of sheet-like and tubular crystals. In terms of resolution, sheets diffracted better than tubes. The sheet-like crystals used for 3D map reconstruction were obtained from a dialysis buffer containing 200 mM NaCl combined with using C. glutamicum lipids. Electron microscopic images were taken from frozen-hydrated crystals using a helium-cooled JEOL 300 SFF microscope or a liquid nitrogen-cooled FEI Tecnai G2 microscope at 300 kV, which allowed optimal data collection and minimized radiation damage to the sample. More than 1000 images of tilt angles up to 50° were taken and evaluated using optical diffraction of a laser beam. The best 200 images were processed with the MRC image processing software package, and 79 images from different tilt angles were merged to the final data set used for calculation of a 3D map at a planar resolution of 8 Å. The structure shows BetPdeltaC45 as a trimer with each monomer consisting of 12 transmembrane alpha-helices. Protein termini and loop regions could not be determined due to the limited resolution of the map. Six of the twelve helices line a central cavity forming a potential substrate-binding chamber. Each monomer shows a central cavity in different sizes and shapes. Thus, the constitutively active BetPdeltaC45 thus forms an unusual asymmetric homotrimer. BetP most likely reflects three different conformational states of secondary transporters: the cytoplasmically open (C), the occluded (O), and the periplasmically open (P) states. The C and O states are similar to BetP WT projection structure, while the P state is discrepant and highly flexible due to the shape and size of the central cavity as well as the lowest intensity of the density. The observation of the P state corresponds well to the constitutively active property of BetPdeltaC45. For the high resolution structure of the C and O states are available, this work presents the first structural information of the P state of a secondary transporter.
The volume changes of lithium and sodium under pressure are discussed with respect to the packing density of the atoms and their valence. In densely packed Li I (bcc), Li II (fcc), and Li III (alpha-Hg ype), valence increases from 1 at ~ 5 GPa to ~ 2.5 at 40 GPa. The maximum valence 3 is attained in Li IV (body-centered cubic, 16 atoms per cell, packing density q = 0.965) at 47 GPa. In densely packed Na I (bcc) a linear increase of valence from 1 at ~ 10 GPa to 2.9 at 65 GPa is found which continues in Na II (fcc) up to 4.1 at 103 GPa.
The volume changes of solid iodine under pressure are discussed with respect to the packing density of the atoms and to valence. The packing density of solid iodine which is 0.805 under ambient pressure increases to 0.976 in monoatomic iodine-II, 0.993 in iodine-III, and 1 in fcc iodine-IV. Simultaneously, the valence increases from 1 in the free molecule to 1.78 in the crystal structure under ambient pressure, 2.72 – 2.81 in iodine-II, 2.86 – 2.96 in iodine-III, and 3 in fcc iodine-IV. The valence then remains constant up to about 180 GPa and rises moderately to 3.15 at the highest investigated pressure of 276 GPa. Parameters for calculating bond numbers, valences and atomic volumes of densely packed halogens, hydrogen, oxygen, and nitrogen are given.
The volume changes of cesium under pressure are discussed with respect to the packing density of the atoms and valence. The element is univalent in densely packed Cs I and Cs II. Valence increases in Cs III (packing density q = 0.973), in Cs IV (q = 0.943), in Cs V (q ~ 0.99), and in close packed Cs VI. The diminuition of volume beyond ~ 15 GPa is caused by this increase only which implies that electrons of the fifth shell act as valence electrons.
Relationships between bond lengths and bond numbers and also between atomic volumes and valencies are derived and parameters for their calculation are given for the s-block, p-block, and d-block metals. From the atomic volumes under pressure, the valencies of three solid lanthanoids have been confirmed or redetermined: La 3; Ce 2. 3. and 4; Yb 2 and 3.
Metallic radii rm are correlated with the ionic radii ri by linear relationships. For groups 1 up to 7 as well as for Al, Ga, In, Tl, Sn, and Pb the ionic radii refer to the maximum valences (oxidation states) as known from compounds according to rm ~ 1.16 x (ri + 0.64) [A° ]. For groups 8 up to 12, rm ~ 0.48 x (ri + 2.26) [°A] with valences W = 14 - G (G = group number). These valences are considered regular (Wr). For groups 1 up to 12, they obey the equation Wr = 7 - |G - 7|. According to this equation all outer s electrons and the unpaired d electrons should be involved in chemical bonding, i.e. in the cohesion of the element in the solid state. From the melting temperatures and the atomic volumes it is concluded, however, that only 19 out of the 30 d-block elements have regular valences, namely the elements of groups 3, 5, 6, 10, 11 as well as Os, Ir, Zn, Cd, and possibly Ru. All of the non-regular valences are lower than the regular ones. Four of them are integers: Mn 3; Fe, Co 4; Re 6.
[Nachruf] Walter Sterzel
(2014)
In recent publications Otto Hahn, last president of the Kaiser-Wilhelm-Gesellschaft, is charged with having favoured the Nazi regime, before World War II by politically purging institutes and suppressing Lise Meitner’s contribution to the discovery of nuclear fission, and during the war by contributing to the German war efforts, mainly to the development of nuclear weapons. These charges, however, which partly concern also the Kaiser-Wilhelm-Gesellschaft and some of their institutes are based on ignorance or disregard of the historical sources.
It is considered whether Fermat’s so called Last Theorem can be understood by substituting variables by polynomials and discussing their properties. The same substitution yields a survey of the Pythagorean Triples.
Background: Immigration has a strong impact on the development of health systems, medicine and science worldwide. Therefore, this article provides a descriptive study on the overall research output.
Methods: Utilizing the scientific database Web of Science, data research was performed. The gathered bibliometric data was analyzed using the established platform NewQIS, a benchmarking system to visualize research quantity and quality indices.
Findings: Between 1900 and 2016 a total of 6763 articles on immigration were retrieved and analyzed. 86 different countries participated in the publications. Quantitatively the United States followed by Canada and Spain were prominent regarding the article numbers. On comparing by additionally taking the population size into account, Israel followed by Sweden and Norway showed the highest performance. The main releasing journals are the Public Health Reports, the Journal of Immigrant and Minority Health and Social Science & Medicine. Over the decades, an increasing number of Public, Environmental & Occupational Health articles can be recognized which finally forms the mainly used subject area.
Conclusion: Considerably increasing scientific work on immigration cannot only be explained by the general increase of scientific work but is also owed to the latest development with increased mobility, worldwide crises and the need of flight and migration. Especially countries with a good economic situation are highly affected by immigrants and prominent in their publication output on immigration, since the countries’ publication effort is connected with the appointed expenditures for research and development. Remarkable numbers of immigrants throughout Europe compel medical professionals to consider neglected diseases, requires the public health system to restructure itself and finally promotes science.
We examine the photoinduced excited state dynamics of pyrene modified adenosine, a versatile probe for folding and hybridization of ribonucleic acids. Measurements in different solvents revealed complex ultrafast dynamics, but high robustness since the overall fluorescence quantum yield (Φf) is hardly affected. The result is a strong fluorescent RNA-probe whose spectral properties change in a defined way upon environmental changes.
In dieser Arbeit wurden zwei Themenblöcke bearbeitet. Zum einen der Aufbau und die Charakterisierung des Kerr-Schalters, einer Anlage zur Messung von Fluoreszenz mit einer Zeitauflösung im Femtosekundenbereich. Zum anderen die Charakterisierung von pyrenmodifizierten Nukleobasen, sowie deren Anwendung in einem neomycinbindenden Aptamer.
The radiative lifetimes of the C3Il-X3II transition of the CSi radical have been calculated from highly correlated electronic wavefunctions and compared with available experimental data. For this transition, the Franck-Condon approximation fails due to the strong R-dependency of the transition moment function.
The tetraaryl μ‐hydridodiborane(4) anion [2H]− possesses nucleophilic B−B and B−H bonds. Treatment of K[2H] with the electrophilic 9‐H‐9‐borafluorene (HBFlu) furnishes the B3 cluster K[3], with a triangular boron core linked through two BHB two‐electron, three‐center bonds and one electron‐precise B−B bond, reminiscent of the prominent [B3H8]− anion. Upon heating or prolonged stirring at room temperature, K[3] rearranges to a slightly more stable isomer K[3 a]. The reaction of M[2H] (M+=Li+, K+) with MeI or Me3SiCl leads to equimolar amounts of 9‐R‐9‐borafluorene and HBFlu (R=Me or Me3Si). Thus, [2H]− behaves as a masked [:BFlu]− nucleophile. The HBFlu by‐product was used in situ to establish a tandem substitution‐hydroboration reaction: a 1:1 mixture of M[2H] and allyl bromide gave the 1,3‐propylene‐linked ditopic 9‐borafluorene 5 as sole product. M[2H] also participates in unprecedented [4+1] cycloadditions with dienes to furnish dialkyl diaryl spiroborates, M[R2BFlu].
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.
Chelidamic acid (4-hydroxypyridine-2,6-dicarboxylic acid) and 2,6-diaminopyridine react to form the title salt, C5H8N3+·C7H4NO5-; there are two formula units in the asymmetric unit. The pyridine N atom of 2,6-diaminopyridine is protonated whereas chelidamic acid is deprotonated at both carboxylate groups but protonated at the N atom; the reaction involves intra- and intermolecular proton transfer. In the crystal, each 2,6-diaminopyridinium cation participates in five strong N-H...O hydrogen bonds (including one bifurcated hydrogen bond). The crystal structure also features strong O-H...O hydrogen bonds between the chelidamate anions, leading to chains along the a axis.
The title co-crystal, C9H9NO2·C6H6O2, is composed of one 2,6-diacetylpyridine molecule and one resorcinol molecule as the asymmetric unit. In the 2,6-diacetylpyridine molecule, the two carbonyl groups are antiperiplanar to the pyridine N atom. In the crystal, the 2,6-diacetylpyridine and resorcinol molecules are connected by two O-H...O hydrogen bonds, forming planar chains of alternating components running along [120].
The soluble loop BC region guides, but not dictates, the assembly of the transmembrane cytochrome b6
(2017)
Studying folding and assembly of naturally occurring α-helical transmembrane proteins can inspire the design of membrane proteins with defined functions. Thus far, most studies have focused on the role of membrane-integrated protein regions. However, to fully understand folding pathways and stabilization of α–helical membrane proteins, it is vital to also include the role of soluble loops. We have analyzed the impact of interhelical loops on folding, assembly and stability of the heme-containing four-helix bundle transmembrane protein cytochrome b6 that is involved in charge transfer across biomembranes. Cytochrome b6 consists of two transmembrane helical hairpins that sandwich two heme molecules. Our analyses strongly suggest that the loop connecting the helical hairpins is not crucial for positioning the two protein “halves” for proper folding and assembly of the holo-protein. Furthermore, proteolytic removal of any of the remaining two loops, which connect the two transmembrane helices of a hairpin structure, appears to also not crucially effect folding and assembly. Overall, the transmembrane four-helix bundle appears to be mainly stabilized via interhelical interactions in the transmembrane regions, while the soluble loop regions guide assembly and stabilize the holo-protein. The results of this study might steer future strategies aiming at designing heme-binding four-helix bundle structures, involved in transmembrane charge transfer reactions.
Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway.
Biacetylbis(methylimine) (1) is obtained by formic acid catalyzed condensation of biacetyl and methylamine. Photoelectron- and UV spectra, H NMR and 13C NMR data are compared with those of the new compound biacetylbis(isopropylimine) (2) and glyoxalbis(isopropylimine) (3).
Membrane proteins play vital role in a variety of cellular processes, such as signal transduction, transport and recognition. In turn they are involved in numerous human diseases and currently represent one of the most prevalent drug targets. A comprehensive understanding of the mechanisms mediated by membrane proteins requires information about their structures at near-atomic resolution, although structural studies of membrane proteins remain behind those of soluble proteins. A bottleneck in the study of membrane proteins resides in the difficulties that are encountered during their high-level production in cell based systems. However, many toxic effects attributed to the over production of membrane proteins are eliminated by cell-free expression, as viable host cells are no longer required. Therefore, the objective of this study was to obtain adequate amounts of selected membrane transport proteins for their structural studies using a cell-free expression system. For the establishment of the cell-free system for membrane proteins, the transporters YbgR and YiiP from Salmonella typhimurium LT2, PF0558 and PF1373 from Pyrococcus furiosus, from the cation diffusion family (CDF), BetP from Corynebacterium glutamicum from the betaine/carnitine/choline transporter (BCCT) family and Aq-2030 from Aquifex aeolicus VF5 from the monovalent cation/proton antiporter-2 (CPA2) family were selected. An Escherichia coli S-30 extract based cellfree system was established by generating the best expression constructs of the target proteins, preparing T7 RNA polymerase and an S-30 extract with high translation efficiency. The functionality of the S-30 extract was shown by the cell-free expression of correctly folded Green Fluorescent Protein (GFP). Essential factors of the cell-free system such as the Mg2+ concentration, the bacterial S-30 extract proportion in the reaction mixture and the time-course of cell-free reactions have been optimized. For the cell-free production of membrane proteins in soluble form, the possibility to supplement cell-free reactions with detergents was explored. A wide range of non-ionic or zwitterionic detergents, were found to be compatible with cell-free synthesis, while ionic detergents and non-ionic detergents at high concentrations had an inhibitory effect. Moreover, high concentrations of polyoxyethylene-alkyl-ethers (Brij) detergents were found to have enhancing effect on the production levels as well as on the solubility of cell-free produced proteins. As membrane proteins tend to misfold and aggregate in a membrane-free translation system, the possibility to supplement the cell-free reactions with inner membrane vesicles (IMVs) to obtain correctly folded target transport proteins was explored. All the target proteins were successfully produced in the batch cell-free reactions and were found to be incorporated in the IMVs. A continuous exchange cell-free (CECF) system was established, where consumable substrates (amino acids, nucleotides and energy regenerating compounds) were supplied to the cell-free reaction mixture through a dialysis membrane, which in consequence resulted in high-level production of target proteins compared to the batch system. The osmosensing and osmoregulated sodium-coupled symporter BetP from C. glutamicum was chosen for the large scale production in CECF set-up. The protein is easily produced in E. coli and is functional as assayed by its transport activity, after purification and reconstitution in liposomes. It is therefore possible to compare in-vivo and cell-free production. High-level cell-free production of BetP was achieved in CECF mode in different forms: (i) as precipitate, (ii) as soluble form in detergent, and (iii) incorporated in IMVs. Cell-free production of BetP resulted in the yield of about 0.5 mg of purified BetP from 1 ml of CECF reaction. The yield of purified BetP was increased to 1.6 fold by addition of 1% polyoxyethylene-(20)-cetyl-ether (Brij58) detergent in the reaction mixture. Moreover, the high level cell-free production of BetP (0.5 mg purified BetP/ml reaction mixture) incorporated in IMVs was shown for the first time in this work.However, it was observed that oligomerization of BetP was not efficient in the cell-free system. Factors that can promote the folding of membrane proteins such as lipids and chaperones were investigated. Addition of lipids and molecular chaperone GroE facilitated correct folding of BetP resulting in increased yield and stability of cell-free produced BetP. The results obtained indicate that most of the cell-free produced BetP exists in functional oligomeric form. The possibility of obtaining milligram amounts of BetP, a 12 trans-membrane protein from the cell-free reactions holds promise for structural and functional studies of other membrane proteins. In any case, the strategies adapted in this study should prove extremely valuable for the production of membrane proteins in the E. coli cell-free expression system.
Im Zuge der steigenden Bedeutung der Proteomforschung und der »Molekularisierung« der Medizin werden neue, effizientere Plattformen zur Untersuchung von Proteinen und deren Wechselwirkungen notwendig. Hier bietet die Nanotechnologie, eine Wissenschaft mit Ursprüngen in der Physik und der Halbleiterindustrie, attraktive Lösungsperspektiven. Ein Bereich der Forschung am Institut für Biochemie der Universität Frankfurt um Prof. Dr. Robert Tampé widmet sich den Aspekten der Nanotechnologie zur Entwicklung von Protein-Chips für die Proteomforschung und Erzeugung von Mustern im Kleinstformat.
First milestone of this Ph.D. thesis was the successful extension of conventional NTA/His-tag technique to self-assembling, multivalent chelator thiols for high-affinity recognition as well as stable and uniform immobilization of His-tagged proteins on chip surfaces. Bis-NTA was linked via an oligoethylene glycol to alkyl thiols by an efficient modular synthesis strategy yielding a novel, multivalent compound for formation of mixed SAMs with anti-adsorptive matrix thiols on gold. Multivalent chelator chips allow a specific, high-affinity, reversible, long-term immobilization of His-tagged proteins. In AFM studies reversibility of the specific protein immobilization process was visualized at single molecule level. The entire control over the orientation of the immobilized protein promotes this chip surface to an optimal platform for studies focusing on research targets at single molecule level and nanobiotechnology. Based on the constructed protein chip platform above and a novel AFM mode (contact oscillation mode, COM) – developed during the current Ph.D. work – protein nanolithography under physiological conditions enabling fabrication of active biomolecular patterns in countless variety has been established. Reversible COM-mediated nanostructuring is exceptionally suitable for multiplexed patterning of protein assemblies in situ. The first selfassembled protein layer acts as a biocompatible and ductile patterning material. Immobilized proteins can be replaced by the AFM tip applying COM, and the generated structures can be erased and refilled with different proteins, which are immobilized in a uniform and functional manner. Multi-protein arrays can be systematically fabricated by iterative erase-and-write processes, and employed for protein-protein interaction analysis. Fabrication of two-dimensionally arranged nanocatalytic centres with biological activity will establish a versatile tool for nanobiotechnology. As an alternative chip fabrication approach, the combined application of methodologies from surface chemistry, semiconductor technology, and chemical biology demonstrated successfully how pre-patterned templates for micro- and nanoarrays for protein chips are fabricated. The surface physical, as well the biophysical experiments, proved the functionality of this technology. The promises of such process technology are fast and economic fabrication of ready-to-use nanostructured biochips at industrial scale. Membrane proteins are complicated in handling and hence require sophisticated solutions for chip technological application. A silicon-on-insulator (SOI) chip substrate with microcavities and nanopores was employed for first technological investigation to construct a protein chip suitable for membrane proteins. The formation of an artificial lipid bilayer using vesicle fusion on oxidized SOI cavity substrates was verified by CLSM. Future AFM experiments will give further insights into the chip architecture and topography. This will provide last evidence of the sealing of the cavity by the lipid bilayer. Transmembrane proteins will be employed for reconstitution experiments on this membrane protein chip platform. Highly integrated microdevices will find application in basic biomedical and pharmaceutical research, whereas robust and portable point-of-care devices will be used in clinical settings.
Lineare sowie zyklische 3-Alkylpyridinalkaloide sind vor allem in Schwämmen der Ordnung Haplosclerida, zu der auch Haliclona viscosa zählt, weit verbreitet. Die Synthese der zuvor von C. Volk isolierten Haliclamine C und D, des Viscosamins und des Viscosalin C bildete den Ausgangspunkt dieser Arbeit.[1-4] Sie erfolgte ausgehend von den bekannten Synthesen der Cyclostellettamine und Haliclamine[5-7] und gliedert sich in drei Abschnitte: erstens Synthese eines ω-Hydroxyalkylpyridins aus einem Bromalkohol, zweitens Funktionalisierung der Monomere in Abhängigkeit der gewählten Methode zur Di- bzw. Trimerisierung und drittens Verknüpfung und gegebenenfalls Zyklisierung. Durch Anwendung und Weiterentwicklung der bekannten Synthesewege wurden so insgesamt 14 lineare Monomere, zwei zyklische Monomere, 16 Cyclostellettamine, zwei Isocyclostellettamine, sieben Haliclamine, fünf Viscosaline sowie Viscosamin[8] und ein Analogon mit Heptylkette hergestellt. Dieser synthetische Zugang ermöglichte es, sowohl den finalen Strukturbeweis für die zuvor isolierten Verbindungen zu erbringen, als auch durch die Analyse der Fragmentierungs-muster von synthetischen und natürlichen Verbindungen mehr über das Verhalten dieser Verbindungen unter MS-Bedingungen zu erfahren. Die so gewonnenen Erkenntnisse führten dazu, dass drei unbekannte Verbindungen ohne Isolierung der Reinsubstanz mit einer Kombination von MS- und HPLC-Daten identifiziert werden konnten. So konnten das erste monozyklische 3-Alkylpyridinalkaloid marinen Ursprungs und zwei neue Haliclamine identifiziert und synthetisiert werden Des Weiteren gelang es, für die von C. Volk isolierten, jedoch nicht identifizierten Verbindungen Strukturen zu ermitteln bzw. auf Grund der MS-Daten Strukturvorschläge zu machen. Die durch den synthetischen Zugang große Anzahl verfügbarer 3-Alkylpyridinalkaloide ermöglichte außerdem eine systematische Untersuchung über den Zusammenhang von biologischer Aktivität und Struktur. Die Ergebnisse der am Helmholtz Institut für Infektionsforschung durchgeführten Experimente zu den antibakteriellen sowie cytotoxischen Eigenschaften von natürlichen wie auch rein synthetischen 3-Alkylpyridinalkaloiden zeigten, dass die Aktivität sich schon beim Addieren bzw. Subtrahieren einer Methylengruppe in einer Alkylkette signifikant ändert. [1] C. A. Volk, M. Köck, Org. Lett. 2003, 5, 3567-3569. [2] C. A. Volk, M. Köck, Org. Biomol. Chem. 2004, 2, 1827-1830. [3] C. A. Volk, H. Lippert, E. Lichte, M. Köck, Eur. J. Org. Chem. 2004, 3154-3158. [4] C. A. Volk, Dissertation, Johann Wolfgang Goethe Universität (Frankfurt am Main), 2004. [5] A. Grube, C. Timm, M. Köck, Eur. J. Org. Chem. 2006, 1285-1295 und Referenzen darin. [6] J. E. Baldwin, D. R. Spring, C. E. Atkinson, V. Lee, Tetrahedron 1998, 54, 13655-13680. [7] A. Kaiser, X. Billot, A. Gateau-Olesker, C. Marazano, B. C. Das, J. Am. Chem. Soc. 1998, 120, 8026-8034. [8] C. Timm, M. Köck, Synthesis 2006, 2580-2584.
A new pseudopolymorph of perchlorinated neopentasilane: the benzene monosolvate Si(SiCl3)4·C6H6
(2020)
A new pseudopolymorph of dodecachloropentasilane, namely a benzene monosolvate, Si5Cl12·C6H6, is described. There are two half molecules of each kind in the asymmetric unit. Both Si5Cl12 molecules are completed by crystallographic twofold symmetry. One of the benzene molecules is located on a twofold rotation axis with two C—H groups located on this rotation axis. The second benzene molecule has all atoms on a general position: it is disordered over two equally occupied orientations. No directional interactions beyond normal van der Waals contacts occur in the crystal.
Background: Simple peak-picking algorithms, such as those based on lineshape fitting, perform well when peaks are completely resolved in multidimensional NMR spectra, but often produce wrong intensities and frequencies for overlapping peak clusters. For example, NOESY-type spectra have considerable overlaps leading to significant peak-picking intensity errors, which can result in erroneous structural restraints. Precise frequencies are critical for unambiguous resonance assignments.
Results: To alleviate this problem, a more sophisticated peaks decomposition algorithm, based on non-negative matrix factorization (NMF), was developed. We produce peak shapes from Fourier-transformed NMR spectra. Apart from its main goal of deriving components from spectra and producing peak lists automatically, the NMF approach can also be applied if the positions of some peaks are known a priori, e.g. from consistently referenced spectral dimensions of other experiments.
Conclusions: Application of the NMF algorithm to a three-dimensional peak list of the 23 kDa bi-domain section of the RcsD protein (RcsD-ABL-HPt, residues 688-890) as well as to synthetic HSQC data shows that peaks can be picked accurately also in spectral regions with strong overlap.
Adequate digital resolution and signal sensitivity are two critical factors for protein structure determinations by solution NMR spectroscopy. The prime objective for obtaining high digital resolution is to resolve peak overlap, especially in NOESY spectra with thousands of signals where the signal analysis needs to be performed on a large scale. Achieving maximum digital resolution is usually limited by the practically available measurement time. We developed a method utilizing non-uniform sampling for balancing digital resolution and signal sensitivity, and performed a large-scale analysis of the effect of the digital resolution on the accuracy of the resulting protein structures. Structure calculations were performed as a function of digital resolution for about 400 proteins with molecular sizes ranging between 5 and 33 kDa. The structural accuracy was assessed by atomic coordinate RMSD values from the reference structures of the proteins. In addition, we monitored also the number of assigned NOESY cross peaks, the average signal sensitivity, and the chemical shift spectral overlap. We show that high resolution is equally important for proteins of every molecular size. The chemical shift spectral overlap depends strongly on the corresponding spectral digital resolution. Thus, knowing the extent of overlap can be a predictor of the resulting structural accuracy. Our results show that for every molecular size a minimal digital resolution, corresponding to the natural linewidth, needs to be achieved for obtaining the highest accuracy possible for the given protein size using state-of-the-art automated NOESY assignment and structure calculation methods.
The adaptive immune system is able to detect and destroy cells that are malignantly transformed or infected by intracellular pathogens. Specific immune responses against these cells are elicited by antigenic peptides that are presented on major histocompatibility complex class I (MHC I) molecules and recognized by cytotoxic T lymphocytes at the cell surface. Since these MHC I-presented peptides are generated in the cytosol by proteasomal protein degradation, they can be metaphorically described as a window providing immune cells with insights into the state of the cellular proteome. A crucial element of MHC I antigen presentation is the peptide-loading complex (PLC), a multisubunit machinery, which contains as key constituents the transporter associated with antigen processing (TAP) and the MHC I-specific chaperone tapasin (Tsn). While TAP recognizes and shuttles the cytosolic antigenic peptides into the endoplasmic reticulum (ER), Tsn samples peptides in the ER for their ability to form stable complexes with MHC I, a process called peptide proofreading or peptide editing. Through its selection of peptides that improve MHC I stability, Tsn contributes to the hierarchy of immunodominant peptide epitopes. Despite the fact that it concerns a key event in adaptive immunity, insights into the catalytic mechanism of peptide proofreading carried out by Tsn have only lately been gained via biochemical, biophysical, and structural studies. Furthermore, a Tsn homolog called TAP-binding protein-related (TAPBPR) has only recently been demonstrated to function as a second MHC I-specific chaperone and peptide proofreader. Although TAPBPR is PLC-independent and has a distinct allomorph specificity, it is likely to share a common catalytic mechanism with Tsn. This review focuses on the current knowledge of the multivalent protein–protein interactions and the concomitant dynamic molecular processes underlying peptide-proofreading catalysis. We do not only derive a model that highlights the common mechanistic principles shared by the MHC I editors Tsn and TAPBPR, and the MHC II editor HLA-DM, but also illustrate the distinct quality control strategies employed by these chaperones to sample epitopes. Unraveling the mechanistic underpinnings of catalyzed peptide proofreading will be crucial for a thorough understanding of many aspects of immune recognition, from infection control and tumor immunity to autoimmune diseases and transplant rejection.
Members of the ATP‐binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP‐binding cassette in the nucleotide‐binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs:
The title compound, C(21)H(18)ClN, was synthesized by an enanti-oselective Brønsted acid-catalysed transfer hydrogenation reaction. The six-membered heterocycle adopts a half-chair conformation. It has the biphenyl residue in an axial position. The two rings of the biphenyl residue are almost coplanar [dihedral angle = 2.65 (9)°]. The crystal packing is stabilized by N-H⋯Cl hydrogen bonds, which connect the mol-ecules into chains running along the a axis.
Die vorliegende Arbeit befasst sich mit der Entwicklung von neuen enantioselektiven und diastereoselektiven Brønsted-Säure katalysierten Reaktionen. Das Aktivierungsprinzip entspricht dabei einer klassischen Säure-Base-Reaktion, in der eine Brønsted-Säure einen Elektronenpaar-Donor protoniert, woraus die Bildung eines Ionenpaares resultiert. Erweitert man dieses Konzept durch den Einsatz einer chiralen Protonenquelle und verwendet als Base ein prochirales Substrat, wie ein Imin, so entsteht durch dessen Protonierung ein chirales Ionenpaar, wodurch das Substrat einerseits aktiviert wird und anderseits asymmetrische Induktion über das chirale Anion erfährt. Greift in dem darauf folgenden Schritt ein Nucleophil selektiv über eine Seite des positiv geladenen Elektrophils an, so bildet sich enantioselektiv ein neues Stereozentrum. Die Natur nutzt dieses Prinzip zum Aufbau von optisch reinen α-Aminosäuren. So katalysiert die Glutamatdehydrogenase (GDH) die Darstellung von Glutaminsäure durch Protonierung des entsprechenden α-Iminoglutarats, wodurch der nachfolgende Hydrid-Angriff mittels Nicotinamidadenindinukleotid (NADH) selektiv die (L)-Aminosäure liefert. Dieses Konzept konnte während der eigenen Diplomarbeit auf die enantioselektive Brønsted-Säure katalysierte Transferhydrierung von Ketiminen übertragen werden. Dabei simuliert eine chirale Protonenquelle 1 das Enzym (GDH) und das Reduktionsmittel NADH wird durch ein synthetisches Analogon, das Hantzsch Dihydropyridin 8a ersetzt ... Die vorliegende Arbeit ist kumulativ verfasst. Der größte Teil der hier vorgestellten Ergebnisse ist bereits veröffentlicht oder zur Publikation eingereicht. Die experimentellen Daten sind Bestandteil der in Kapitel 10 aufgeführten Publikationen und werden nicht gesondert diskutiert. Folgende Teile dieser Arbeit wurden bereits veröffentlicht: Highly Enantioselective Organocatalytic Carbonyl-Ene Reaction with strongly Acid, Chiral Brønsted Acids as Efficient Catalysts Rueping M., Theissmann T., Kuenkel A., Koenigs R.M., Angewandte Chemie International Edition 2008, 47, 6798, Angewandte Chemie 2008, 120, 6903. Asymmetric counterion pair catalysis: An enantioselective Brønsted acid-catalyzed protonation Rueping M., Theissmann T., Raja S., Bats J.W., Advanced Synthesis & Catalysis 2008, 350, 1001. An enantioselective chiral brønsted acid catalyzed imino-azaenamine reaction Rueping M., Sugiono E., Theissmann T., Kuenkel A., Köckritz A., Pews-Davtyan A., Nemati N., Beller M., Organic Letters 2007, 9, 1065. Remarkably low catalyst loading in Brønsted acid catalyzed transfer hydrogenations: Enantioselective reduction of benzoxazines, benzothiazines, and benzoxazinones Rueping M., Antonchick A.P., Theissmann T., Angewandte Chemie International Edition 2006, 45, 6751, Angewandte Chemie 2006, 118, 6903. A highly enantioselective brønsted acid catalyzed cascade reaction: Organocatalytic transfer hydrogenation of quinolines and their application in the synthesis of alkaloids Rueping M., Antonchick A.P., Theissmann T., Angewandte Chemie International Edition 2006, 45, 3683, Angewandte Chemie 2006, 118, 3765. Metal-free Brønsted acid catalyzed transfer hydrogenation - New organocatalytic reduction of quinolines Rueping M., Theissmann, T., Atonchick A.P., Synlett 2006, 1071. The twinned crystal structure of diiodobis(triphenylphosphine) palladium(II) dichloromethane disolvate at 173 K Theissmann T., Bolte M., Acta Crystallographica Section E, 2006, E62, 1056. Folgende Manuskripte wurden zur Veröffentlichung eingereicht: First Enantioselective Chiral Brønsted Acid Catalyzed Synthesis of 4´-Substituted Tetrahydroquinolines Rueping M., Theissmann T., Stoeckel M., Atonchick A.P. Asymmetric Organocatalytic Reductions in the Enantioselective Synthesis of Fluoroquinolones, Flumiquine and Levofloxacin Rueping M, Stoeckel M., Theissmann T., Haack K. Synthesis and Structural Investigations of H8-BINOL-derived N-triflylphosphoramides Rueping M., Nachtsheim B.J., Koenigs R., Ieawsuwan W., Theissmann T. Buchbeitrag: Metal-free Brønsted Acid Catalyzed Transfer-Hydrogenation: Enantioselective Synthesis of Tetrahydroquinolines Rueping M., Theissmann T., Atonchick A.P., Catalysts for Fine Chemical Industry, Vol. 5, 2006
The field of dynamic nuclear polarization has undergone tremendous developments and diversification since its inception more than 6 decades ago. In this review we provide an in-depth overview of the relevant topics involved in DNP-enhanced MAS NMR spectroscopy. This includes the theoretical description of DNP mechanisms as well as of the polarization transfer pathways that can lead to a uniform or selective spreading of polarization between nuclear spins. Furthermore, we cover historical and state-of-the art aspects of dedicated instrumentation, polarizing agents, and optimization techniques for efficient MAS DNP. Finally, we present an extensive overview on applications in the fields of structural biology and materials science, which underlines that MAS DNP has moved far beyond the proof-of-concept stage and has become an important tool for research in these fields.