Struktur- und Reaktionsaufklärung durch Kombination von Theorie und Experiment

Die vorliegende Dissertation beinhaltet Anwendungen der Quantenchemie und methodische Entwicklungen im Bereich der "Coupled-Cluster"-Theorie zu den folgenden Themen: 1.) Die Bestimmung von Geometrieparametern in wasserstoffverbrückten Komplexen mit Pikometer-Genauigkeit durch Kopplung von NMR-Experimenten und quantenchemischen Rechnungen wird an zwei Beispielen dargelegt. 2.) Die hierin auftretenden Unterschiede in Theorie und Experiment werden diskutiert. Hierzu wurde die Schwingungsmittelung des Dipolkopplungstensors implementiert, um Nullpunkt-Effekte betrachten zu können. 3.) Ein weiterer Aspekt der Arbeit behandelt die Strukturaufklärung an diskotischen Flüssigkristallen. Die quantenchemische Modellbildung und das Zusammenspiel mit experimentellen Methoden, vor allem der Festkörper-NMR, wird vorgestellt. 4.) Innerhalb dieser Arbeit wurde mit der Parallelisierung des Quantenchemiepaketes ACESII begonnen. Die grundlegende Strategie und erste Ergebnisse werden vorgestellt. 5.) Zur Skalenreduktion des CCCSD(T)-Verfahrens durch Faktorisierung wurden verschiedene Zerlegungen des Energienenners getestet. Ein sich hieraus ergebendes Verfahren zur Berechnung der CCSD(T)-Energie wurde implementiert. 6.) Die Reaktionsaufklärung der Bildung von HSOH aus di-tert-Butyl-Sulfoxid wird vorgestellt. Dazu wurde die Thermodynamik der Reaktionsschritte mit Methoden der Quantenchemie berechnet.

Na +-coupled betaine symporters involved in osmotic stress response in prokaryotes and eukaryotes

The betaine/GABA transporter BGT1 is one of the most important osmolyte transporters in the kidney. BGT1 is a member of the neurotransmitter sodium symporter (NSS) family, facilitates Na+/Cl--coupled betaine uptake to cope with hyperosmotic stress. Betaine transport in kidney cells is upregulated under hypertonic conditions by a yet unknown mechanism when increasing amounts of intracellular BGT1 are inserted into the plasma membrane. Re-establishing isotonicity results in ensuing depletion of BGT1 from the membrane. BGT1 phosphorylation on serines and threonines might be a regulation mechanism. In the present study, four potential PKC phosphorylation sites were mutated to alanines and the responses to PKC activators, phorbol 12-myristate acetate (PMA) and dioctanoyl-sn-glycerol (DOG) were determined. GABA-sensitive currents were diminished after 30 min preincubation with these PKC activators. Staurosporine blocked the response to DOG. Three mutants evoked normal GABA-sensitive currents but currents in oocytes expressing the mutant T40A were greatly diminished. [3H]GABA uptake was also determined in HEK-293 cells expressing EGFP-tagged BGT1 with the same mutations. Three mutants showed normal upregulation of GABA uptake after hypertonic stress, and downregulation by PMA was normal compared to EGFP-BGT1. In contrast, GABA uptake by the T40A mutant showed no response to hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in MDCK cells, grown on glass or filters, revealed that T40A was present in the cytoplasm after 24 h hypertonic stress while the other mutants and EGFP-BGT1 were predominantely present in the plasma membrane. All four mutants co-migrated with EGFP-BGT1 on Western blots suggesting they are full-length proteins. In conclusion, T235, S428, and S564 are not involved in downregulation of BGT1 due to phosphorylation by PKC. However, T40 near the N-terminus may be part of a hot spot important for normal trafficking or insertion of BGT1 into the plasma membrane. Additionally, a link between substrate transport regulation, insertion of BGT1 into the plasma membrane and N-glycosylation in the extracellular loop 2 (EL2) could be revealed. The functional importance of two predicted N-glycosylation sites, which are conserved in EL2 within the NSS family were investigated for trafficking, transport and regulated plasma membrane insertion by immunogold-labelling, electron microscopy, mutagenesis, two-electrode voltage clamp measurements in Xenopus laevis oocytes and uptake of radioactive-labelled substrate into MDCK cells. Trafficking and plasma membrane insertion of BGT1 was clearly promoted by proper N-glycosylation in both, oocytes and MDCK cells. De-glycosylation with PNGase F or tunicamycin led to a decrease in substrate affinity and transport rate. Mutagenesis studies revealed that in BGT1 N183 is the major N-glycosylation site responsible for full protein activity. Replacement of N183 with aspartate resulted in a mutant, which was not able to bind N-glycans suggesting that N171 is a non-glycosylated site in BGT1. N183D exhibited close to WT transport properties in oocytes. Surprisingly, in MDCK cells plasma membrane insertion of the N183D mutant was no longer regulated by osmotic stress indicating unambiguously that association with N-glycans at this position is linked to osmotic stress-induced transport regulation in BGT1. The molecular transport mechanism of BGT1 remains largely unknown in the absence of a crystal structure. Therefore investigating the structure-function relationship of BGT1 by a combination of structural biology (2D and 3D crystallization) and membrane protein biochemistry (cell culture, substrate transport by radioactive labeled GABA uptake into cells and proteoliposomes) was the aim of this work. While the functional assays are well established, structure determination of eukaryotic membrane transporters is still a challenge. Therefore, a suitable heterologous expression system could be defined, starting with cloning and overexpression of an optimized gene. The achieved expression levels in P. pastoris were high enough to proceed with isolation of BGT1. Furthermore, purification protocols could be established and resulted in pure protein, which could even be reconstituted in an active form. The quality and homogeneity of the protein allowed already 2D and 3D crystallization, in which initial crystals could be obtained. Interestingly, the striking structural similarity of BGT1 to the bacterial betaine transporter BetP, which became a paradigm for osmoregulated betaine transport, provided information on substrate coordination in BGT1. The structure of a BetP mutant that showed activity for GABA was solved to 3.2Å in complex with GABA in an inward facing open state. This structure shed some light into the molecular transport mechanisms in BGT1 and might help in future to design conformationally locked BGT1 to enforce the on-going structure determination.

Structural characterization of intermediate and metastable phases by electron microscopy

Structure characterization of nanocrystalline intermediates and metastable phases is of primary importance for a deep understanding of synthetic processes undergoing solid-to-solid state phase transitions. Understanding the evolution from the first nucleation stage to the final synthetic product supports not only the optimization of existing processes, but might assist in tailoring new synthetic paths. A systematic investigation of intermediates and metastable phases is hampered because it is impossible to produce large crystals and only in few cases a pure synthetic product can be obtained. Structure investigation by X-ray powder diffraction methods is still challenging on nanoscale, especially when the sample is polyphasic. Electron diffraction has the advantage to collect data from single nanoscopic crystals, but is limited by data incompleteness, dynamical effects and fast deterioration of the sample under the electron beam. Automated diffraction tomography (ADT), a recently developed technique, making possible to collect more complete three-dimensional electron diffraction data and to reduce at the same time dynamical scattering and beam damage, thus allowing to investigate even beam sensitive materials (f.e. hydrated phases and organics). At present, ADT is the only technique able to deliver complete three-dimensional structural information from single nanoscopic grains, independently from other surrounding phases. Thus, ADT is an ideal technique for the study of on-going processes where different phases exist at the same time and undergo several structural transitions. In this study ADT was used as the main technique for structural characterization for three different systems and combined subsequently with other techniques, among which high-resolution transmission electron microscopy (HRTEM), cryo-TEM imaging, X-ray powder diffraction (XRPD) and energy disperse X-ray spectroscopy (EDX).rnAs possible laser host materials, i.e. materials with a broad band emission in the near-infrared region, two unknown phases were investigated in the ternary oxide system M2O-Al2O3-WO3 (M = K, Na). Both phases exhibit low purity as well as non-homogeneous size distribution and particle morphology. The structures solved by ADT are also affected by pseudo-symmetry. rnSodium titanate nanotubes and nanowires are both intermediate products in the synthesis of TiO2 nanorods which are used as additives to colloidal TiO2 film for improving efficiency of dye-sensitized solar cells (DSSC). The structural transition from nantubes to nanowires was investigated in a step by step time-resolved study. Nanowires were discovered to consist of a hitherto unknown phase of sodium titanate. This new phase, typically affected by pervasive defects like mutual layer shift, was structurally determined ab-initio on the basis of ADT data. rnThe third system is related with calcium carbonate nucleation and early crystallization. The first part of this study is dedicated to the extensive investigations of calcium carbonate formation in a step by step analysis, up to the appearance of crystalline individua. The second part is dedicated to the structure determination by ADT of the first-to-form anhydrated phase of CaCO3: vaterite. An exhaustive structure analysis of vaterite had previously been hampered by diffuse scattering, extra periodicities and fast deterioration of the material under electron irradiation. rn

Determination of trace elemental species in periostracum of Cypraea shells

n this work, three Cypraea species (C. talpa, C. tigris and C. zebra) were exhaustively studied. The shells have been separated in the structural layers. The mineralogy, ultra- and micro-structure of each layer were analyzed by Confocal Laser Scanning Microscopy (CLSM), Scanning Electron Microscopy (SEM), X-Ray Diffractometry (XRD) and Raman Spectroscopy (RS). The presence of biologically relevant trace metals (Mn, Co, Fe, Zn, Cr, etc.) has been investigated using Instrumental Neutron Activation Analysis (INAA) and Inductively Coupled Plasma – Mass Spectrometry (ICP-MS) as detection tool. A new method has been developed and optimized to extract and analyze the soluble organic matrix (SOM) of the shell. Although the molecular nature of the SOM is not really known, it contains at least large protein fraction, if not only consists of proteins. The extracted matrices were compared between layers and species using Size Exclusion High Performance Liquid Chromatography coupled with Ultra Violet Spectrometry (SE-HPLC-UV), Gel electrophoresis (GE) and protein quantification tests. For the first time to our knowledge the association of trace elements to the protein in the SOM of the shell was studied using hyphenated on line as well as combined off line techniques and validated through inter-comparison tests between the different methods applied. Interesting correlations between the trace element concentration, the microstructure and the protein content were directly and indirectly detected. The metals Cu, Ni, Co and Zn have shown to bind to the SOM extracted from C. talpa, C. tigris and C. zebra shells. Within the conclusions of this work it was demonstrated that these protein-metal-complexes (or metal containing proteins) change from one layer to the other and are different between the three snails analyzed. In addition, the complexes are clearly related only to certain protein fractions of the SOM, and not to the whole SOM observed. These fractions and show not to be very metal-specific (i.e. some of these fractions bind two or three different metals).

Nuclear charge radius determination of 7,10 Be and the one-neutron halo nucleus 11 Be

The only nuclear model independent method for the determination of nuclear charge radii of short-lived radioactive isotopes is the measurement of the isotope shift. For light elements (Z < 10) extremely high accuracy in experiment and theory is required and was only reached for He and Li so far. The nuclear charge radii of the lightest elements are of great interest because they have isotopes which exhibit so-called halo nuclei. Those nuclei are characterized by a a very exotic nuclear structure: They have a compact core and an area of less dense nuclear matter that extends far from this core. Examples for halo nuclei are 6^He, 8^He, 11^Li and 11^Be that is investigated in this thesis. Furthermore these isotopes are of interest because up to now only for such systems with a few nucleons the nuclear structure can be calculated ab-initio. In the Institut für Kernchemie at the Johannes Gutenberg-Universität Mainz two approaches with different accuracy were developed. The goal of these approaches was the measurement of the isotope shifts between (7,10,11)^Be^+ and 9^Be^+ in the D1 line. The …first approach is laser spectroscopy on laser cooled Be^+ ions that are trapped in a linear Paul trap. The accessible accuracy should be in the order of some 100 kHz. In this thesis two types of linear Paul traps were developed for this purpose. Moreover, the peripheral experimental setup was simulated and constructed. It allows the efficient deceleration of fast ions with an initial energy of 60 keV down to some eV and an effcient transport into the ion trap. For one of the Paul traps the ion trapping could already be demonstrated, while the optical detection of captured 9^Be^+ ions could not be completed, because the development work was delayed by the second approach. The second approach uses the technique of collinear laser spectroscopy that was already applied in the last 30 years for measuring isotope shifts of plenty of heavier isotopes. For light elements (Z < 10), it was so far not possible to reach the accuracy that is required to extract information about nuclear charge radii. The combination of collinear laser spectroscopy with the most modern methods of frequency metrology …finally permitted the …first-time determination of the nuclear charge radii of (7,10)^Be and the one neutron halo nucleus 11^Be at the COLLAPS experiment at ISOLDE/ CERN. In the course of the work reported in this thesis it was possible to measure the absolute transition frequencies and the isotope shifts in the D1 line for the Be isotopes mentioned above with an accuracy of better than 2 MHz. Combination with the most recent calculations of the mass effect allowed the extraction of the nuclear charge radii of (7,10,11)^Be with an relative accuracy better than 1%. The nuclear charge radius decreases from 7^Be continuously to 10^Be and increases again for 11^Be. This result is compared with predictions of ab-initio nuclear models which reproduce the observed trend. Particularly the "Greens Function Monte Carlo" and the "Fermionic Molecular Dynamic" model show very good agreement.

Determination of effective charge of flexible polyelectrolytes by light scattering

The most important property controlling the physicochemical behaviour of polyelectrolytes and their applicability in different fields is the charge density on the macromolecular chain. A polyelectrolyte molecule in solution may have an effective charge density which is smaller than the actual charge density determined from its chemical structure. In the present work an attempt has been made to quantitatively determine this effective charge density of a model polyelectrolyte by using light scattering techniques. Flexible linear polyelectrolytes with a Poly(2-Vinylpyridine) (2-PVP) backbone are used in the present study. The polyelectrolytes are synthesized by quaternizing the pyridine groups of 2-PVP by ethyl bromide to different quaternization degrees. The effect of the molar mass, degree of quaternization and solvent polarity on the effective charge is studied. The results show that the effective charge does not vary much with the polymer molar mass or the degree of quaternization. But a significant increase in the effective charge is observed when the solvent polarity is increased. The results do not obey the counterion condensation theory proposed by Manning. Based on the very low effective charges determined in this study, a new mechanism for the counterion condensation phenomena from a specific polyelectrolyte-counterion interaction is proposed