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In the paper by Bolte [Acta Cryst. (2006), E62, m1609-m1610], the chemical name in the title and the chemical diagram are incorrect. The correct title is {5-[4'-(2,2,5,5-Tetramethyl-3-pyrroline-1-oxyl-3-carbonyloxy)biphenyl-4-ylethynyl]-2,3,7,8,12,13,17,18-octaethylporphyrinato}copper(II) benzene solvate' and the correct diagram is given below.
Unlimited self-renewal is an absolute prerequisite for any malignancy, and is the ultimate arbiter of the continuous growth and metastasis of tumors. It has been suggested that the self-renewal properties of a tumor are exclusively contained within a small population, i.e., the so-called cancer stem cells. Enhanced self-renewal potential plays a pivotal role in the development of leukemia. My data have shown that APL associated translocation products PML/RARalpha and PLZF/RARalpha increased the replating efficiency of mouse lin-/Sca1+ hematopoietic stem cells (HSCs). This effect is partly mediated by induction of gamma–catenin which is an important mediator of the Wnt signaling pathway and has been shown to be up regulated by the AML associated translocation products(AATPs). Suppression of gamma–catenin by siRNA can abrogate the increased replating efficiency induced by AATPs. Transduction of gamma–catenin in lin-/Sca1+ HSCs led to increased replating efficiency and the expression of stem cell markers Sca1 and c-kit. Additionally it induced accelerated cell cycle progression of mouse bone marrow HSCs. Transduction/transplantation mouse models have shown that ectopic expression of gamma–catenin in HSCs led to acute myeloid leukemia without maturation. These data suggest important roles of Wnt signaling pathway in the leukemogenesis induced by PML/RARalpha, PLZF/RARalpha and AML1/ETO. In contrast to AATPs, CML and Ph+-ALL associated translocation products p185(BCR-ABL) and p210(BCR-ABL) did not affect the self-renewal potential of hematopoietic stem/progenitor cells. However my studies indicated that their reciprocal translocation products p40(ABL/BCR) and p96(ABL/BCR) actually increased the replating efficiency of hematopoietic stem/progenitor cells. The effect is stronger when induced by p96(ABL/BCR) than by p40(ABL/BCR). It is very intriguing that p96(ABL/BCR) can activate Wnt signaling and up regulate the expression of HoxB4. Transduction/transplantation mouse model has shown that p40(ABL/BCR) and p96(ABL/BCR) both have their own leukemogenic potential. Given the fact that leukemic stem cells maintain the growth of tumor and are the origin of relapse, the cure of leukemia is dependent on the eradication of the leukemic stem cell and abrogation of aberrantly regulated self-renewal capability. Both t-RA and As2O3 have been shown to induce complete remission in APL patients with PML/RARalpha translocation product. However, t-RA as a single agent achieves completeremission (CR) but not complete molecular remissions (CMR). Therefore, virtually all patients will experience a relapse within a few months. In contrast to t-RA, As2O3 as a single agent is able to induce CR as well as CMR followed by long-term relapse-free survival in about 50% of APL patients even if relapsed after treatment with t-RA-containing chemotherapy regimens. Nothing is known about the mechanisms leading to the complete different clinical outcomes by the two compounds although both have been shown to induce differentiation of blast cells, proliferation arrest, induction of apoptosis and degradation of PML/RARalpha. We investigated the effect of t-RA and arsenic on PML/RARalpha-expressing cell population with stem cell capacity derived from the APL cell line NB4 as well as Sca1+/lin- murine bone marrow cells. We found that t-RA did not reduce the replating efficiency in PML/RARalpha- and PLZF/RARalpha-infected Sca1+/lincells whereas it selected small compact colonies representing very early progenitor cells. T-RA was unable to reduce the capacity to form colony forming units-spleen (CFU-S) of Sca1+/lin-cells expressing PML/RARalpha, additionally t-RA did not impair the capability of engraftment of NB4 cells in NOD/SCID mouse. On the contrary to t-RA, As2O3 abolished the aberrant self-renewal potential of Sca1+/lin- cells expressing PML/RARalpha. As2O3 not only abolished the replating efficiency of PML/RARalpha positive cells but also completely abrogated the ability of PML/RARalpha-positive HSC to produce CFU-S in vivo. On the contrary to As2O3, t-RA increased the absolute cell number and the percentage of cells in the side population with respect to the whole cell population in NB4 cells. Taken together these data suggest that arsenic but not all-trans retinoic acid overcomes the aberrant stem cell capacity of PML/RARalpha positive leukemic stem cells. My data prove for the first time that there is a direct relationship between the capacity of compounds to effectively target the LSC and their capacity to eradicate the leukemia, and, thereby, to induce complete molecular remission and long-term relapse-free survival. Thus, in order to increase the curative potential of leukemia therapies, future studies need to include the effect of given compounds on the stem cell compartment to determine their ability to eradicate the LSC.
Riboswitches are highly structured elements in the 50-untranslated regions (50-UTRs) of messenger RNA that control gene expression by specifically binding to small metabolite molecules. They consist of an aptamer domain responsible for ligand binding and an expression platform. Ligand binding in the aptamer domain leads to conformational changes in the expression platform that result in transcription termination or abolish ribosome binding. The guanine riboswitch binds with high-specificity to guanine and hypoxanthine and is among the smallest riboswitches described so far. The X-ray-structure of its aptamer domain in complex with guanine/ hypoxanthine reveals an intricate RNA-fold consisting of a three-helix junction stabilized by longrange base pairing interactions. We analyzed the conformational transitions of the aptamer domain induced by binding of hypoxanthine using highresolution NMR-spectroscopy in solution. We found that the long-range base pairing interactions are already present in the free RNA and preorganize its global fold. The ligand binding core region is lacking hydrogen bonding interactions and therefore likely to be unstructured in the absence of ligand. Mg2+-ions are not essential for ligand binding and do not change the structure of the RNA-ligand complex but stabilize the structure at elevated temperatures. We identified a mutant RNA where the long-range base pairing interactions are disrupted in the free form of the RNA but form upon ligand binding in an Mg2+-dependent fashion. The tertiary interaction motif is stable outside the riboswitch context.
Eine große Zahl natürlicher sekundärer Metabolite sind kleine und strukturell oft sehr verschiedene Polypeptide und Polyketide. Diese bioaktiven Substanzen haben im allgemeinen ein breit aufgestelltes therapeutisches Potential und werden von verschiedenen bakteriellen Stämmen und Pilzen biosynthetisiert. Sie sind sowohl biologisch, als auch therapeutisch wichtig als Cytostatika, Immunsuppressiva und Antibiotika mit einem sehr großen antibakteriellen und antiviralen Potential. Diese oft äußerst komplexen Polypeptide und Polyketide werden von modular aufgebauten Megaenzymen in mehrstufigen Mechanismen synthetisiert. Für die Synthese dieser Peptide sind sehr große Proteincluster verantwortlich, die meistens aus einer begrenzten Anzahl sehr großer, Multidomänen umfassenden, Superenzyme aufgebaut werden. Diese Proteincluster mit einem Molekulargewicht bis in den Bereich von MegaDalton werden als nicht-ribosomale Peptidsynthetasen (NRPS) und Polyketidsynthetasen (PKS) bezeichnet. Die NRPS Systeme zeichnen sich dadurch aus, daß für die biosynthetisierten Polypeptide keine Information in Form von Nukleinsäuren wie DNA oder RNA kodiert (Walsh, C.T., 2004; Sieber & Marahiel, 2005). Für die Synthese der Polypeptide ist eine Aktivierung der einzelnen Bausteine, der Aminosäuren, durch Amino-acyl-adenylierung notwendig. Im Anschluß an die Aktivierung, wird die aktivierte Aminosäure über einen Thioester gebunden weitertransportiert. Die Thioesterbildung erfolgt an Cysteaminthiolgruppen intrinsischer 4’-Phosphopantethein-kofaktoren. Eine Modul einer NRPS stellt eine geschlossene Einheit zum Einbau einer Aminosäure mit einer hohen Spezifität für das Substrat und die biosynthetische Reaktion dar. Diese Module sind aus Domänen aufgebaut, die definierte Funktionen haben und mittels flexibler Linker miteinander verbunden sind. Die Domänen werden nach ihrer Funktion unterschieden. Die Acyl-adenylierung oder Aktivierung eines Substrates, beispielsweise einer Aminosäure, erfolgt durch die A-Domänen. Die Peptidyl- oder Acyltransportfunktion der aktivierten Substrate wird durch Thioester-domänen (T-Domäne), auch PCP (peptidyl carrier domain) genannt, bewältigt. Die Biosynthese der Kopplungsreaktion, beispielsweise die Ausbildung der Peptidbindung in NRPS Systemen, erfolgt an den Kondensations-Domänen (C-Domäne). Für die Substratspezifität eines Synthesemoduls sind die A-Domänen verantwortlich, welche die Aktivierung eines Substrat durch ATP-Hydrolyse ermöglichen. In NRPS Systemen sind auch Zyklisierungsreaktionen, durchgeführt von Cyclase-Domänen (Cy-Domänen), L/D-Epimerase-funktionen (E-Domänen) und N-Methylierungen (M-Domänen) beschrieben. So wird in Tyrocidin A an zwei Positionen spezifisch Phenylalanin in die D-Form epimerisiert und anschließend in der Peptidbiosynthese verwendet. Die Interaktion und Erkennung zwischen den multi-modularen Superenzymen, zum korrekten Aufbau der kompletten Synthetase, wurden in letzter Zeit Kommunikations-Domänen (COM-Domänen) beschrieben. Wie die aufgebaute Synthetase die korrekte Sequenz der biosynthetischen Reaktionsschritte sicherstellt ist nicht bekannt. Die enorme Diversität biosynthetischer Reaktionen in NRPS Systemen und die hohe Substratvielfalt in den verschiedensten Synthetasen unterschiedlicher Stämme eröffnet ein weites Feld für mögliche Neukombinationen von Modulen und Modifikationen von Produkten, um neue bioaktive Polypeptide mit antibiotischen Eigenschaften durch die Gestaltung neuer biosynthetischer Reaktionswege zu erhalten. Die Biosyntheseprodukte der NRPS und PKS Systeme lassen sich Gruppen kategorisieren wie Peptidantibiotika, beispielsweise beta-Lactame und makrozyklischer Polypeptide. Weitere Gruppen sind die makrozyklischen Lactone, beispielsweise Polyene und Makrolide, aromatische Verbindungen, wie Chloramphenicol, und Chinone (Tetracyclin). Die näher diskutierten Beispiele sind die antibakteriellen Polypeptide Surfactin und Tyrocidin A. Surfactin ist ein antibakteriell wirkendes makrozyklisches Lipoheptapeptid, welches von Bacillus subtilis synthetisiert wird und ein enormes antivirales Potential besitzt. Tyrocidin A ist ein antibakteriell wirkendes makrozyklisches Decapeptid und wird von Bacillus brevis und Brevisbacillus parabrevis synthetisiert. Zusätzlich werden viele bakterielle Toxine ebenfalls durch solche Systeme multi-modularer Synthetasen erzeugt. Ein Beispiel ist das Polyketid Vibriobactin, das Toxin des humanpathogenen Bakterium Vibrio cholerae. Ein zunehmendes Problem der wachsenden Weltbevölkerung moderner Gesellschaften und in den Entwicklungsländern ist die wachsende Zahl multiresistenter Bakterienstämme. Die starke Progression in der Entwicklung von Resistenzen gegen Antibiotika ist auch Gegenstand des aktuellen WHO-Reports (2006). Alarmierend ist die beschleunigte Resistenzentwicklung gegen die sogenannten Reserveantibiotika Vancomycin und Ceftazidim. Ein umfangreicheres Verständnis der Interaktion zwischen Domänen in einem Modul und zwischen Modulen eines NRPS Systems ist Grundlage für die Neukombination unterschiedlicher Module zur erfolgreichen Gestaltung neuer Biosynthesen. Da die meisten dieser Biosynthesen oder die Synthese alternativer Substanzen nicht in der Organischen Chemie zu realisieren sind oder die Produkte zu teuer wären, um diese in großen Mengen zu erzeugen, muß das Ziel sein die NRPS und PKS Systeme in ihrem modularen Aufbau und ihre Interaktion zu verstehen, um alternative Antibiotika biosynthetisch herzustellen. Peptidyl Carrier Proteine (PCPs) sind kleine zentrale Transport-Domänen, integriert in den Modulen nicht-ribosomaler Peptidsynthetasen (NRPSs). PCPs tragen kovalent über eine Phosphoesterbindung einen aus dem Protein herausragenden 4’-phosphopantetheinyl (4’-PP) Kofaktor. Der 4’-PP Kofaktor ist an der Seitenkette eines hochkonservierten Serins gebunden, welche ein zentraler Bestandteil der Phosphopantethein-Erkennungs-Sequenz ist. Die Erkennungssequenz ist homolog in vielen Proteinen mit ähnlicher Funktion, inklusive Acyl Carrier Proteinen (ACPs) der Fettsäuresynthetasen (FAS) und der Polyketidsynthetasen (PKS). Die Thiolgruppe des 4’-PP Kofaktors dient zum aktiven Transport der Substrate und der Intermediate der NRPS Systeme. Die generelle Organisation und die Kontrolle der exakt aufeinander folgenden Reaktionsschritte in der Peptidsynthetase, ist die entscheidende Frage für die Funktion des Proteinclusters (assembly line mechanism). In Modulen der NRPS Systeme folgen die PCP-Domänen C-terminal auf die Adenylierungsdomänen (A-Domäne). Die Aufgabe der A-Domänen ist die Selektion and die Aktivierung einer spezifischen Aminosäure für die „assembly line“. Die eigentliche Bildung der Peptidbindung erfolgt an der Kondensations-Domäne (C-Domäne). Der Transfer der Peptidintermediate und der aktivierten Aminosäuren zwischen A-Domänen und C-Domänen ist Aufgabe der PCPs. Um diese Funktion erfüllen zu können, ist eine große Bewegung in PCPs, bzw. des 4’-PP Kofaktors notwendig, welche als „swinging arm model“ (Weber et al., 2001) beschrieben wurde. Die PCPs koordinieren damit die Peptidbiosynthese während sie mit diversen Domänen der Synthetasen spezifisch wechselwirken müssen. Die molekularen Mechanismen des Transportes wurden bisher allerdings nicht untersucht. Eine Dynamik der Transport-Domänen wurde bereits postuliert (Kim & Prestegard, 1989; Andrec et al., 1995), konnte bisher aber nicht gezeigt werden (Weber et al., 2001). Interessanterweise zeigt sowohl apo-PCP (ohne den kovalent gebundenen 4’-PP Kofaktor) also auch holo-PCP langsamen chemischen Austausch, der als jeweils zwei stabile Konformationen beschrieben werden konnte. Diese jeweils zwei stabilen Zustände, welche sich im Austausch befinden, wurden als A und A*, für apo-PCP, und entsprechend H und H* für holo-PCP bezeichnet. Während der A- und der H-Zustand sich sowohl voneinander als auch von den entsprechenden A* und H*-Zuständen unterscheiden und spezifisch für die apo- und die holo-Form von PCP sind, ist die kalkulierte Struktur vom A*-Zustand größten Teils identisch mit der des H*-Zustandes. Die erhaltenen NMR-Strukturen des A-Zustandes, des H-Zustandes und des gemeinsamen A/H-Zustandes beschreiben in ihrer Gesamtheit ein neues Modell für ein allosterie-kontrolliertes System dualer konformationeller Zwei-Zustands-Dynamik. Zu dem beobachteten konformationellen Austausch der PCP-Domäne, konnte die Bewegung des 4’-PP Kofaktors koordiniert werden. Die Bewegung des 4’-PP Kofaktors in Verbindung mit dem konformationellen Austausch der PCP-Domäne charakterisiert die Interaktion mit katalytischen Domänen eines NRPS Moduls. Des weiteren konnte mit Hilfe des Modells die Wechselwirkung mit externen Interaktionspartnern, wie der Thioesterase II und der 4’-PP Transferase, untersucht werden. Die externe Thioesterase II der Surfactin-Synthetase (SrfTEII) von Bacillus subtilis ist ein separat expremiertes 28 KDa Protein. Sie gehört zur Familie der alpha/beta-Hydrolasen und ist verantwortlich für die Regenerierung falsch beladener 4’- PP Kofaktoren der Peptidyl Carrier Domänen. Die SrfTEII wurde mittels Lösungs-NMR untersucht, die Resonanzen wurden zugeordnet, erste strukturelle Modelle konnte berechnet werden und das Interaktionsverhalten mit verschiedenen modifizierten Kofaktoren und PCPs wurde analysiert. Die Spezifität der Substraterkennung durch die SrfTEII kann beschrieben werden. Interessanterweise zeigt auch die SrfTEII Doppelpeaks für einzelne Aminosäuren, diese können als Indikator für eine spezifische Substraterkennung durch das Enzym verwendet werden und helfen den funktionellen Unterschied zwischen der SrfTEI-Domäne und SrfTEII zu verstehen.
In the first part of the present work (Chapter 3), EPR spectroscopy at different microwave frequencies, namely at 9 GHz (X-band), 34 GHz (Q-band) and 180 GHz (G-band), was employed to resolve the g-values and the HFCs of a putative radical intermediate involved in the reduction of benzoyl-CoA catalyzed by benzoyl-CoA reductase. In particular, the effect of 33S-labeling on the EPR line shape was studied at X- and Q-band frequencies in order to gain further evidence for a sulfur centered radical proposed to be the electron donor in the reduction or the aromatic ring of BCoA [I]. The spectral components observed at X-, Q- and G-band were overall consistent and showed at least three overlapping EPR signals. The signal postulated to be due to a disulfide radical anion showed no resolved g-values and a relaxation behaviour faster than expected for such a radical species. These observations together with the simulations suggest that the signal could arise from a radical exchange coupled to an [4Fe-4S] cluster located nearby. In the future, pulsed EPR and ENDOR spectroscopy on the 57Fe-labeled enzyme could help to solve this question. The potential of high-field ENDOR in combination with 13C- and 31P-labeling for investigating the structure at the active site in proteins could be verified in the studies of the ligation sphere of the cofactor Mn2+ in Ras as reported in Chapter 4 [2]. Therein, high-field ENDOR performed at 94 GHz (W-band) was used to detect the hyperfine interactions between the electron spin mainly located on the metal ion and the phosphorous nuclei of the bound GDP and GppNHp as well as the carbon nuclei of bound amino acids in the wild-type Ras protein and its oncogenic mutant G12V. These studies aimed at searching for an additional free phosphate ion or amino acid ligand bound to the metal center in the wild type GDP-bound protein with respect to its oncogenic mutant. Rom the 13C- and 31P-ENDOR spectra, the hyperfine couplings of directly bound amino acids and the bound nucleotides were compatible with the hyperfine couplings obtained from DFT calculations based on the crystal structure data. No differences in the 13C- and 31P-ENDOR spectra could be found for the wild-type GDP-bound protein in comparison to its oncogenic mutant in frozen solution. Therefore, no evidence for binding of an additional free phosphate ion or amino acid ligand in the wild-type GDP-bound protein was found. The distances between the detected nuclei and the meta1 ion were in agreement with the ones extracted from crystal structures reported in the literature. Future 35C1-ENDOR studies could clarify whether a chloride ion from the buffer solution could be the ligand replacing one water molecule in the wild type GDP-bound Ras. In Chapter 5, the implementation of a high-field ENDOR setup into a homebuilt pulsed EPR spectrometer operating at 180 GHz is reported and its performance for 1H-ENDOR demonstrated on the model system BDPA. Mims and Davies ENDOR spectra were also obtained for Ras(wt).Mn2+.DP. The increased nuclear Zeeman resolution at 180 GHz may be further exploited in the future by extending the setup for studying hyperfine couplings of low-y nuclei such as 33S, 15N , 17O or 2H. In the present work, the advantages of performing EPR and ENDOR experiments at high fields and frequencies could be nicely demonstrated with the 94 GHz ENDOR studies of Ras. Furthermore, the complementing information obtained at X- and Q-band frequencies in the multifrequency EPR studies on BCR demonstrated that the analysis of EPR spectra can be greatly facilitated by simulating the spectra measured at different MW frequencies with the same set of parameters consistent with a proposed radical. Overall, it could be shown that the use of different experimental techniques at multiple fields and frequencies renders EPR spectroscopy a powerfull tool for structural studies in biological systems.
One of the most important tasks in chemistry and especially in structural biology has always been the elucidation of three-dimensional molecular structures - either of small molecules or large biopolymers. Among the (bio)physical methods to acquire structural data at atomic resolution electron paramagnetic resonance (EPR) spectroscopy is the most valuable technique for obtaining structural information about many different kinds of paramagnetic species. In biological systems, either paramagnetic metal ions/clusters, transient paramagnetic intermediates in electron transfer processes or artificially attached stable spin labels can be found. The usual approach to interpret EPR spectra is to perform simulations based on the so-called spin Hamiltonian (SH). This means that the well-defined numerical parameters (tensors) in the SH representing different types of interaction are obtained by fitting the experimental data. The SH parameters include electronic g-values, hyperfine coupling (HFC) and quadrupole coupling (&C) constants, zero-field splittings and constants to describe exchange and dipolar interactions between electron spin systems. However, since the SH only contains spin degrees of freedom, a direct translation of the SH EPR parameters into structural information is not straightforward. Therefore, methods to predict such SH interaction parameters starting from molecular structures are required. In this thesis it was investigated whether quantum chemical calculations of EPR parameters based on density functional theory (DFT) methods may be employed to overcome these problems thus enabling a correlation of experimental EPR data with molecular structure. It was the central goal of this work to point out the potential of a fruitful interplay between quantum chemistry and experiment and to study how both can benefit from each other. For this purpose DFT methods were applied to a variety of organic radical or transition metal systems to calculate different EPR parameters. Using the 'broken symmetry' formalism it was possible to compute the exchange coupling constant for a nitroxide biradical and furthermore decompose the exchange mechanism in different through-bond and through-space interactions. Spin density distributions, 14N and 1H HFC constants as well as dipole moments and polarizabilities were computed for a number of aromatic nitroxides to examine their properties and select promising candidates which may serve as DNA-intercalating spin labels. Systematic investigations of the influence of hydrogen bond geometry on the 14N QC parameters for imidazole-water and methylimidazole-benzosemiquinone complexes lead to the conclusion that especially the imidazole amino nitrogen &C parameters are very sensitive probes of the bond geometry, in particular of the hydrogen bond length. The results of this study may be applied to biological systems, e.g. to gain structural information about quinone binding sites. Moreover, quantum chemical methods were applied to elucidate the structure of a nitrogen-centered radical intermediate in the inhibition process of ribonucleotide reductase (RNR). It was possible to find a molecular structure in accordance with all experimentally available data, thus revealing the longsought structure of the No radical and providing evidence for the trapping of a 3'-ketonucleotide in the reduction process catalyzed by RNR. To test the capability of modern DFT methods to predict g- and molybdenum HFC tensors for MoV complexes, validation studies were carried out. Comparison of computed EPR parameters of a number of MoV compounds with corresponding experimental values showed that g- and HFC tensors could be predicted in good accuracy, although some systematic errors of the computational methods have to be considered for such heavy 4d1 transition meta1 systems. Furthermore, DFT calculations on a Mn2+ binding site model of the hammerhead ribozyme allowed to conclude that the structure of the binding site as studied by EPR spectroscopy in frozen solution is very likely to be identical to the site found occupied by Mn2+ in crystals. Finally, computational methods were employed to aid in the structural characterization of the Mn2+ binding site in Ras (rat sarcoma protein) by providing accurate starting parameters for spectral simulations and furthermore helping to interpret the experimental data. In conclusion, it was demonstrated in this thesis that the combination of sophisticated experimental and quantum chemical methods represents a powerful approach in the field of EPR spectroscopy and that it may be essential to employ EPR parameter computations to extract the full information content from EPR spectra. Therefore, great potential lies in future applications of DFT methods to the large number of systems where detailed and reliable experimental data is available but where an unequivocal correlation of these data with structural information is still lacking.
Summary and Outlook The aim of this work was the investigation of the Mn2+ binding sites in hammerhead and the Diels-Alder ribozymes. This project consists of three main topics. In the first part quantification and structural characterization of Mn2+ binding sites in the m- and the tsHHRz using Electron Paramagnetic Resonance (EPR) spectroscopy are described. The second part summarizes the newest results obtained for the cleavage activity of both mand tsHHRzs in the presence of different Mg2+ and Mn2+ and Na+ ion concentrations using the new method with fluorescent-labeled RNAs. Here the influence of neomycin B on the structure of Mn2+ binding pockets and on the catalytic activity of both HHRzs is discussed. In addition, a possible role of Mn2+ ions is suggested from correlation of the EPR data with the kinetic results. The last chapter is devoted to quantification and differentiation of Mn2+ binding sites of the Diels-Alder ribozyme using continuous wave (cw) EPR experiments in solution. In this work EPR spectroscopy was used to study the binding of Mn2+ ions to the cis tsHHRz and to compare it with the binding to the trans mHHRz and to the Diels-Alder ribozyme. Cw EPR measurements showed that the tsHHRz possesses a single highaffinity Mn2+ binding site with a KD of < 10 nM at a NaCl concentration of 0.1 M. This dissociation constant is three orders of magnitude smaller than the KD determined for the single high-affinity Mn2+ site in the mHHRz (KD = 4.4 μM). The measurements of catalytic activity have been performed using fluorescent-labeled RNAs. Compared to the mHHRz, the cis tsHHRz cleaves up to 20-fold faster in the presence of Mg2+/Mn2+ ions with no saturation of the cleavage rates at high metal(II) ion concentrations. This is in good agreement with the last investigations on the trans tsHHRz (Nelson et al. 2005). Thus, the much stronger Mn2+ binding and higher cleavage activity were attributed to the interaction between the two external loops of the tsHHRz which reduces the RNA dynamics and traps the Mn2+ in the tightly folded conformation. Intriguingly, according to the EPR studies the binding constants for Mn2+ ions are several orders higher than the concentration of Mn2+ ions required for the catalytic activity (mHHRz: KD = 4.4 ± 0.5 μM and the Mn2+ concentration required to achieve half of the maximum cleavage rate [Mn2+]1/2 = 4.1 ± 0.6 mM respectively). Therefore, strongly bound Mn2+ ions seem to be needed for the folding of the HHRz, whereas weakly bound metal(II) ions are required to achieve full catalytic activity, and may be directly involved in catalysis. A comparison between the Electron Spin Echo Envelope Modulation (ESEEM) and Hyperfine Sublevel Correlation (HYSCORE) spectra of m- and tsHHRz demonstrates that both binding sites in HHRzs are structurally very similar. This suggests that the Mn2+ is located in both ribozymes between the bases A9 and G10.1 of the sheared G•A tandem basepair, as shown previously and in detail for the mHHRz (Vogt and DeRose 1998, Schiemann et al. 2003). However, the hyperfine spectra of the tsHHRz with 15N labeled G10.1 revealed no difference in comparison with the ones with 14N. This leads to an interpretation that the Mn2+ binding sites in both ribozymes are not identical. In addition, aminoglycoside antibiotic neomycin B inhibits the cleavage activity of both despite of the fact that it displaces the high-affinity Mn2+ ion only from the mHHRz. Hence, binding of neomycin B to the m- and the tsHHRzs probably occurs at different sites and neomycin B displaces only loosely bound Me2+ ions from the tsHHRs, whereas in the mHHRz both the high-affinity ion and the weakly bound ions are replaced. Therefore, it cannot be excluded that weakly bound Mg2+/Mn2+ ions, together with looploop interactions, induce a structural rearrangement which brings the high-affinity ion closer to the cleavage site. In the case of the Diels-Alder ribozyme it possesses five Mn2+ binding sites with KD = 0.6 ± 0.2 μM in solution under conditions where it is catalytically active. The competition experiment with Cd2+ allows to distinguish three different types of Mn2+ binding sites in the Diels-Alder ribozyme including inner-sphere monomeric Mn2+, monomeric Mn2+ bound through water-mediated contacts and electronically coupled dimeric Mn2+. Three Mn2+ ions are more strongly bound to the ribozyme via inner-sphere contacts, whereas two other Mn2+ ions form water-mediated outer-sphere contacts with the nucleotides of the ribozyme. The inner-sphere Mn2+ with the highest affinity and the fourth Mn2+ ions added to the ribozyme form a dimer with a Mn2+-Mn2+ distance of ~6 Å (as arises from simulations). Moreover, an addition of the product analog inhibitor (AMDA) to the [Diels-Alder ribozymes/ Mn2+] complex shows no conformational changes in the Mn2+ binding pockets. This is in good agreement with the recent studies which suggest that the Diels-Alder ribozyme is preorganized (Keiper et al. 2004). Some considerations on the evolution of the project (Outlook) There may be several venues of continuation of this project, which exploit on unique combination of EPR experiments and biochemical studies on RNA. This combination may allow us to significantly contribute to understanding of metal role in HHRz catalysis. Since the tsHHRz possesses the high affinity Mn2+ binding site (Kd < 10 nM) it creates a possibility to find conditions where the structural site is occupied by Mn2+, while catalytic sites are occupied by Mg2+ ions. If these conditions will be established by EPR titration, a set of standard biochemical experiments may be designed to look at the kinetic of cleavage and differentiate the “structural” and catalytic effects. The other experiment would be to look at the Mn2+ binding site in the tsHHRz in comparison with P1 and P1/P2 complexes and compare the results with the ones for the mHHRz. No matter the answer, P1 can be used as a simpler model to study the effect of tertiary structure on Mn2+ binding. A set of the tsHHRz mutants can be created to observe the mutations affect on Mn2+ binding sites, Mn2+ affinity and correlate the data with the kinetic analysis. FRET-based kinetic assay with fluorophore pairs on P1 and P2 can be designed for the kinetic experiments. Having this system one will be able to perform kinetic measurements 100-fold faster comparing to standard gel procedures (everything will be done in 96-wells). By manipulating the lengths and the sequence of P2 we most likely will be able to use FRET assay for the chemical step analysis (provided Kd > k2), and measure it using stop-flow system with time resolution of microseconds. And finally, one will be able to quantitatively measure the effect of neomycin B on the tsHHRz. Another interesting possibility would be to look at the state of metal(II) in the tsHHRz – enzyme alone (dissociated product) and in the enzyme-product complex and compare with the full-length tsHHRz. It will provide the information about the local rearrangements upon catalysis and the role of metal(II) ions. Furthermore, additional pulse-EPR experiments using 15N labeling have to be performed in order to reveal the location of the high-affinity Mn2+ binding site in the tsHHRz. Additionally, paramagnetic Mn2+ ions can be localized within the global fold of HHRzs using PELDOR and site-directed spin labeling. Further characterization of the high-affinity binding site in the tsHHRz can be performed using high-field ENDOR measurements in order to obtain the 14N and 31P tensors.
P2X receptors are ligand (ATP)-gated ion channels that open an intrinsic cation permeable pathway in response to extracellular ATP released from both neuronal and non-neuronal cells. P2X receptors are abundantly distributed and mediate a wide variety of physiological functions, ranging from fast synaptic transmission in the central, peripheral, and enteric nervous system, to proinflammatory cytokine release from immune cells. The primary aim of this work was to elucidate the pathway that leads to the finally assembled trimeric P2X receptors, including the assessment of a possible role of ER chaperones and folding factors in this process. Additionally, the study was conducted to investigate the various ER quality control processes involved in the selection of “properly folded and assembled” P2X receptors that are suitable for the surface expression.
The mitochondrial respiratory chain consists of NADH:ubiquinone oxidoreductase (Complex-I), succinate:ubiquinone reductase (Complex-II), ubiquinol:cytochrome c reductase (Complex-III), cytochrome c oxidase (Complex-IV) and cytochrome c as an electron mediator between Complex-III and Complex-IV. Paracoccus denitrificans membranes were used as a model system for the association of the mitochondrial respiratory chain. More than 50 years ago, a model was given for a supercomplex assembly formed by stable associations between these complexes. This model gradually shifted by the model of random diffusion given by Hackenbrock et al. 1986 Different independent approaches were used to further analyze this situation in a native membrane environment, thus avoiding any perturbation caused by detergent solubilization: (a) measuring the distance and orientation of the different complexes by multi-frequency EPR Spectroscopy we started to analyze simple system, the interaction between CuA fragment derived from P. denitrificans and various c type cytochrome by Pulsed X band and G band (180 GHz) EPR. Partner proteins for the CuA (excess negative surface charge) were (i) horse heart cytochrome c which contain a large number of positive charges in heme crevice,(ii) the cytochrome c552 soluble fragment (physiological electron donor and have positive charges), and as a control (iii) the cytochrome c1 soluble fragment (negative surface potential, derived from bc1 complex) The measurements were performed at several magnetic field positions varying temperature between 5 to 30 K. Both the X band and the high-field measurements show the existence of a strong relaxation enhancement of the CuA by the specific binding of the P. denitrificans cytochrome c552 and horse heart cytochrome c. This relaxation enhancement is dependent on temperature and provides information about the distance and relative orientation of the two interacting spins within this protein-protein complex. (b) For quantitative information about lateral diffusion of cytochrome c oxidase in the native membrane Fluorescence Correlation Spectroscopy (FCS) was used. In this experiment, diffusion coefficients for oxidase differ in the case of supercomplex for wild type membrane and for two deletion mutants lacking either Complex-I or Complex-III. (c) The optical absorption spectroscopy at microsecond level resolution was tried for the translational mobility of oxidase in membrane vesicles. Due to the presence of different hemes in the native membrane, carbon monoxide (CO) used as a probe for the experiment. The optimization of the experimental conditions were carried out to get the optimal signal.
Background: Particle Swarm Optimization (PSO) is an established method for parameter optimization. It represents a population-based adaptive optimization technique that is influenced by several "strategy parameters". Choosing reasonable parameter values for the PSO is crucial for its convergence behavior, and depends on the optimization task. We present a method for parameter meta-optimization based on PSO and its application to neural network training. The concept of the Optimized Particle Swarm Optimization (OPSO) is to optimize the free parameters of the PSO by having swarms within a swarm. We assessed the performance of the OPSO method on a set of five artificial fitness functions and compared it to the performance of two popular PSO implementations. Results: Our results indicate that PSO performance can be improved if meta-optimized parameter sets are applied. In addition, we could improve optimization speed and quality on the other PSO methods in the majority of our experiments. We applied the OPSO method to neural network training with the aim to build a quantitative model for predicting blood-brain barrier permeation of small organic molecules. On average, training time decreased by a factor of four and two in comparison to the other PSO methods, respectively. By applying the OPSO method, a prediction model showing good correlation with training-, test- and validation data was obtained. Conclusion: Optimizing the free parameters of the PSO method can result in performance gain. The OPSO approach yields parameter combinations improving overall optimization performance. Its conceptual simplicity makes implementing the method a straightforward task.