Refine
Year of publication
- 2016 (81) (remove)
Document Type
- Article (46)
- Doctoral Thesis (25)
- Book (6)
- Contribution to a Periodical (3)
- Other (1)
Has Fulltext
- yes (81)
Is part of the Bibliography
- no (81)
Keywords
Institute
- Biochemie und Chemie (81) (remove)
A chiral analog of the bicyclic guanidine TBD : synthesis, structure and Brønsted base catalysis
(2016)
Starting from (S)-β-phenylalanine, easily accessible by lipase-catalyzed kinetic resolution, a chiral triamine was assembled by a reductive amination and finally cyclized to form the title compound 10. In the crystals of the guanidinium benzoate salt the six membered rings of 10 adopt conformations close to an envelope with the phenyl substituents in pseudo-axial positions. The unprotonated guanidine 10 catalyzes Diels–Alder reactions of anthrones and maleimides (25–30% ee). It also promotes as a strong Brønsted base the retro-aldol reaction of some cycloadducts with kinetic resolution of the enantiomers. In three cases, the retro-aldol products (48–83% ee) could be recrystallized to high enantiopurity (≥95% ee). The absolute configuration of several compounds is supported by anomalous X-ray diffraction and by chemical correlation.
As a centerpiece of antigen processing, the ATP-binding cassette transporter associated with antigen processing (TAP) became a main target for viral immune evasion. The herpesviral ICP47 inhibits TAP function, thereby suppressing an adaptive immune response. Here, we report on a thermostable ICP47-TAP complex, generated by fusion of different ICP47 fragments. These fusion complexes allowed us to determine the direction and positioning in the central cavity of TAP. ICP47-TAP fusion complexes are arrested in a stable conformation, as demonstrated by MHC I surface expression, melting temperature, and the mutual exclusion of herpesviral TAP inhibitors. We unveiled a conserved region next to the active domain of ICP47 as essential for the complete stabilization of the TAP complex. Binding of the active domain of ICP47 arrests TAP in an open inward facing conformation rendering the complex inaccessible for other viral factors. Based on our findings, we propose a dual interaction mechanism for ICP47. A per se destabilizing active domain inhibits the function of TAP, whereas a conserved C-terminal region additionally stabilizes the transporter. These new insights into the ICP47 inhibition mechanism can be applied for future structural analyses of the TAP complex.
The geminal frustrated Lewis pair tBu2PCH2B(Fxyl)2 (1; Fxyl=3,5-(CF3)2C6H3) is accessible in 65 % yield from tBu2PCH2Li and (Fxyl)2BF. According to NMR spectroscopy and X-ray crystallography, 1 is monomeric both in solution and in the solid state. The intramolecular P⋅⋅⋅B distance of 2.900(5) Å and the full planarity of the borane site exclude any significant P/B interaction. Compound 1 readily activates a broad variety of substrates including H2, EtMe2SiH, CO2/CS2, Ph2CO, and H3CCN. Terminal alkynes react with heterolysis of the C−H bond. Haloboranes give cyclic adducts with strong P−BX3 and weak R3B−X bonds. Unprecedented transformations leading to zwitterionic XP/BCX3 adducts occur on treatment of 1 with CCl4 or CBr4 in Et2O. In less polar solvents (C6H6, n-pentane), XP/BCX3 adduct formation is accompanied by the generation of significant amounts of XP/BX adducts. FLP 1 catalyzes the hydrogenation of PhCH=NtBu and the hydrosilylation of Ph2CO with EtMe2SiH.
This thesis is concerned with protein structures determined by nuclear magnetic resonance (NMR), and the text focuses on their analysis in terms of accuracy, gauged by the correspondence between the structural model and the experimental data it was calculated from, and in terms of precision, i.e. the degree of uncertainty of the atomic positions. Additionally, two protein structure calculation projects are described...
Phenytoin (PHT), valproic acid, and modern antiepileptic drugs (AEDs), eg, remacemide, loreclezole, and safinamide, are only effective within a maximum of 70%-80% of epileptic patients, and in many cases the clinical use of AEDs is restricted by their side effects. Therefore, a continuous need remains to discover innovative chemical entities for the development of active and safer AEDs. Ligands targeting central histamine H3 receptors (H3Rs) for epilepsy might be a promising therapeutic approach. To determine the potential of H3Rs ligands as new AEDs, we recently reported that no anticonvulsant effects were observed for the (S)-2-(4-(3-(piperidin-1-yl)propoxy)benzylamino)propanamide (1). In continuation of our research, we asked whether anticonvulsant differences in activities will be observed for its R-enantiomer, namely, (R)-2-(4-(3-(piperidin-1-yl)propoxy)benzylamino)propaneamide (2) and analogs thereof, in maximum electroshock (MES)-, pentylenetetrazole (PTZ)-, and strychnine (STR)-induced convulsion models in rats having PHT and valproic acid (VPA) as reference AEDs. Unlike the S-enantiomer (1), the results show that animals pretreated intraperitoneally (ip) with the R-enantiomer 2 (10 mg/kg) were moderately protected in MES and STR induced models, whereas proconvulsant effect was observed for the same ligand in PTZ-induced convulsion models. However, animals pretreated with intraperitoneal doses of 5, 10, or 15 mg/kg of structurally bulkier (R)-enantiomer (3), in which 3-piperidinopropan-1-ol in ligand 2 was replaced by (4-(3-(piperidin-1-yl)propoxy)phenyl)methanol, and its (S)-enantiomer (4) significantly and in a dose-dependent manner reduced convulsions or exhibited full protection in MES and PTZ convulsions model, respectively. Interestingly, the protective effects observed for the (R)-enantiomer (3) in MES model were significantly greater than those of the standard H3R inverse agonist/antagonist pitolisant, comparable with those observed for PHT, and reversed when rats were pretreated with the selective H3R agonist R-(α)-methyl-histamine. Comparisons of the observed antagonistic in vitro affinities among the ligands 1-6 revealed profound stereoselectivity at human H3Rs with varying preferences for this receptor subtype. Moreover, the in vivo anticonvulsant effects observed in this study for ligands 1-6 showed stereoselectivity in different convulsion models in male adult rats.
Das Hauptziel dieser Dissertation lag in der Verbesserung einzelner Schritte im Prozess der automatischen Proteinstrukturbestimmung mittels Kernmagnetischer Resonanz (NMR). Dieser Prozess besteht aus einer Reihe von sequenziellen Schritten, welche zum Teil bereits erfolgreich automatisiert wurden. CYANA ist ein Programmpaket, welches routinemäßig zur automatischen Zuordnung der chemischen Verschiebungen, der Nuclear Overhauser Enhancement (NOE) Signalen und der Strukturrechnung von Proteinen verwendet wird. Einer der Schritte, der noch nicht erfolgreich automatisiert wurde, stellt die Signalidentifizierung von NMR Spektren dar. Dieser Schritt ist besonders wichtig, da Listen von NMR-Signalen Grundlage aller Folgeschritte sind. Fehler in den Signallisten pflanzen sich in allen Folgeschritten der Datenauswertung fort und können am Ende in falschen Strukturen resultieren. Daher war ein Ziel dieser Arbeit, einen robusten und verlässlichen Algorithmus zur Signalidentifizierung von NMR Spektren in CYANA zu implementieren. Dieser Algorithmus sollte mit dem in FLYA implementierten Ansatz zur automatischen Resonanzzuordnung, der automatischen NOE-Zuordnung und der Strukturrechnung mit CYANA kombiniert werden. Der in CYANA implementierte CYPICK Algorithmus ahmt den von Hand durchgeführten Ansatz nach. Bei der manuellen Methode schaut sich der Wissenschaftler zweidimensionale Konturliniendarstellungen der NMR Spektren an und entscheidet anhand verschiedener Geomtrie- und Ähnlichkeitskriterien, ob es sich um ein Signal des Proteins oder um einen Artefakt handelt. Proteinsignale sind ähnlich zu konzentrischen Ellipsen und erfüllen bestimmte geometrische Kriterien, wie zum Beispiel ungefähr kreisförmiges Aussehen nach entsprechender Skalierung der spektralen Achsen und gänzlich konvexe Formen, die Artefakte nicht aufzeigen. CYPICK bewertet die Konturlinien lokaler Extrema nach diesen Bedingungen und entscheidet anhand dieser, ob es sich um ein echtes Signal handelt oder nicht. Das zweite Ziel dieser Arbeit war es ein Maß zur Quantifizierung der Information von strukturellen NMR Distanzeinschränkungen zu entwickeln. Der sogenannte Informationsgehalt (I) ist vergleichbar mit der Auflösung in der Röntgenkristallographie. Ein weiteres Projekt dieser Dissertation beschäftigte sich mit der strukturbasierten Medikamentenentwicklung (SBDD). SBDD wird meist von der Röntgenkristallographie durchgeführt. NMR hat jedoch einige Vorteile gegenüber der Röntgenkristallographie, welche interessant für SBDD sind. Daher wurden Strategien entwickelt, die NMR für SBDD zugänglicher machen sollen.
Gastric cancer is one of the most common malignancies and a leading cause of cancer death worldwide. The prognosis of stomach cancer is generally poor as this cancer is not very sensitive to commonly used chemotherapies. Epigenetic modifications play a key role in gastric cancer and contribute to the development and progression of this malignancy. In order to explore new treatment options in this target area we have screened a library of epigenetic inhibitors against gastric cancer cell lines and identified inhibitors for the BET family of bromodomains as potent inhibitors of gastric cancer cell proliferations. Here we show that both the pan-BET inhibitor (+)-JQ1 as well as a newly developed specific isoxazole inhibitor, PNZ5, showed potent inhibition of gastric cancer cell growth. Intriguingly, we found differences in the antiproliferative response between gastric cancer cells tested derived from Brazilian patients as compared to those from Asian patients, the latter being largely resistant to BET inhibition. As BET inhibitors are entering clinical trials these findings provide the first starting point for future therapies targeting gastric cancer.
Weltweit sind ca. 130–180 Millionen Menschen mit HCV infiziert und jährlich sterben etwa 500.000 Menschen an dessen Folgen. Die neuartigen Therapien versprechen zwar eine sehr hohe Heilungsrate, sind aber aufgrund ihrer enorm hohen Kosten nur in Industrieländern verfügbar. Noch immer gibt es keine prophylaktische Vakzinierung gegen HCV. Deshalb ist es wichtig, den HCV-Lebenszyklus und die Interaktion zwischen Wirtszelle und Virus detailliert zu verstehen, um die Entwicklung von Therapien und Impfungen zu ermöglichen. Außerdem kann ein fundiertes Wissen von HCV translatiert werden und auf neuartige Erreger der Familie der Flaviviridae, wie Denguevirus und Zikavirus, angewendet werden. Während der Zelleintritt und die Replikation von HCV relativ gut charakterisiert sind, bleiben die Assemblierung und Freisetzung der viralen Partikel schlecht verstandene Schritte des HCV-Lebenszyklus. In dieser Arbeit sollte die Rolle des zellulären Proteins α-Taxilin im Lebenszyklus von HCV untersucht werden. In einer späteren Phase der Arbeit wurde der endosomale Freisetzungsweg von HCV untersucht. Dazu wurden HCV Varianten generiert und charakterisiert, die Fluoreszenz-Proteine im NS5A- und E1-Protein enthalten, durch die es möglich ist, den Replikationskomplex und die Viruspartikel zu visualisieren und zu quantifizieren und den viralen Lebenszyklus dadurch besser untersuchen zu können...
Für die Optimierung sowie Entwicklung lichtsteuerbarer Systeme für biologische Anwendungen oder neue Materialien ist ein detailliertes Verständnis der zugrunde liegenden komplexen, lichtinduzierten Prozesse eine Voraussetzung. Die Verwendung von Photoschaltern in Makromolekülen ermöglicht eine zeitliche und örtliche Kontrolle über strukturelle Änderungen sowie die entsprechend folgenden (biologischen) Funktionen durch die Verwendung von Licht als externem Auslöser.
Ein wichtiger Bestandteil dieser Arbeit befasst sich mit der Entwicklung eines auf Licht reagierenden Riboschalters, welcher die gezielte Kontrolle über Genexpression ermöglicht. Hierzu wurde eine spektroskopische Charakterisierung von verschiedenen Photoschaltern bezüglich einer Verwendung als biologischer Ligand sowie der Wechselwirkungen zwischen Azobenzolen und RNA, auch hinsichtlich ihrer Bindungsdynamiken durchgeführt. Zunächst wurde die hohe Abhängigkeit der (photo-)chemischen Eigenschaften der Azobenzole von der Wahl der Substituenten untersucht, wobei besonders die Anwendung in wässrigem Milieu betrachtet wurde. In einer detaillierten (zeitaufgelösten) Studie wurde der positionsabhängige Einfluss der Hydroxy-Substitution von Azobenzolen auf die Photoisomerisierung in wässriger Lösung untersucht. Für eine ortho-Substitution ergab sich hierbei ein alternativer Deaktivierungskanal nach Photoanregung, welcher stärker ausgeprägt ist als die Isomerisierung. Hierbei wird ein intramolekularer Protontransfer im angeregten Zustand (ESIPT) beobachtet, welcher mit einer Zeitkonstante von 0.3 ps beschrieben werden kann und in einer Keto-Spezies resultiert. Eine Keto-Enol-Tautomerie konnte für die para-Hydroxy-Substitution schon im Grundzustand beobachtet werden. Somit können beide Spezies gezielt adressiert werden. Durch Acetylierung der Hydroxygruppe verlangsamt sich die thermische Relaxation des cis-Isomer zu dem entsprechenden trans-Isomer signifikant ohne die Isomerisierung zu beeinträchtigen. Dementsprechend ermöglicht eine solche Acetylierung die Verwendung von bekannten Azobenzolderivaten als Photoschalter.
Zudem werden in dieser Arbeit zwei verschiedene Herangehensweisen in der Entwicklung eines Riboschalters beschrieben, welcher sich durch Licht regulieren lässt.
Diese sind durch kovalentes bzw. nicht-kovalentes Einbringen eines Azobenzolderivats in die RNA Struktur charakterisiert. Ein neuer Linker, welcher auf einer Desoxyribose-Struktur beruht, wird für die kovalente Anbindung des Azobenzols an den RNA Strang präsentiert, welcher eine licht-induzierte Dehybridisierung ermöglichen soll. Eine außergewöhnlich hohe Schaltamplitude mit einem cis-Gehalt von etwa 90% konnte für das Azobenzol im RNA Einzelstrang schon bei Raumtemperatur ermittelt werden. Zudem wurde der Einfluss des Photoschalters sowie der benachbarten Nukleotide in der RNA auf die Stabilität der RNA Doppelhelix untersucht. Die zweite Vorgehensweise beruht auf einer nicht-kovalenten Bindung zwischen einem Azobenzolderivat und einem RNA-Aptamer, welche lediglich für eines der Photoisomere ermöglicht wird, wodurch eine örtliche und zeitliche Kontrolle der Ligandenbindung der RNA erfolgt. Im Rahmen dieser Arbeit war es möglich zwei verschiedene photoschaltbare RNA Aptamere zu identifizieren und zu untersuchen, welche eine hohe Spezifität und Affinität aufweisen. Zudem wurde die Photoisomerisierung des Azobenzols innerhalb der RNA-Struktur sowie daraus resultierende lichtinduzierte Konformationsänderungen der RNA mittels zeitaufgelöster Anreg-/Abtastspektroskopie untersucht. Die daraus resultierende Dynamik der photoinduzierten Ligandenbindung sollte eine weitere gezielte Optimierung lichtschaltbarer biologischer Systeme erlauben.
Der zweite Teil dieser Arbeit beschäftigt sich mit der zeitaufgelösten Untersuchung eines photoschaltbaren Foldamers. Speziell wurde der strukturelle Übergang des OmPE-Foldamers 10-5 zwischen einer definierten helikalen und einer ungefalteten Konformation auf Grund der Photoisomerisierung der, in das Rückgrat integrierten, Azobenzole untersucht.
Dabei konnten die frühen (Ent-)Faltungsmechanismen des Foldamers im sub-Nanosekunden-Zeitbereich beobachtet werden, welche durch quantenmechanische Rechnungen unterstützt werden konnten. Darüberhinaus, war es möglich einen Anregungsenergietransfer vom PE-Rückgrat des Foldamers auf die Azobenzole nachzuweisen, welcher die Lebensdauer der angeregten Zustände des Systems signifikant verkürzt.
Diese Arbeit liefert wichtige Informationen zu den Reaktionspfaden, den gezielten Wechselwirkungen zwischen Photoschaltern und größeren organischen Molekülen, sowie den daraus resultierenden lichtinduzierten strukturellen Änderungen durch die Anwendung einer Vielzahl an (zeitaufgelösten) spektroskopischen Methoden. Diese Ergebnisse tragen zum weiteren Verständnis komplexer Prozesse in biologischem sowie nicht-biologischem Zusammenhang und somit zu einer weiterführenden Entwicklung neuer Systeme bei.
Metal-organic frameworks (MOFs) have emerged as a promising class of crystalline porous inorganic-organic hybrid materials showing a wide range of applications. In order to realize the integration of MOFs into specific devices, this thesis mainly focuses on the controlled growth and the properties of highly oriented surface-mounted metal-organic frameworks (SURMOFs).
The stepwise layer-by-layer (LbL) growth method exhibits vast advantages for the controllable growth of SURMOFs regarding the crystallite orientation, film thickness and homogeneity. However, up to date, only a few MOFs have been demonstrated to be suited for this protocol. So the first project of this thesis was designed to extend the applicability of the LbL growth. To this end, a semi-rigid linker based [Cu2(sdb)2(bipy)] (sdb = 4,4’-sulfonylbiphenyl dicarboxylate; bipy = 4,4’-bipyridine) MOF was chosen. Employing the LbL growth, [Cu2(sdb)2(bipy)] SURMOFs were successfully grown onto both pyridyl- and carboxyl-terminated surfaces at the temperature range of 15-65 °C. Interestingly, the orientation of the SURMOFs largely depends on temperature on both surfaces. At low temperatures (below 40 °C), SURMOFs with exclusive [010] orientation are obtained. In contrast, at high temperatures (40-65 °C), [001] oriented SURMOF growth is favored. A novel growth mode was demonstrated, which is, instead of surface chemistry, the temperature-induced ripening processes and the tendency to minimize surface energies can dominate the SURMOF growth.
Inspired by the advantages of LbL deposition of isoreticular SURMOFs, the second project was conceived to grow multivariate SURMOFs (MTV-SURMOFs) using mixed dicarboxylate linkers. We advance a hypothesis that linker acidity (expressed by the pKa values) may have an influence on the oriented growth of MTV-SURMOFs. To test the hypothesis, seven isoreticular [Cu2L2(dabco)] (L = single kind of dicarboxylate linker; dabco = 1,4-diazabicyclo[2.2.2]octane) SURMOFs were grown onto pyridyl-terminated surfaces at 60 °C. The quality of [001] orientation is greatly affected by the acidity of the linkers. With this observation, we deposited a series of [Cu2Lm2(dabco)] (Lm = mixed dicarboxylate linkers) SURMOFs under the same conditions. [Cu2Lm2(dabco)] SURMOFs with exclusive [001] orientation are obtained when the growth solution contains two linkers of relatively high pKa value or more than two kinds of linkers (independent of the pKa values), while the mixtures of ligands with relatively low pKa values or a high content of low pKa valued linkers can result in mis-oriented growth of SURMOFs with unexpected [100] orientation.
Moreover, the LbL growth shows enormous potential in the rational construction of functional SURMOFs. Therefore, the third project of this thesis was devised to deposit SURMOFs containing redox-active species. For this, the 4,4’-biphenyldicarboxylic acid (H2(bpdc)) linker was functionalized with ferrocene (Fc) and dimethyl ferrocene (Me2Fc) moieties. [Cu2(bpdc-amide-Fc)2(dabco)] SURMOF (Fc-SURMOF) is perfectly grown along the [100] direction, while mis-oriented growth of [Cu2(bpdc-amide-Me2Fc)2(dabco)] SURMOF (Me2Fc-SURMOF) was observed. Surprisingly, Fc-SURMOF shows excellent electrochemical properties due to the reversible oxidation and reduction of the ferrocene moieties in the oriented pores, while the Me2Fc-SURMOF was found to be a closely packed insulating layer since no extensive charge transfer is observed. A diffusion controlled mechanism of redox reaction is proposed, where the diffusion of the counter anions in the pores limits the current.
Besides the LbL growth protocol, the spin-coating technique is also promising for the oriented growth of SURMOFs. Driven by the specific applications, the fourth project of this thesis was planned to grow functional SURMOFs containing catalytically active units. The Keggin-type polyoxometalates (POMs) with high catalytic activities were chosen to functionalize the HKUST-1 SURMOFs. Combining the technique with methanol vapor induced growth, a series of POM functionalized HKUST-1 SURMOFs (denoted as POM@HKUST-1 SURMOFs) were controllably deposited onto pyridyl-terminated surfaces. The SURMOFs exhibit great potential as electrocatalysts in electrochemical devices due to the excellent redox properties of POMs. In addition, the PTA@HKUST-1 (PTA = phosphotungstic acid) SURMOF can be employed as an ideal platform for the selective loading of methylene blue (MB) dye with high efficiency. Owing to the strong binding between the dye molecules and the framework, the MB dye cannot be desorbed by ion exchange and MB loaded PTA@HKUST-1 SURMOF shows reliable redox properties under inert conditions, further confirming the application potential in electrochemical devices.
ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters.
CryoEM at IUCRJ: a new era
(2016)
The title fluorinated bisbenzoxazine, C18H18F2N2O2, crystallizes with one half-molecule in the asymmetric unit, which is completed by inversion symmetry. The fused oxazine ring adopts an approximately half-chair conformation. The two benzoxazine rings are oriented anti to one another around the central C-C bond. The dominant intermolecular interaction in the crystal structure is a C-H...F hydrogen bond between the F atoms and the axial H atoms of the OCH2N methylene group in the oxazine rings of neighbouring molecules. C-H...[pi] contacts further stabilize the crystal packing.
The title benzoxazine molecule, C18H18Br2N2O2, was prepared by a Mannich-type reaction of 4-bromophenol with ethane-1,2-diamine and formaldehyde. The title compound crystallizes in the monoclinic space group C2/c with a centre of inversion located at the mid-point of the C-C bond of the central CH2CH2 spacer. The oxazinic ring adopts a half-chair conformation. The structure is compared to those of other functionalized benzoxazines synthesized in our laboratory. In the crystal, weak C-H...Br and C-H...O hydrogen bonds stack the molecules along the b-axis direction.
Crystal structure of 1,3-bis(3-tert-butyl-2-hydroxy-5-methylbenzyl)-1,3-diazinan-5-ol monohydrate
(2016)
In the title hydrate, C28H42N2O3·H2O, the central 1,3-diazinan-5-ol ring adopts a chair conformation with the two benzyl substituents equatorial and the lone pairs of the N atoms axial. The dihedral angle between the aromatic rings is 19.68 (38)°. There are two intramolecular O-H...N hydrogen bonds, each generating an S(6) ring motif. In the crystal, classical O-H...O hydrogen bonds connect the 1,3-diazinane and water molecules into columns extending along the b axis. The crystal structure was refined as a two-component twin with a fractional contribution to the minor domain of 0.0922 (18).
The title Schiff base, C19H22N2O3, was synthesized via the condensation reaction of 1,3-diaminopropan-2-ol with 4-methoxybenzaldehyde using water as solvent. The molecule exists in an E,E conformation with respect to the C=N imine bonds and the dihedral angle between the aromatic rings is 37.25 (15)°. In the crystal, O-H...N hydrogen bonds link the molecules into infinite C(5) chains propagating along the a-axis direction. The packing of these chains is consolidated by C-H...O interactions and C-H...[pi] short contacts, forming a three-dimensional network.
In the title compound, C7H14N4·2C6H5ClO, which crystallized with two crystallographically independent 4-chlorophenol molecules and one 1,3,6,8-tetraazatricyclo[4.3.1.13,8]undecane (TATU) molecule in the asymmetric unit, the independent components are linked by two O-H...N hydrogen bonds. The hydrogen-bond acceptor sites are two non-equivalent N atoms from the aminal cage structure, and the tricyclic system distorts by changing the C-N bond lengths. In the crystal, these hydrogen-bonded aggregates are linked into chains along the c axis by C-H...N hydrogen bonds. The crystal structure also features C-H...[pi] contacts.
The excellent results of dispersion‐corrected density functional theory (DFT‐D) calculations for static systems have been well established over the past decade. The introduction of dynamics into DFT‐D calculations is a target, especially for the field of molecular NMR crystallography. Four 13C ss‐NMR calibration compounds are investigated by single‐crystal X‐ray diffraction, molecular dynamics and DFT‐D calculations. The crystal structure of 3‐methylglutaric acid is reported. The rotator phases of adamantane and hexamethylbenzene at room temperature are successfully reproduced in the molecular dynamics simulations. The calculated 13C chemical shifts of these compounds are in excellent agreement with experiment, with a root‐mean‐square deviation of 2.0 ppm. It is confirmed that a combination of classical molecular dynamics and DFT‐D chemical shift calculation improves the accuracy of calculated chemical shifts.
The arachidonic acid cascade is a key player in inflammation, and numerous well-established drugs interfere with this pathway. Previous studies have suggested that simultaneous inhibition of 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH) results in synergistic anti-inflammatory effects. In this study, a novel prototype of a dual 5-LO/sEH inhibitor KM55 was rationally designed and synthesized. KM55 was evaluated in enzyme activity assays with recombinant enzymes. Furthermore, activity of KM55 in human whole blood and endothelial cells was investigated. KM55 potently inhibited both enzymes in vitro and attenuated the formation of leukotrienes in human whole blood. KM55 was also tested in a cell function-based assay. The compound significantly inhibited the LPS-induced adhesion of leukocytes to endothelial cells by blocking leukocyte activation.
Development and implementation of novel optogenetic tools in the nematode Caenorhabditis elegans
(2016)
Optogenetics, though still only a decade old field, has revolutionized research in neurobiology. It comprises of methods that allow control of neural activity by light in a minimally-invasive, spatio-temporally precise and genetically targeted manner. The optogenetic actuators or the genetically encoded light sensitive elements mediate light driven manipulation of membrane potential, intracellular signalling, neuronal network activity and behaviour (Fenno et al. 2011; Dugué et al. 2012). These techniques have been particularly useful for dissecting neural circuits and behaviour in the transparent and genetically amenable nematode model system Caenorhabditis elegans (Husson et al. 2013; Fang-yen et al. 2015).
In fact, C. elegans was the first living organism in which microbial rhodopsin based optogenetic tools (Channelrhodopsin-2 or ChR2, and Halorhodopsin or NpHR) were successfully implemented and bimodal 'remote' control of behaviour was achieved (Nagel et al. 2005; Zhang et al. 2007). Since then it has been a prominent model for the development and application of novel optogenetic tools and techniques, especially in the nervous system which comprises of 302 neurons and is organised in a hierarchical organization. The environmental stimuli are sensed by the sensory neurons, leading to the processing of information by the downstream interneurons, that relay to motor neurons which in-turn synapse onto muscles that drive the movement-based responses.
The microbial rhodopsins like ChR2 and NpHR mediate light driven depolarization and hyperpolarization, respectively and thereby activate or inhibit neural activity. However, they do not allow local control of membrane potential as they are expressed all over the plasma membrane of the cell rather than being restricted to specific domains, for example synaptic sites. Moreover, they completely over-ride the intrinsic activity of the cell, completely bypassing the signal transduction processes inside the cell. Thus, in order to study intracellular signalling and to answer questions pertaining to the endogenous role of receptors and channels in an in-vivo context, the optogenetic tool-kit needs to be expanded.
This thesis aimed at developing and implementing novel optogenetic tools in C. elegans that allow for sub-cellular signalling control as well as endogenous receptor control. These are: two light activated guanylyl cyclases (bPGC and BeCyclOp) to modify cyclic guanosine monophosphate (cGMP) mediated signalling in the sensory neurons, as well as attempts towards rendering endogenous C. elegans receptors - glutamate receptor (GLR-3/-6), acetylcholine receptor (ACR-16), glutamate gated chloride channel (GLC-1) light switchable and to understand their biological function in-vivo.
Organisms respond to sensory cues by activation of a primary receptor followed by relay of information downstream to effector targets by secondary signalling molecules. cGMP is a widely used 2nd messenger in cellular signaling, acting via protein kinase G or cyclic nucleotide gated (CNG) channels. In sensory neurons, cGMP allows for signal modulation and amplification, before depolarization. Chemo-, thermo-, and oxygen-sensation in C. elegans involve sensory neurons that use cGMP as the main 2nd messenger. For example, ASJ is the pheromone sensing neuron regulating larval development, AWC is the chemosensory neuron responding to volatile odours and BAG senses oxygen and carbon dioxide in the environment. In these neurons, cGMP acts downstream of the GPCRs and functions by activating cationic TAX-2/-4 CNG channels, thereby depolarising the sensory neuron. Manipulating cGMP levels is required to access signalling between sensation and sensory neuron depolarization, thereby provide insights into signal encoding. We achieve this by implementing two photo-activatable guanylyl cyclases - 1) a mutated version of Beggiatoa sp. bacterial light-activated adenylyl cyclase, with specificity for GTP (Ryu et al. 2010), termed BlgC or bPGC (Beggiatoa photoactivated guanylyl cyclase) and 2) guanylyl cyclase rhodopsin (Avelar et al. 2014) from Blastocladiella emersonii (BeCyclOp).
bPGC is a BLUF (blue light sensing using flavin) domain containing cyclase which uses FAD as the co-factor and catalyses the synthesis of cGMP from GTP upon activation by blue light. Prior to implementation in sensory neurons, a simpler heterologous system with co-expression of the TAX-2/-4 CNG channel in C. elegans body wall muscle (BWM) was used. The cGMP generated by the light activated cyclases activates the CNG channel leading to the muscle depolarization, thereby causing changes in body length which can be easily scored.
The discovery of antibiotics represented a key milestone in the history of medicine. However, with the rise of these life-saving drugs came the awareness that bacteria deploy defense mechanisms to resist these antibiotics, and they are good at it. Today, we appear at a crossroads between discovery of new potent drugs and omni-resistant superbugs. Moreover, the misuse of antibiotics in different industries has increased the rate of resistance development by providing permanent selective pressure and, subsequently, enrichment of multidrug resistant pathogens. As a result, antimicrobial resistance has now become an urgent threat to public health worldwide (http:// www.who.int/drugresistance/documents/surveillancereport/en/). The development of multidrug resistance (MDR) in an increasing number of pathogens, including Pseudomonas, Acinetobacter, Klebsiella, Salmonella, Burkholderia, and other Gram-negative bacteria is a serious issue. Membrane efflux pump complexes of the Resistance-Nodulation-Division (RND) superfamily play a key role in the development of MDR in these bacteria. These pumps, together with other transporters, contribute to intrinsic and acquired resistance of bacteria toward most, if not all, of the compounds available in our antimicrobial arsenal. Given the enormous drug polyspecificity of MDR efflux pumps, studies on their mechanism of action are extremely challenging, and this has negatively impacted both on the development of new antibiotics that are able to evade these efflux pumps and on the design of pump inhibitors. The collection of articles in this eBook, published as a Research Topic in Frontiers in Microbiology, section of Antimicrobials, Resistance, and Chemotherapy, aims to update the reader about the latest advances on the structure and function of RND efflux transporters, their roles in the overall multidrug resistance phenotype of Gram-negative pathogens, and on the strategies to inhibit their activities. ...
The development of single-photon-counting detectors, such as the PILATUS, has been a major recent breakthrough in macromolecular crystallography, enabling noise-free detection and novel data-acquisition modes. The new EIGER detector features a pixel size of 75 × 75 µm, frame rates of up to 3000 Hz and a dead time as low as 3.8 µs. An EIGER 1M and EIGER 16M were tested on Swiss Light Source beamlines X10SA and X06SA for their application in macromolecular crystallography. The combination of fast frame rates and a very short dead time allows high-quality data acquisition in a shorter time. The ultrafine φ-slicing data-collection method is introduced and validated and its application in finding the optimal rotation angle, a suitable rotation speed and a sufficient X-ray dose are presented. An improvement of the data quality up to slicing at one tenth of the mosaicity has been observed, which is much finer than expected based on previous findings. The influence of key data-collection parameters on data quality is discussed.
Im Rahmen dieser Arbeit wurden zum Vergleich die Strukturen der ATP-Synthasen von Arabidopsis thaliana, Asparagus officinalis, Allium cepa, Helianthus annus, Solanum tuberosum, Bos taurus und Saccharomyces cerevisiae gelöst. Die ATP-Synthase von S. cerevisiae konnte mit einer Auflösung von 19 Å gelöst werden. Der Winkel zwischen den zwei ATP-Synthase-Monomeren in dem ATP-Synthase-Dimer hatte für jede Spezies einen bestimmten Wert. Dieser Winkel änderte sich innerhalb einer Spezies nur wenig im Gegensatz zu Untersuchungen mit Einzelpartikelanalyse.
Die ATP-Synthase-Dimere aus den untersuchten Spezies besitzen unterschiedliche Winkel zwischen 78˚ und 122˚. Der Winkel des ATP-Synthase-Dimers aus S. tuberosum (122˚) viel größer als der in anderen Pflanzen (~98˚), B. taurus (105˚) und S. cerevisiae (78˚). Die Proben von S. tuberosum und B. taurus waren jedoch dünner, was den Winkel eventuell beeinflussen könnte. Um dies auszuschließen müssen in Zukunft weitere Untersuchungen durchgeführt werden.
Des Weiteren wurde im peripheren Stiel der ATP-Synthasen von allen Pflanzenspezies eine Dichte entdeckt, die in B. taurus und S. cerevisiae nicht vorhanden ist. Die Dichte könnte durch eine zusätzliche Untereinheit oder veränderte Untereinheit im Vergleich zu B. taurus und S. cerevisiae kommen.
Weiterhin wurde die Bildung von Reihen aus ATP-Synthase-Dimeren untersucht. Es wurden ATP-Synthase-Dimere von Polytomella sp. gereinigt und in Lipid rekonstituiert. Es wurde das ATP-Synthase-Dimer von Polytomella sp. verwendet, da dieses besonders stabil ist und während der Reinigung nicht zum ATP-Synthase-Monomer zerfällt. Zur Rekonstitution wurde die milde GRecon-Methode verwendet. Hierbei werden Membranproteine in einem Zuckergradienten gleichzeitig in Lipid rekonstituiert und nach ihrer Dichte getrennt. Abhängig von der Dichte der Proteoliposomen ist die Konzentration an Membranproteinen unterschiedlich. In Proteoliposomen mit einer hohen Konzentration bilden sich dünne Schichten in denen die ATP-Synthase-Dimeren Zickzack-Muster formen. Dies deutet darauf hin, dass das ATP-Synthase-Dimer die Membran verformt. In Proteoliposomen mit einer niedrigeren Konzentration an ATP-Synthase-Dimeren wurden runde Vesikel detektiert, in denen die ATP-Synthase-Dimere lange Reihen bilden und die Membran innerhalb jedes ATP-Synthase-Dimer ebenfalls verformt ist. Molekulare Simulationen bestätigen dieses Ergebnis.
Zudem wurde das ATP-Synthase-Dimer in zwei verschiedene Lipide ohne Cardiolipin rekonstituiert, da Cardiolipin ein Lipid ist welches in der bakteriellen und mitochondrialen Membran gefunden wurde und in hohen Konzentrationen in Membrankrümmungen lokalisiert ist (Huang et al., 2006), wie auch die ATP-Synthase-Dimere. Ohne Cardiolipin ist die Rekonstitution nicht geglückt beziehungsweise sind die ATP-Synthase-Dimere weniger gut zueinander angeordnet. Das deutet auf die Wichtigkeit von Cardiolipin in der Stabilisierung der Reihen von ATP-Synthase-Dimeren hin. Weitere Experimente mit verschiedenen ATP-Synthase-Dimeren in verschiedenen Lipiden sind nötig um dies zu untermauern.
Ein weiteres Ziel dieser Arbeit war es ein klonierbares Label zu etablieren, um ein bestimmtes Protein in Kryo-Elektronentomogramme zu identifizieren. Das Label sollte klein sein, um das zu identifizierbare Protein nicht zu beeinflussen und groß genug um in Kryo-Elektronentomogramme identifizierbar zu sein. In Einzelbildern wurde das 6 kDa große Metallothionein gebunden mit Gold identifiziert, wenn zwei Metallothioneine an dem gewünschten Protein kloniert wurden. Metallothionein besteht zu 33 % aus Cysteinen, welche Schwermetalle binden.
In meinen Studien habe ich bewiesen, dass drei Metallothioneine, gebunden mit Gold, in Kryo-Elektronentomogramme detektiert werden können. Jedoch tritt bei der Verwendung von Metallothionein durch die hohe Anzahl an Cysteinen vermehrt Aggregation auf. Bei meinen Untersuchungen fand ich heraus, dass auch das Maltose-Binde-Protein (MBP) ein Signal gleicher Intensität erzeugt. Durch Verwendung von MBP tritt aber keine Aggregation auf und man kann MBP auch zum Reinigen des Proteins verwenden.
Entwicklung neuer Multikomponentenreaktionen zur Synthese von Amin- und α-Aminosäurederivaten
(2016)
Die Entwicklung neuer Synthesemethoden ist von enormer Bedeutung hinsichtlich der Darstellung von neuen Verbindungen mit speziellen, anwendungsorientierten Eigenschaften und in Bezug auf die Suche nach ökologisch verträglicheren und effizienteren Herstellungsmethoden. Multikomponentenreaktionen (MCRs) bieten hierbei eine gute Ansatzmöglichkeit. Gegenüber den klassischen, linear verlaufenden 2-Stufen-Reaktionen weisen MCRs eine hohe Atom-Ökonomie und effiziente Bindungsbildung auf, können zur Minimierung von Zeit-, Energie-, Material- und Kostenaufwand sowie zur geringeren Generierung von Abfallmengen beitragen und ermöglichen einen schnellen Aufbau diverser Molekülstrukturen. Vor diesem Hintergrund gelang im Rahmen der vorliegenden Arbeit die Entwicklung mehrerer 3-Komponentenreaktionen basierend auf der nukleophilen Addition von Arylboronsäuren an in situ gebildete N-Acyl- bzw. N-Sulfonylimine, womit die Synthese von diversen alpha-substituierten Amiden, chiralen, alpha-substituierten Sulfonamiden, chiralen alpha-Arylglycinen sowie von Arylmethylsulfonamiden erfolgte. Der Schlüssel zu einer erfolgreichen Umsetzung hinsichtlich der Methode mit Amiden war die Verwendung eines dualen Katalysatorsystems aus Lewis-Säure und Pd(II) sowie die Anwesenheit von Wasser. Die enantioselektiven Varianten konnten mittels Sulfonamide anstelle der Amide sowie unter Einsatz von Pd(II) und einem chiralen Oxazolin-Liganden erreicht werden. Die neuen Methoden sind einfach in der Durchführung, weisen einen breiten Substrat-bereich auf und im Falle der asymmetrischen Varianten hohe Enantioselektivitäten.
Allerdings besitzt die Reaktionsführung über Organoboronsäuren zwei entscheidende Nachteile: Zum einen bedarf es der Verwendung vorfunktionalisierter Boronsäuren und zum anderen werden stöchiometrische Mengen borhaltiger Abfälle erzeugt. Daher wurden im Rahmen dieser Arbeit auch Prozesse untersucht, bei denen hinsichtlich der Atom-Ökonomie keine unnötig vorfunktionalisierten Startmaterialien eingesetzt werden und bei denen keine oder nur ökologisch vollkommen unbedenkliche Nebenprodukte entstehen. Ein erster Ansatz in diese Richtung gelang dabei mit der Entwicklung einer neuen 3-Komponentenreaktion basierend auf einer Brønsted-Säurekatalysierten, benzylischen C–H-Bindungsfunktionalisierung von 2-Alkylazaarenen.
The ability of endothelial cells to properly adapt to changes in a dynamic blood perfused environment is essential to maintain the physiological function of the vascular system and of the organs. Epigenetic control of gene expression is believed to be the mechanism controlling cell-fate determination and cell-phenotype maintenance. In the thesis, two JmjC demethylases were screened for their function in endothelial biology. Both of them were proved to play a central role in angiogenesis.
The histone 3 lysine 4 demethylase JARID1B was identified as the most highly expressed demethylase in endothelial cell. Knockdown of JARID1B in human umbilical vein endothelial cells (HUVEC) attenuated cell migration, angiogenic sprouting and tube formation. Jarid1b null mice exhibited attenuated retinal angiogenesis and reduced endothelial sprout outgrowth from aortic segments. Microarray data identified that the antiangiogenic transcription factor HOXA5 was suppressed by JARID1B. Consistently, chromatin immunoprecipitation experiment revealed that JARID1B occupies and reduces the histone 3 lysine 4 methylation levels at the HOXA5 promoter, demonstrating a direct function of JARID1B in endothelial HOXA5 gene regulation. Hence, as a highly expressed JmjC protein in endothelial cells, JARID1B fundamentally maintains endothelial angiogenic phenotypes perhaps through suppression of HOXA5.
As second enzyme it was identified that the histone plant homeodomain finger protein 8 (PHF8) plays a role in endothelial angiogenic sprouting as well as tube formation and cell migration. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. To characterize the underlying mechanism, E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4, but not E2F1, expression in endothelial cells. Likewise, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Thus, it is proposed that PHF8 maintains endothelial function by controlling E2F4 expression. On the other hand, microarray and subsequent qPCR validation revealed that the expressions of small nuclear RNAs (snRNAs) were regulated by PHF8. Co-immunoprecipitation experiment demonstrated that PHF8 interacts with spliceosome related proteins SNRP70 and SRPK1 as well as snRNA. Indeed, PHF8 contributed to splicing: GLS and VEGF-A displayed alternative splicing in PHF8 depleted cells. In addition, c-FOS introns were showed to be retained after knockdown of PHF8 in endothelial cells. These results demonstrated that, by controlling angiogenic mRNA splicing, PHF8 could affect endothelial properties.
Collectively, the results uncover the important roles of JARID1B and PHF8 in endothelial cells in the control of angiogenesis. Changing histone modifiers appears as an attractive concept for pro- and antiangiogenic therapy. The present work adds JARID1B and PHF8 as novel potential targets to this emerging field.
Orientation-selective DEER (Double Electron-Electron Resonance) measurements were conducted on a series of rigid and flexible molecules containing Cu(II) ions. A system with two rigidly held Cu(II) ions was afforded by the protein homo-dimer of copper amine oxidase from Arthrobacter globiformis. This system provided experimental DEER data between two Cu(II) ions with a well-defined distance and relative orientation to assess the accuracy of the methodology. Evaluation of orientation-selective DEER (os DEER) on systems with limited flexibility was probed using a series of porphyrin-based Cu(II)–nitroxide and Cu(II)–Cu(II) model systems of well-defined lengths synthesized for this project. Density functional theory was employed to generate molecular models of the conformers for each porphyrin-based Cu(II) dimer studied. Excellent agreement was found between DEER traces simulated using these computed conformers and the experimental data. The performance of different parameterised structural models in simulating the experimental DEER data was also investigated. The results of this analysis demonstrate the degree to which the DEER data define the relative orientation of the two Cu(II) ions and highlight the need to choose a parameterised model that captures the essential features of the flexibility (rotational freedom) of the system being studied.
Super-resolution fluorescence microscopy revolutionizes cell biology research and provides novel insights on how proteins are organized at the nanoscale and in the cellular context. In order to extract a maximum of information, specialized tools for image analysis are necessary. Here, we introduce the LocAlization Microscopy Analyzer (LAMA), a comprehensive software tool that extracts quantitative information from single-molecule super-resolution imaging data. LAMA allows characterizing cellular structures by their size, shape, intensity, distribution, as well as the degree of colocalization with other structures. LAMA is freely available, platform-independent and designed to provide direct access to individual analysis of super-resolution data.
Functional dynamics of ribonucleic acids : development and application of spectroscopic tools
(2016)
Im Rahmen der vorliegenden Dissertation wird der Aufbau eines zeitaufgelösten Fluorimeters, die photophysikalische Grundcharakterisierung der drei 2-(Pyrenylethinyl)-Adenosine (PyAs) und das Wechselwirkungsgeflecht des tetracyclinbindenden Aptamers (TC-Aptamer) mit seinem Liganden Tetracycyclin (TC) und Mg2+ dargestellt.
Das zeitaufgelöste Fluorimeter basiert auf der experimentellen Technik des zeitkorrelierten Einzelphotonenzählens. Es verfügt über zwei Anregungsquellen: gepulste UV-LEDs und einen frequenzverdoppelten titandotierten Saphirlaser. Diese Quellen Decken einen Wellenlängenbereich von (310 - 550) nm ab. Das Spektrometer kann unter günstigen Umständen eine Zeitauflösung von 50 ps erreichen bei einer zeitlichen Messungenauigkeit von weniger als 0,02 %.
Die Leistungsfähigkeit des Aufbaus wird anhand einer umfangreichen Studie an den drei PyAs demonstriert.
Die drei PyAs 2-(1-Pyrenylethinyl)-Adenosine (1PyA), 2-(2-Pyrenylethinyl)-Adenosine (2PyA) und 2-(4-Pyrenylethinyl)-Adenosine (4PyA) sind eine Gruppe fluoreszierender RNA-Nukleosidanaloga, welche die Gesamtheit aller möglichen Konfigurations-isomere der Grundverbindung PyA umfassen. Ihre zeitabhängigen Fluoreszenzzerfallseigenschaften werden ergänzt von stationären Absorptions- und Fluoreszenzspektren, ultraschneller transienter Absorptionsspektroskopie und quantenchemischen Rechnungen. Die Fluoreszenz von 1PyA und 4PyA gehorchen der Regel von Kasha, wohingegen 2PyA einen triexponentiellen Zerfall mit ausgeprägter Abhängigkeit von der Anregungswellenlänge zeigt. Die transienten Absorptionsspektren aller drei Isomere weisen im gesamten Spektrum dominante, wenig strukturierte Absorptionsbanden des ersten angeregten Zustands auf, welche im nahen UV in unterschiedlichem Maße vom Grundzustandsbleichen und stimulierter Emission überlagert werden. 2PyA zeigt eine deutlich ausgeprägte Signatur für eine interne Umwandlung hin zum S1, wenn es in höhere angeregte Zustände angeregt wird.
Das Fluoreszenzverhalten von 2PyA wird mithilfe eines lokal angeregten (LE) und zweier intramolekularer Ladungstransferzustände, von denen einer der koplanaren Orientierung von Pyren und Adenin (MICT) und der andere einer um 90 ° verdrehten Orientierung (TICT) entspricht. Der LE-Zustand ist hierbei verknüpft mit dem S2 von 2PyA, welcher einer rein pyrenlokalisierten Anregung entspricht. Dieser Zustand existiert so in 1PyA und 4PyA nicht. Der verdrehte TICT-Zustand ist nur in 2PyA bevölkerbar, weil für 2PyA die Barriere zur Bildung von Rotameren am niedrigsten ist und das Molekül nach Anregung daher in diese Geometrie kommen kann und dann durch die stärkere elektrostatische Anziehung stabilisiert wird. 1PyA und 4PyA emittieren hingegen nur aus dem MICT-Zustand.
Die Komplexbildung des TC-Aptamers mit seinem Liganden TC in Lösung wird empfindlich beeinflusst durch die-Konzentration von Magnesiumkationen. Dies wird untersucht durch Bindungs- und Faltungs- und Denaturierungsstudien mit verschiedenen Mg- und Harnstoffkonzentrationen. Als experimentelle Observable dienen hierbei die konformationsabhängige Nukleobasenabsorption und ihr Zirkulardichroismus im fernen UV, die Fluoreszenz des Liganden TC und die freiwerdende Wärme der exothermen Bindungsreaktion des Aptamers mit Mg in An- und Abwesenheit von TC.
Ohne Mg ist eine Interaktion des TC-Aptamers mit TC nicht nachweisbar. Dies liegt daran, dass Mg die notwendige elektrostatische Abschirmung der negativen elektrischen Ladung am RNA-Rückgrat zur Verfügung stellt. Die Abschirmung erlaubt es dem Aptamer kompakte Strukturen mit tertiären Kontakten auszubilden. Wenn die Mg-Konzentration die Faltung des Aptamers vollständig unterstützt (> 1 mM), so befindet sich das Aptamer weitgehend in einer vorgefalteten Konformation, welche der bindungskompetenten stark ähnelt. In diesem Zustand kann das Aptamer seinen Liganden extrem schnell, nämlich annähernd diffusionslimitiert binden. Unter diesen Bedingungen hat TC kaum Einfluss auf die Konformation seines Aptamers.
Bei physiologischen Mg-Konzentrationen (0,2 - 0,8 mM) kann das Aptamer kompakte Konformationen mit tertiären Strukturen einnehmen. Diejenige Konformation, welche der bindenden sehr stark ähnelt, dominiert das konformationelle Gleichgewicht jedoch noch nicht vollständig, es ist lediglich eine Konformation von vielen möglichen. Daher eröffnen physiologische Mg-Konzentrationen dem TC-Aptamer Teile des Konformationsraumes, welche andernfalls nicht zugänglich wären und TC stabilisiert selektiv die native Konformation. Diese konformationelle Verschiebung liefert kann hierbei zur robusten Signalgebung für die Funktion als Riboschalter dienen.
We investigate complexes of two paramagnetic metal ions Gd3+ and Mn2+ to serve as polarizing agents for solid-state dynamic nuclear polarization (DNP) of 1H, 13C, and 15N at magnetic fields of 5, 9.4, and 14.1 T. Both ions are half-integer high-spin systems with a zero-field splitting and therefore exhibit a broadening of the mS = −1/2 ↔ +1/2 central transition which scales inversely with the external field strength. We investigate experimentally the influence of the chelator molecule, strong hyperfine coupling to the metal nucleus, and deuteration of the bulk matrix on DNP properties. At small Gd-DOTA concentrations the narrow central transition allows us to polarize nuclei with small gyromagnetic ratio such as 13C and even 15N via the solid effect. We demonstrate that enhancements observed are limited by the available microwave power and that large enhancement factors of >100 (for 1H) and on the order of 1000 (for 13C) can be achieved in the saturation limit even at 80 K. At larger Gd(III) concentrations (≥10 mM) where dipolar couplings between two neighboring Gd3+ complexes become substantial a transition towards cross effect as dominating DNP mechanism is observed. Furthermore, the slow spin-diffusion between 13C and 15N, respectively, allows for temporally resolved observation of enhanced polarization spreading from nuclei close to the paramagnetic ion towards nuclei further removed. Subsequently, we present preliminary DNP experiments on ubiquitin by site-directed spin-labeling with Gd3+ chelator tags. The results hold promise towards applications of such paramagnetically labeled proteins for DNP applications in biophysical chemistry and/or structural biology.
The high selectivity of biological transformations taking place in Nature have long inspired synthetic chemists to develop analogous chemical processes. Similarly, transient intermediates identified in chemical transformations often provide a basis to understand biological processes. Therefore, new insights gained in biological studies are often useful for chemistry and vice versa.
Proteins, and catalytically active enzymes, are among the most essential units of living cells. Metalloproteins or -enzymes, i.e., proteins or enzymes that contain transition metal ions such as copper, nickel, iron or zinc are often involved in processes like (1) metal-ion storage and transport, (2) exchange of electrons with the environment in catalysis and electron transfer reactions, and (3) dioxygen storage, transport, and metabolization.
For decades, copper-mediated biological oxidations have spurred a great deal of interest among synthetic and catalytic chemists. Copper enzymes such as dopamine β-monooxygenase (DβM), peptidylglycine α-hydroxylating monooxygenase (PHM),particulate methane monooxygenase (pMMO) and tyrosinase activate molecular oxygen (O2) and incorporate one of the oxygen atoms selectively into C−H bonds yielding hydroxylated organic substrates. Remarkable progress in bioinorganic research has led to the development of a large number of copper-based model systems supported by various nitrogen donor ligands that bind O2, cleave the O−O bond, and/or afford hydroxylation reactions similar to copper enzymes. These synthetic model systems have helped to understand the structureactivity relationships of their biological role models and supporting theoretical studies have contributed substantially to the development of the field. Specifically, several density functional theory (DFT) studies have provided detailed mechanistic insights into coppermediated aliphatic and aromatic hydroxylation reactions. Until to date, however, pertinent quantum chemical research still suffers from severe problems as to identify sufficiently accurate and efficient methods for mechanistic studies, and conflicting literature reports have created confusions within the scientific community. Therefore, the first aim of this thesis is to identify a DFT method well suited to describe copper-mediated hydroxylation reactions. With this method at hand a number of interesting hydroxylation reactions is investigated aiming at a detailed understanding of the underlying reaction mechanisms.
The thesis is divided into four chapters of which the first, the introductory chapter, is further divided into three sections (1) copper proteins and enzymes, (2) copper-O2 reactivity in enzymes and (3) biomimetic Cu/O2 chemistry. The first section gives a brief overview of a number of copper enzymes. The second section provides a concise introduction to the biochemical transformations brought about by those copper enzymes that perform aliphatic and aromatic hydroxylation reactions. It is shown that such copper enzymes carry different types of active sites which are responsible for their specific biological functions. These copper enzymes with their biological function are the role models for synthetic chemistry. In the third section, biomimetic Cu/O2 chemistry, the insights gathered in the past 35 years of extensive research on copper-based synthetic model systems that mimic various aspects of copper-enzyme reactivity are reviewed. Various types of active copper sites have been realized in these synthetic model systems and a brief introduction to the respective reactivities towards C−H bonds is presented. We will specifically focus on isomerization processes of dinuclear active Cu2O2 sites and the specific reactivity aspects of these isomers, as these phenomena have been the subject of enormous research efforts aiming at the understanding of the function of the enzyme tyrosinase.
Theory has been integral part of this research and density functional theory (DFT) has effectively taken over the role as a working horse in most studies. Therefore, the second chapter is devoted to an exposition of earlier DFT applications in mechanistic studies of Cu/O2 chemistry. We specifically highlight the problems related to the use of DFT in this field and illustrate the present state of knowledge.
The third chapter of this thesis provides results and discussion of (1) DFT benchmark studies and (2) mechanistic studies. In the first section, the results of a careful benchmark study on the performance of various DFT methods to study the μ-η2:η2-peroxodicopper(II)/bis(μ-oxo)dicopper(III) core isomerization and the C–H hydroxylation processes are compared with available experimental reference data. We provide an assessment of the effects of relativity, counteranions, and dispersion on the reference reactions. The most suitable DFT method evolving from this study, BLYP-D/def2-TZVP including solvent and relativistic corrections, is applied in the next sections to investigate the mechanistic scenario underlying three copper-dioxygen mediated hydroxylation reactions of aliphatic and aromatic C–H bonds. Our mechanistic studies show that bis(μ-oxo)dicopper(III) complexes are capable of achieving selective aliphatic and aromatic C–H hydroxylations. The study of substituent effects in these reactions has further shown that the bis(μ-oxo)dicopper complex acts as an electrophile in hydroxylation.
The fourth chapter presents the conclusions of our investigations. Part of the work presented in this thesis has been published in a peer reviewed journal and enclosed in appendix 1. Further research work, not presented in chapters 1-4, was conducted during my PhD time. This has led to two publications which are added in the appendix.
Cytochrome c oxidases (CcOs), members of the heme-copper containing oxidase (HCO) superfamily, are the terminal enzymes of aerobic respiratory chains. The cbb3-type cytochrome c oxidases (cbb3-CcO) form the C-family and have only the central catalytic subunit in common with the A- and B-family HCOs. In Pseudomonas stutzeri, two cbb3 operons are organized in a tandem repeat. The atomic structure of the first cbb3 isoform (Cbb3-1) was determined at 3.2 Å resolution in 2010 (S. Buschmann, E. Warkentin, H. Xie, J. D. Langer, U. Ermler, and H. Michel, Science 329:327-330, 2010, http://dx.doi.org/10.1126/science.1187303). Unexpectedly, the electron density map of Cbb3-1 revealed the presence of an additional transmembrane helix (TMH) which could not be assigned to any known protein. We now identified this TMH as the previously uncharacterized protein PstZoBell_05036, using a customized matrix-assisted laser desorption ionization (MALDI)-tandem mass spectrometry setup. The amino acid sequence matches the electron density of the unassigned TMH. Consequently, the protein was renamed CcoM. In order to identify the function of this new subunit in the cbb3 complex, we generated and analyzed a CcoM knockout strain. The results of the biochemical and biophysical characterization indicate that CcoM may be involved in CcO complex assembly or stabilization. In addition, we found that CcoM plays a role in anaerobic respiration, as the ΔCcoM strain displayed altered growth rates under anaerobic denitrifying conditions.om Pseudomonas stutzeri, a bacterium closely related to the human pathogen Pseudomonas aeruginosa.
Identifizierung des vertebraten-spezifischen Proteins C7orf43 als neue TRAPPII Komplexuntereinheit
(2016)
Bei den transport protein particle (TRAPP) Komplexen handelt es sich um eine Familie von Protein Komplexen, die jeweils aus mehreren Untereinheiten bestehen. In der vorliegenden Arbeit konnte das Protein C7orf43 als neue potenzielle TRAPPII Untereinheit identifiziert werden, die - wie auch die beiden anderen TRAPPII-spezifischen Komponenten TRAPPC9 und TRAPPC10 - sowohl für die Erhaltung von ERGIC, Golgi-Apparat und COPI Vesikel als für den ER zu Golgi Transportweg benötigt wird.
Investigating three-dimensional (3D) structures of proteins in living cells by in-cell nuclear magnetic resonance (NMR) spectroscopy opens an avenue towards understanding the structural basis of their functions and physical properties under physiological conditions inside cells. In-cell NMR provides data at atomic resolution non-invasively, and has been used to detect protein-protein interactions, thermodynamics of protein stability, the behavior of intrinsically disordered proteins, etc. in cells. However, so far only a single de novo 3D protein structure could be determined based on data derived only from in-cell NMR. Here we introduce methods that enable in-cell NMR protein structure determination for a larger number of proteins at concentrations that approach physiological ones. The new methods comprise (1) advances in the processing of non-uniformly sampled NMR data, which reduces the measurement time for the intrinsically short-lived in-cell NMR samples, (2) automatic chemical shift assignment for obtaining an optimal resonance assignment, and (3) structure refinement with Bayesian inference, which makes it possible to calculate accurate 3D protein structures from sparse data sets of conformational restraints. As an example application we determined the structure of the B1 domain of protein G at about 250 μM concentration in living E. coli cells.
Introduction: Interferon alpha (IFNα) is routinely used in the clinical practice for adjuvant systemic melanoma therapy. Understanding the molecular mechanism of IFNα effects and prediction of response in the IFNα therapy regime allows initiation and continuation of IFNα treatment for responder and exclusion of non-responder to avoid therapy inefficacy and side-effects. The transporter protein associated with antigen processing-1 (TAP1) is part of the MHC class I peptide-loading complex, and important for antigen presentation in tumor and antigen presenting cells. In the context of personalized medicine, we address this potential biomarker TAP1 as a target of IFNα signalling.
Results: We could show that IFNα upregulates TAP1 expression in peripheral blood mononuclear cells (PBMCs) of patients with malignant melanoma receiving adjuvant high-dose immunotherapy. IFNα also induced expression of TAP1 in mouse blood and tumor tissue and suppressed the formation of melanoma metastasis in an in vivo B16 tumor model. Besides its expression, TAP binding affinity and transport activity is induced by IFNα in human monocytic THP1 cells. Furthermore, our data revealed that IFNα clearly activates phosphorylation of STAT1 and STAT3 in THP1 and A375 melanoma cells. Inhibition of Janus kinases abrogates the IFNα-induced TAP1 expression. These results suggest that the JAK/STAT pathway is a crucial mediator for TAP1 expression elicited by IFNα treatment.
Conclusion: We suppose that silencing of TAP1 expression provides tumor cells with a mechanism to escape cytotoxic T-lymphocyte recognition. The observed benefit of IFNα treatment could be mediated by the shown dual effect of TAP1 upregulation in antigen presenting cells on the one hand, and of TAP1 upregulation in ‘silent’ metastatic melanoma cells on the other hand. In conclusion, this work contributes to a better understanding of the mode of action of IFNα which is essential to identify markers to predict, assess and monitor therapeutic response of IFNα treatment in the future.
A growing body of evidence suggests that macrophage polarization dictates the expression of iron-regulated genes. Polarization towards iron sequestration depletes the microenvironment, whereby extracellular pathogen growth is limited and inflammation is fostered. In contrast, iron release contributes to cell proliferation, which is important for tissue regeneration. Moreover, macrophages constitute a major component of the infiltrates in most solid tumors. Considering the pivotal role of macrophages for iron homeostasis and their presence in association with poor clinical prognosis in tumors, we approached the possibility to target macrophages with intracellular iron chelators. Analyzing the expression of iron-regulated genes at mRNA and protein level in primary human macrophages, we found that the iron-release phenotype is a characteristic of polarized macrophages that, in turn, stimulate tumor cell growth and progression. The application of the intracellular iron chelator (TC3-S)2 shifted the macrophage phenotype from iron release towards sequestration, as determined by the iron-gene profile and atomic absorption spectroscopy (AAS). Moreover, whereas the addition of macrophage supernatants to tumor cells induced tumor growth and metastatic behavior, the supernatant of chelator-treated macrophages reversed this effect. Iron chelators demonstrated potent anti-neoplastic properties in a number of cancers, both in cell culture and in clinical trials. Our results suggest that iron chelation could affect not only cancer cells but also the tumor microenvironment by altering the iron-release phenotype of tumor-associated macrophages (TAMs). The study of iron chelators in conjunction with the effect of TAMs on tumor growth could lead to an improved understanding of the role of iron in cancer biology and to novel therapeutic avenues for iron chelation approaches.
Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (∼1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy.
Die NADPH-Oxidasen stellen eine wichtige Quelle für reaktive Sauerstoffspezies (Reactive oxygen species; ROS) im Organismus dar. Hierbei dienen die NADPH-Oxidasen nicht nur der Pathogenabwehr, sondern haben einen Einfluss auf eine Vielzahl an oxidativen, physiologischen Prozessen. Unter den NADPH-Oxidasen ist NOX4 einzigartig, da es hauptsächlich im endoplasmatischen Retikulum (ER) lokalisiert ist, konstitutiv aktiv ist und Wasserstoffperoxid (H2O2) produziert. Wir vermuten, dass diese besonderen Eigenschaften eine Konsequenz aus der Interaktion mit bislang unentdeckten NOX4-interagiereden Proteinen ist.
Zweidimensionale blau-native Polyacrylamid-Gelelektrophorese (BN-PAGE) kombiniert mit SDS-PAGE zeigte NOX4 in makromolekularen Komplexen. Interagierende Proteine wurden durch eine quantitative SILAC (stable isotope labeling of amino acids in cell culture)-Co-immunopräzipitation (Co-IP) in NOX4-überexprimierenden HEK293-Zellen gescreent. Hierdurch konnten verschiedene interagierende Proteine identifiziert werden, wobei Calnexin die robusteste Interaktion aufwies. Calnexin konnte zudem in NOX4-haltigen Komplexen durch Complexome Profiling der BN-PAGE oder gleichzeitiger Antikörperfärbung nachgewiesen werden. Die Calnexin-NOX4-Interaktion konnte mittels reverser Co-IP und Proximity ligation assay bestätigt werden, während NOX1, NOX2 und NOX5 nicht mit Calnexin interagierten. Calnexin-Defizienz, untersucht in embryonalen Mausfibroblasten oder durch shRNA gegen Calnexin, reduzierte die NOX4-Proteinexpression und ROS-Bildung, wobei die mRNA-Expression unverändert blieb. Des Weiteren wurde untersucht, ob der bekannte Interaktionspartner von NADPH-Oxidasen, p22phox, wirklich essentiell für die Expression oder Aktivität von NOX4 ist, da es nur in manchen der NOX4-Co-IPs nachgewiesen wurde. Um den Einfluss von p22phox für NOX4 aufzuklären wurde ein CRISPR/Cas9 Knockdown in NOX4-überexprimierenden HEK293 Zellen etabliert. p22phox zeigte keinen Einfluss auf die NOX4-Expression, jedoch war die NOX4-abhängige ROS-Produktion in p22phox-Knockout Zellen verschwunden.
Unsere Ergebnisse deuten darauf hin, dass endogenes NOX4 makromolekulare Komplexe mit Calnexin ausbildet, welches für die korrekte Reifung, Prozessierung und Funktion von NOX4 im ER nötig ist. Darüber hinaus ist p22phox nicht für die Reifung von NOX4, aber für dessen Aktivität nötig. Diese Ergebnisse zeigen eine vielfältige Regulation von NOX4 auf Proteinebene.
FUSE Binding Protein 1 (FUBP1) is a transcriptional regulator, which is overexpressed in various cancer entities, including hepatocellular carcinoma (HCC) and colorectal cancer (CRC). It fulfills pro-proliferative and anti-apoptotic functions in cancer cells, resulting in increased proliferation and reduced sensitivity towards apoptotic stimuli.
Previously, camptothecin (CPT) and its clinically used analog 7-ethyl 10hydroxycamptothecin (SN-38) were shown to inhibit FUBP1 in biophysical interaction displacement assays (AlphaScreen; surface plasmon resonance, SPR), and first insights into the cellular effects of FUBP1 inhibition were obtained. CPT and SN-38 are known to potently inhibit topoisomerase 1 (TOP 1), and until today, these inhibitors were thought to be specific for this target. This could be disproved by our FUBP1 binding studies. An open issue, which is addressed in this thesis, was the contribution of FUBP1 inhibition to SN-38-mediated apoptosis apoptosis.
During this thesis, a low micromolar efficacy of CPT/SN-38-induced inhibition of FUBP1 binding to the Far Upstream Sequence Element (FUSE) oligonucleotide of p21 was determined. Furthermore, FUBP1 was for the first time shown to directly interact with a potential FUSE sequence upstream of the transcription start in pro-apoptotic gene BIK. In proof of-principle experiments, an effective inhibition of the binding of FUBP1 to the FUSE BIK DNA by CPT/SN-38 was verified.
One of the main goals of this thesis was to further elucidate the contribution of cellular FUBP1-inhibition by CPT/SN-38 to the anti-cancer potential of these substances. For this purpose, the TOP 1 mutant and TOP 1 wild type colorectal cancer sub-cell lines HCT116 G7 and HCT116 S were used. CPT/SN-38 was shown to induce apoptosis in single and combinatorial treatments with mitomycin c (MMC), independently of the TOP 1 mutation status of the cells. Furthermore, a prominent induction of a FUBP1 target gene signature was observed upon treatment of both cell lines with CPT/SN-38. Consequently, CPT/SN-38 was able to fulfill its anticancer effects in these cells, although TOP 1 could not be the main target in the mutant cell line.
In a second approach to gain indirect evidence for FUBP1 dependent effects of CPT/SN-38, the TOP 1-specific inhibitors topotecan (TTN) and β lapachone (BL) were used for the treatment of HCC and CRC cell lines. Interestingly, the TOP 1 inhibitors TTN and BL exhibited a reduced potency in apoptosis induction compared to the dual (FUBP1 and TOP 1) inhibitor SN-38.
Finally, two independent screens for a specific FUBP1 inhibitor were performed. In the first approach, a small number of structural and functional CPT-derivatives that exhibited a reduced inhibitory potential against TOP 1, were tested for their ability to interfere with the FUBP1/FUSE binding. Two particular indenoisoquinoline derivatives revealed potent in vitro inhibition of FUBP1 with low micromolar IC50 values.
In a second approach, previously identified candidate FUBP1 inhibitors that had been isolated from the Maybridge Hit Finder library served as lead structures for a structure activity relationship (SAR) study of the inhibition of FUBP1 binding to the FUSE oligonucleotide. After two cycles of optimization, a medium-potent FUBP1 inhibitor was obtained that induced effective deregulation of FUBP1 target genes in cell culture experiments.
Solvent-free treatment of 1,3,6,8-tetraazatricyclo[4.3.1.13,8]undecano (TATU) with 4-chloro-3,5-dimethylphenol led to the formation of the title co-crystal, C7H14N4·2C8H9ClO. The asymmetric unit contains one aminal cage molecule and two phenol molecules linked via two O-H...N hydrogen bonds. In the aminal cage, the N-CH2-CH2-N unit is slightly distorted from a syn periplanar geometry. Aromatic [pi]-[pi] stacking between the benzene rings from two different neighbouring phenol molecules [centroid-centroid distance = 4.0570 (11) Å] consolidates the crystal packing.
Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na+ across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430 kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS.
The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets.
Imaging non-adherent cells by super-resolution far-field fluorescence microscopy is currently not possible because of their rapid movement while in suspension. Holographic optical tweezers (HOTs) enable the ability to freely control the number and position of optical traps, thus facilitating the unrestricted manipulation of cells in a volume around the focal plane. Here we show that immobilizing non-adherent cells by optical tweezers is sufficient to achieve optical resolution well below the diffraction limit using localization microscopy. Individual cells can be oriented arbitrarily but preferably either horizontally or vertically relative to the microscope’s image plane, enabling access to sample sections that are impossible to achieve with conventional sample preparation and immobilization. This opens up new opportunities to super-resolve the nanoscale organization of chromosomal DNA in individual bacterial cells.
Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D. M. (1990) Biochemistry 29, 4031–4037) is postulated to proceed in 2 steps: partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two, and the first three transmembrane (TM) segments, as well as a construct comprising TM1–TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin, A., Cohen, L. S., Arshava, B., Tantry, S., Becker, J. M., Zerbe, O., and Naider, F. (2009) Biophys. J. 96, 3187–3196), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123, and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior.
This thesis deals with the NMR characterization of the structure and the folding dynamics of DNA G quadruplexes as potential therapeutic target in cancer therapy and building block for DNA based nanotechnology.
The first part of this thesis (Chapters 1-5) introduces the reader to the world of G quadruplexes.
The main features of the classic Watson Crick double helix and alternative non B DNA structures are illustrated in Chapter 1. Many different base pairing schemes are possible, besides the canonical Watson Crick motif, thereby expanding the structural complexity of DNA. Non canonical base pairing, such as Hoogsteen hydrogen bonding, enables the assembly of triplets and quartets, which are the building blocks of triplex and quadruplex structures, respectively.
The structural characteristics of DNA G quadruplexes are delineated in detail in Chapter 2.
G quadruplex structures are extremely polymorphic, in terms of strands orientation, loops geometry, grooves width and arrangement of the glycosidic torsion angles. The various structural elements as well as the different cation coordination geometries are here presented, with a special emphasis on the diversity of conformations reported for the telomeric DNA G quadruplexes.
Chapter 3 describes the biological roles of G quadruplex structures in the genome. After introducing the architecture of the telomeric DNA and its interacting proteins, the mechanism of the telomeres elongation catalysed by the telomerase enzyme and its implications for cancer are discussed. The occurrence of G quadruplex structures in functional regions of the genome, such as promoter regions of oncogenes, and their possible roles in regulating the gene transcription are then outlined in the second part of the chapter.
The potential of G quadruplex as a novel anti cancer target is examined in Chapter 4 and the proposed anti cancer mechanisms for a ligand stabilizing G quadruplex structures are discussed.
RNA G quadruplexes and their putative role in gene regulation at the level of translation are briefly illustrated at the end of the chapter.
A general overview on the NMR methods to investigate the G quadruplex structures is presented in Chapter 5. The experimental set up used for the real time NMR studies of the G quadruplex folding is also described.
The second part of the thesis (Chapters 6-8), which is the cumulative part, comprises the original publications grouped in three Chapters according to the topic.
The state of the art on small molecules targeting G quadruplex structures is given at the beginning of Chapter 6, including a summary of the experimental structures of G quadruplexes in complex with ligands available up to date. The publications presented in Chapters 6.1-6.3 are concerned with the elucidation of the interaction modes between DNA G quadruplexes and selected ligands with potential therapeutic applications.
The binding ability of two natural alkaloids (berberine and sanguinarine) to telomeric G quadruplexes is examined in Chapter 6.1. The ability of carbazole and diguanosine derivatives (synthetized in the group of Prof. Dash, IISER, Kolkata) to interact with c-MYC G quadruplex and down regulate c-MYC expression is explored in Chapter 6.2 and Chapter 6.3, respectively.
The energy landscape of human telomeric G quadruplex structures is discussed in Chapter 7, in light of the experimental kinetic studies as well as molecular dynamics simulations reported in literature until now. Up to date there is no general consensus regarding the folding pathway of unimolecular human telomeric G quadruplex, in particular due to the lack of atomic resolution data on the species involved in the folding. Chapter 7.1 presents the first real time NMR study of the human telomeric G quadruplex folding kinetics.
The final chapter of this thesis (Chapter 8) outlines the potential of G-quadruplex structures as building blocks in nanotechnology. After illustrating briefly the additional possibilities offered by alternative non B DNA structures to programme nanomaterials, a number of applications employing G quadruplex structures in different fields of nanotechnology are described. The article presented in Chapter 8.1 investigates the structural and photoswitching properties of a novel intermolecular azobenzene containing G quadruplex synthetized in the group of Prof. Heckel (Goethe University, Frankfurt).
An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca2+-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca2+-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca2+ in response to light.