Thesis (Ph. D.)--Montana State University--Bozeman, 2007. / Typescript. Chairperson, Graduate Committee: David J. Singel. Includes bibliographical references (leaves 114-122).

Pulsed electron-electron double resonance (PELDOR) is an emerging technique for nanometre distance measurements in nano-sized assemblies and between specific sites of molecules. Most commonly nitroxide radicals are used as probes for EPR distance measurements because they are easy to introduce in biological systems such as soluble and membrane proteins or nucleic acids. PELDOR distance measurements currently rely on data processing software which has been proven to accurately extract inter-spin distances from the dipolar coupling between two paramagnetic centres. However, when the dipolar coupling is affected by contributions from other close-by unpaired electrons inaccuracies as broadening effects and artefacts are introduced in the distance distributions derived. This challenge, commonly referred as multi-spin effects, has been affecting the extraction of accurate distance information from PELDOR measurements in chemical and biological systems with multiple spin labels. The aim of this project is to approach, identify and suppress inaccuracies introduced in PELDOR-based distance distributions by multi-spin effects. This is achieved through the synthesis of multiply labelled model systems which would allow for assessment of the impact of multi-spin effects on distance measurements of simple geometries whose behaviour can be easily predicted and modelled. In this work existing methods for suppression of multi-spin effects are tested, together with their efficiency and limitations. The results are used to devise better sets of parameters including alternative settings for extraction of accurate distances from multi-spin systems and to explore their efficiency and limitations. Additional effects influencing distance measurements by pulsed EPR are also examined; in particular the effects of orientation selection and their interplay with multi-spin effects is studied in depth. Studies on rigid symmetric and asymmetric chemical model systems together with heptameric channel membrane proteins allow for outlining of recommendations for PELDOR distance measurements settings on systems presenting similar structural features, including symmetries and inter-spin distances.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 148-174).

This thesis encloses quantum chemical calculations performed in the framework of density functional response theory for evaluating electron paramagnetic resonance (EPR) spin Hamiltonian parameters of various spin-labels in different environments. These parameters are the well known electronic g-tensor and the nitrogen hyperfine coupling constants, which are extensively explored in this work for various systems. A special attention was devoted to the relationships that form between the structural and spectroscopic properties that can be accounted for as an environmental inuence. Such environmental effects were addressed either within a fully quantum mechanical formalism, involving simplified model structures that still capture the physical properties of the extended system, or by employing a quantum mechanics/molecular mechanics (QM/MM) approach. The latter implies that the nitroxide spin label is treated quantum mechanically, while the environment is treated in a classical discrete manner, with appropriate force fields employed for its description. The state-of- the art techniques employed in this work allow for an optimum accounting of the environmental effects that play an important role for the behaviour of EPR properties of nitroxides spin labels. One achievement presented in this thesis includes the first theoretical con_rmation of an empirical assumption that is usually made for inter-molecular distance measurement experiments in deoxyribonucleic acid (DNA), involving pulsed electron-electron double resonance (PELDOR) and site-directed spin labeling (SDSL) techniques. This refers to the fact that the EPR parameters of the spin-labels are not affected by their interaction with the nucleobases from which DNA is constituted. Another important result presented deals with the inuence of a supramolecular complex on the EPR properties of an encapsulated nitroxide spin-label. The enclusion complex affects the hydrogen bonding topology that forms around the R2NO moiety of the nitroxide. This, on the other hand has a major impact on its structure which further on governs the magnitude of the spectroscopic properties. The projects and results presented in this thesis offer an example of successful usage of modern quantum chemistry techniques for the investigation of EPR parameters of spin-labels in complex systems. / <p>QC 20130318</p>

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 217-229).

Photocopy of typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1979. / Bibliography: leaves 174-182. / Microfiche. / xvi, 182 leaves ill. 29 cm

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [115]-118).

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains xi, 121 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 113-115).

Thesis (M.S)--University of Alabama at Birmingham, 2007. / Description based on contents viewed June 12, 2008; title from title screen. Includes bibliographical references (p. 30-32).

The function of a biological molecule is linked to its underlying structure, and determination of that structure can lead to significant insights into its function and how this is performed. There already exist a number of important tools in structural biology, however, the pulsed electron paramagnetic resonance (EPR) technique called pulsed electron-electron double resonance (PELDOR) is the only one capable of accurately measuring isolated distances between attached spin-labels over the range of ~2 to 10 nm, a range which is usually impossible to measure directly with other techniques such as nuclear magnetic resonance (NMR) and X-ray crystallography. This can provide constraints for refinement of structures determined from NMR and X-ray crystallography, or insights into protein docking and protein mechanics. With recent developments in EPR spectrometer instrumentation and spin-labelling it has become possible to conduct PELDOR experiments in the high field EPR regime ( > 3 Tesla) where measurement sensitivity is increased. These experiments can reveal relative orientations of nitroxide spin-labels in complement to their separation, however, analysis and interpretation of these results has been difficult to perform routinely. This thesis presents a characterisation of the high field spectrometer HiPER showing that it is well suited when optimised for making PELDOR experiments. To perform analysis of PELDOR signals from this spectrometer custom signal simulation code has been written. Two case studies are presented. The first relates to the use of the Rx spin label with the PELDOR experiment to derive orientation information from the spin labelled protein Vps75. The recently developed spin label Rx is proposed to attach more rigidly to underlying structure, offering potentially increased accuracy in determination of structure constraints and additional information about relative orientations of different structural features. An orientation selective PELDOR study is presented which compares molecular dynamics (MD) simulations of spin labels attached to sites on the α-helix of the protein Vps75. This has shown great potential for utilising the Rx spin label in a repeatable way on α-helix residue sites for determination of structural constraints. The second case relates to orientation selective PELDOR measurements of spin labelled oligomeric membrane protein structures. High field PELDOR offers great potential in increasing measurement sensitivity and accuracy of structural constraints in oligomeric proteins. A methodology of signal analysis for this class of protein is presented along with measurements of the membrane channel protein MscS. Difficulties of PELDOR measurement on these labelled proteins are discussed and observed relaxation of the spin echo, relevant to pulsed EPR experiments, are investigated and possible mechanisms are presented.