Petrophysical interpretation of 1H NMR relaxation responses from saturated rocks is complicated by paramagnetic species present in fluids. Oxygen dissolved in liquids is one common example. Dipolar interactions of oxygen’s unpaired electron spins with the magnetic moment of fluid nuclei provide a strong relaxation mechanism known as paramagnetic relaxation enhancement (PRE). As a result even low concentrations of dioxygen in its common triplet ground state significantly shorten longitudinal and transverse relaxation times of host fluids. This effect may be employed similarly to any standard tracer technique to study pore connectivity in porous media by detecting a change of oxygen concentration due to diffusion resolved in time and space. Since relaxation enhancement effect is likely stronger in non-wetting phase than in wetting one (where surface relaxation process dominates) this difference can be utilized to study wettability in immiscible multiphase systems. We use a relaxation time contrast between air-saturated and oxygen-free fluids to evaluate oxygen concentration change within two fluid phases saturating rock, to estimate time required to establish equilibrium concentration and to calculate a mutual diffusion coefficient of oxygen. A spatially- and time-resolved T2(z,t) experiment provides the time-dependent oxygen concentration change along the fully- and partially-saturated carbonate core plug exposed to air saturated oil at its inlet. We derive an effective mutual diffusion coefficient of oxygen and accordingly a tortuosity estimate as a function of position along the core and rock saturation. The spatially resolved oxygen diffusion-based tortuosity is compared to simulated conductivitybased tortuosity. The latter is calculated on a high-resolution micro-tomographic image of Mount Gambier limestone by solving the Laplace equation for conductivity.

No description available.

No description available.

Biological membranes form a protective barrier around cells and cellular compartments. A broad range of biochemical processes occur in or at membranes demonstrating that they are not only of structural but also of functional importance. One important class of membrane proteins are membrane-associated glycosyltransferases. WaaG is a representative of this class of proteins; its function is to catalyze one step in the synthesis of lipopolysaccharides, which are outer membrane lipids found in Gram-negative bacteria. To study protein-membrane complexes by biophysical methods, one must employ membrane mimetics, i.e. simplifications of natural membranes. One type of membrane mimetic often employed in solution-state NMR is small isotropic bicelles, obloid aggregates formed from a lipid bilayer that is dissolved in aqueous solvent by detergent molecules that make up the rim of the bicelle. In this thesis, fast dynamics of lipid atoms in bicelles containing lipid mixtures that faithfully mimic plant and bacterial membranes were investigated by NMR relaxation. Lipids were observed to undergo a broad range of motions; while the glycerol backbone was found to be rigid, dynamics in the acyl chains were much more rapid and unrestricted. Furthermore, by employing paramagnetic relaxation enhancements an ‘atomic ruler’ was developed that allows for measurement of the immersion depths of lipid carbon atoms. WaaG is a membrane-associated protein that adopts a GT-B fold. For proteins of this type, it has been speculated that the N-terminal domain anchors tightly to the membrane via electrostatic interactions, while the anchoring of the C-terminal domain is weaker. Here, this model was tested for WaaG. It was found by a set of circular dichroism, fluorescence, and NMR techniques that an anchoring segment located in the N-terminal domain termed MIR-WaaG binds electrostatically to membranes, and the structure and localization of isolated MIR-WaaG inside micelles was determined. Full-length WaaG was also found to bind membranes electrostatically. It senses the surface charge density of the membrane whilst not discriminating between anionic lipid species. Motion of the C-terminal domain could not be observed under the experimental conditions used here. Lastly, the affinity of WaaG to membranes is lower than expected, indicating that WaaG should not be classified as a monotopic membrane protein but rather as a peripheral one. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Manuscript.</p>

No description available.

In der Kernspinresonanzspektroskopie (NMR) treten drei Effekte auf, die paramagnetische und diamagnetische Moleküle in isotroper Lösung unterscheiden: residuale dipolare Kopplung (RDC), Pseudokontaktverschiebung (PCS) und paramagnetische Relaxationsverstärkung (PRE). Alle drei Effekte sind abhängig von intermolekularen Winkeln und Abständen und können daher Informationen über die Struktur und Dynamik des Moleküls liefern. Um diese Informationen zu erhalten, muss das Molekül paramagnetische Eigenschaften aufweisen. Eine der heutzutage gebräuchlichen Methoden verwendet kleine molekulare Tags, die paramagnetische Metallionen koordinieren. Die meisten dieser Tags binden über eine Disulfidbrücke an Cysteine an der Proteinoberfläche. Um diese Methode für DNA anzuwenden werden daher neue Taggingstrategien benötigt. Im Rahmen dieser Arbeit wurde eine modifizierte Nukleobase synthetisiert, mit der ein Schwefelatom in die DNA eingebracht werden kann. Diese Methode erlaubt es, jeden Tag an die DNA zu binden, der als Verbindungsmethode eine Disulfidbrücke nutzt. Mit der Nukleobase wird eine Kohlenstoff-Dreifachbindung in die DNA eingefügt und mit Hilfe einer dipolaren Cycloaddition wird die freie Thiolgruppe eingebracht. Die modifizierte Nukleobase wurde erfolgreich an einem selbstkomplementären DNA-Strang (24 Nukleobasen) getestet. Die Nukleobase wurde während der Synthese der DNA eingefügt und der mit Lutetium, Terbium oder Thulium vorbeladene Cys-Ph-TAHA Tag wurde über eine Disulfidbrücke an die DNA gebunden. Die Beladung des Tags und die Taggingreaktion verliefen hierbei quantitativ. Nach diesem Erfolg war es ein Hauptaspekt dieser Arbeit, eine verlässliche und reproduzierbare Aufreinigungs- und Probenvorbereitungsmethode zu entwickeln. Diesem Punkt kommt besondere Bedeutung zu, da das Phosphatrückgrat der DNA, im Gegensatz zu Proteinen, Metallionen koordinieren kann. Im Theorieteil dieser Arbeit ist eine komplette Herleitung der drei Hauptmerkmale paramagnetischer NMR gegeben. Diese Herleitung beginnt bei Grundbegriffen des Magnetismus und neben den Gleichungen für RDCs, PCSs und PREs werden Ausdrücke für den dipolaren Hamiltonoperator, Kreuzrelaxationsraten, kreuzkorrelierte Relaxationsraten, durch Alignment induzierte RDCs, Korrelationsfunktionen und spektrale Dichten gegeben. Das zweite Thema dieser Arbeit basiert auf einem weiteren paramagnetischen Effekt. Um der reduzierten Empfindlichkeit der Kernspinresonanzspektroskopie verglichen mit anderen Spektroskopiemethoden entgegenzuwirken, wurden viele Methoden entwickelt, die auf eine Erhöhung der Polarisierung der Atomkerne zielen, d.h. um sogenannte hyperpolarisierte Kerne zu erzeugen. Eine dieser Methoden, die photochemisch erzeugte dynamische Kernpolarisierung (photo CIDNP), basiert auf kurzlebigen Radikalen, die durch direkte Laserbestrahlung der Probe im Magneten erzeugt werden. Im Rahmen dieser Arbeit wurde ein photo CIDNP Aufbau entworfen, gebaut und getestet. Die ersten Experimente und Resultate mit Triethylendiamin, L-Tyrosin und 3-Fluor-L-tyrosin zeigen die Vorteile und Grenzen dieser Methode auf. Für 3-Fluor-L-tyrosin wurde eine komplette Analyse des Relaxationsverhaltens, einschließlich der Kreuzrelaxation und der kreuzkorrelierten Relaxation, durchgeführt.

No description available.

No description available.

The human &alpha;-lactalbumin (&alpha;-LA) molten globule formed at low pH is a model for the study of protein folding intermediates. The molten globule lacks native-like side-chain interactions, resulting in a fluctuating ensemble of tertiary structures, characterisation of which has been precluded by severe line-broadening in NMR spectra and a lack of long-range NOEs. Paramagnetic relaxation enhancements (PREs) have been measured in a variant of &alpha;-LA in which all native cysteines have been mutated to alanine (all-Ala &alpha;-LA). Cysteine residues have been mutated into regions of interest and spin labelled with MTSL. These measurements have confirmed that all-Ala &alpha;-LA forms a compact molten globule. Transient, long-range interactions that are stabilising the compact fold have also been identified using PREs measured in urea-denatured states. This has identified several interactions formed by hydrophobic residues from both the &alpha;- and &beta;-domain, which could be important for initiating and driving folding. The molten globule’s 3D topology has been probed by measuring long-range distances between MTSL pairs using Double Electron-Electron Resonance (DEER). Broad distance distributions have been identified between elements of secondary structure, indicative of a fluctuating but compact fold. By contrast, a narrower distance distribution has been measured within one of the major helices, indicative of native-like secondary structure. The surface accessibility of all-Ala &alpha;-LA and that of two other variants ([28-111] &alpha;-LA and 4SS &alpha;-LA) has been probed using solvent PREs obtained using TEMPOL, a paramagnetic co-solute. This has revealed differences in the solvent-exposure of hydrophobic residues due to the removal of disulphide bonds. This method has also identified buried hydrophobic residues that contribute to forming the molten globule’s stable, native-like core.

Macromolecular interactions play very important roles in regulation of all levels of biological processes. Aberrant macromolecular interactions often result in diseases. By applying a combination of spectroscopy, calorimetry, computation and other techniques, the protein-protein interactions in the system of the Shaw2 Kv channel and the protein-RNA interactions in West Nile virus RNA-cellular protein TIAR complex were explored. In the former system, the results shed light on the local structures of the key channel components and their potential interaction mediated by butanol, a general anesthetic. In the later studies, the binding modes of TIAR RRM2 to oligoU RNAs and West Nile virus RNAs were investigated. These findings provided insights into the basis of the specific cellular protein–viral RNA interaction and preliminary data for the development of strategies on how to interfere with virus replication