The topology and dynamics of biomolecules are intricately linked with their biological function. The focus of this thesis is the NMR-based measurement of topology and dynamics in biomolecular systems, and methods of measuring immersion depth and orientation of membrane-associated molecules. In detergent micelles and lipid bilayers, the local concentrations of hydrophobic and hydrophilic molecules are a function of their bilayer immersion depth. For paramagnetic molecular oxygen or metal cations, the magnitudes of the associated paramagnetic isotropic contact shifts and relaxation rate enhancements (PREs) are therefore depth-dependent. NMR measurements of these effects reveal the immersion depth of bilayer- or detergent-associated molecules. This work first explores transbilayer oxygen solubility and thermodynamics, as measured from contact shifts and PREs of the constituent lipid molecules in the presence of 30 bar oxygen. Contact shifts revealed the transmembrane O2 solubility profile spans a factor of seven across the bilayer, while PREs indicated that oxygen partitioning into bilayers and dodecylphosphocholine (DPC) micelles is entropically driven. Next, this work describes how paramagnetic effects from molecular oxygen and Ni(II) cations may be employed to study the immersion depth and topology of drug and protein molecules in DPC micelles. In one study, the positioning of the amphipathic drug imipramine in micelles was determined from O2- and Ni(II)-induced contact shifts. A second study, relying solely on O2-induced PREs, determined the tilt angles and micelle immersion depths of the two alpha helices in a monomeric mutant of the membrane protein phospholamban. A third study utilized 19F NMR to explore the importance of juxtamembraneous tryptophans on the topology of the membrane protein synaptobrevin, via O2-induced contact shifts and solvent-induced isotope shifts of a juxtamembraneous 19F-phenylalanine. Comparison of synaptobrevin constructs with zero, one, and two juxtamembraneous tryptophans revealed that while one tryptophan is sufficient to ‘anchor’ the protein in micelle, the addition of a second tryptophan dampens local dynamics. These solution state NMR studies demonstrate how paramagnetic effects from dissolved oxygen, complemented with measurements of local water exposure, provide detailed, accurate descriptions of membrane immersion depth and topology. These techniques are readily extended to the study of a wide range of biomolecules.

Shaped pulses can be used for uniform spin-1 excitation. The effects of the pulses on spin-1 excitation is seen as distortion of two types: phase distortions and amplitude distortions. By reducing the distortions a spin-1 excitation becomes more uniform. In the case of time-symmetric shaped pulses, spin-1 excitation is free of phase distortions. The spin-1 excitation in that case can be made uniform over a larger frequency bandwidth. The number of possible shaped pulses is so large that a computer-aided search is needed to find the desirable shaped pulses. A theoretical analysis is used to find the connection between a shaped pulse and the corresponding spin-1 excitation. The theoretical analysis in density matrix formalism gives the spin-1 excitation in closed-form expressions that are too complicated. In such a case the connection between a shpaed pulse and spin-1 excitation is not straightforward. A brute-force search for a desirable shaped pulse can consume too much computer time and thus time the scope of the search. By using the formalism of quaternions in the theoretical analysis, spin-1 excitation is presented in simple closed form expressions. It is then shown tht if the choice is limited to time-symmetric shaped pulses then these closed form expressions become much simpler. It is also shown that a spin-1 excitation is free of phase distortions in that case. These simple closed form expressions can be used as the building blocks of a much more concise program code for the computer aided search. As a result a computer aided search for a desirable shaped pulse becomes much faster in speed and larger in scope. More shaped pulses for improved spin-1 can be found. / xiii, 99 leaves : ill. ; 28 cm.

High-resolution one-dimensional 1H, 19F, 31P and 13C MAS NMR experiments were used in a morphological study of solvent-cast and heat-treated poly[bis(trifluoroethoxy)phosphazene] (PBFP). Deconvolution analyses performed on all Nuclear Magnetic Resonance (NMR) spectra are presented. These results suggest the presence of broad and narrow overlapping components at ambient temperature, which were assigned to the crystalline, amorphous and the mesophase regions within the polymer, respectively. The number of signals in the spectra was independently verified using 1H, 19F and 13C Discrimination Induced by Variable Amplitude Minipulses (DIVAM) nutation experiments. Deconvolution analyses showed that heat-treatment increases the overall crystallinity of the solvent-cast PBFP. Further studies conducted on two preparations of the polymer showed significant differences in crystallinity due to variations in the reaction conditions. Magic-Angle Spinning (MAS) NMR spectra of PBFP obtained via living cationic polymerization at ambient temperature indicated that the polymer contains mostly amorphous and mesophase regions with only a small contribution from the crystalline domain. Variable-temperature 31P NMR experiments suggested that the thermotropic transition occurs in a temperature range of 80ºC to 90ºC, where the crystalline signal disappears and a new signal due to a liquid crystalline phase emerges. Spin-lock 31P experiments provided rates of the transverse relaxation in the rotating frame for each signal, showing that the crystalline and the amorphous regions within the polymer are characterized by significantly different mobilities at ambient temperatures, while the v comparable degree of motion occurs between the amorphous and mesophase environments at temperatures above 90ºC. The process of thermal ring-opening polymerization of hexachlorocyclotriphosphazene was monitored using one-dimensional 31P MAS NMR at different stages of the reaction. The ratio between cyclic species and the high molecular weight poly(dichlorophosphazene) was seen to change over time. 31P NMR was seen to be a potentially valuable tool in monitoring rates of chain propagation, branching and cross-linking. Two-dimensional 31P homonuclear Radio-Frequency Driven Recoupling (RFDR) and Incredible Natural Abundance Double Quantum Transfer (INADEQUATE) MAS NMR experiments were first tested on the partially phenoxy-substituted hexachlorocyclotriphosphazene, and subsequently applied in the study of a preparation of the partially trifluoroethoxy-substituted poly(dichlorophosphazene). Very high resolution was obtained in the direct dimension due to the presence of low molecular weight species. Preliminary spectral assignments of all of the observed signals were made on the basis of both known chemical shifts of the related species, and the through-space and through-bond phosphorous-phosphorous connectivities. / xiii, 188 leaves : ill. ; 29 cm

Trimethyltin fluoride (Me3SnF) is a useful fluorinating agent in organometallic chemistry. Its solid-state structure has been investigated by X-ray crystallography showing a polymeric fluorine-bridged structure. Disorder, however, has precluded the accurate refinement of all structural parameters. In order to obtain accurate structural information, trimethyltin fluoride was investigated using high-resolution 13C, 19F, and 119Sn solid-state NMR spectroscopy using a four-channel HFXY capability. The 119Sn{1H} solid-state NMR spectrum agrees with pentacoordination about Sn in this compound. The high-resolution 119Sn{19F, 1H}, 13C{1H,19F} and 19F{1H} NMR spectra offer unambiguous determination of 1J(119Sn-19F) and 1J(119Sn-13C) coupling constants. Furthermore, the analysis of the 119Sn{19F, 1H}, 119Sn{1H}, and 19F{1H} MAS spectra as a function of spinning speed allowed for the determination of the 119Sn CSA and J anisotropy, as well as the 119Sn-19F dipolar couplings. These were determined via SIMPSON simulations of the 13C, 19F, and 119Sn NMR spectra. Finally the 119Sn{19F, 1H} revealed fine structure as the result of 119Sn-117Sn two bond J-coupling, seen here for the first time. Sulfur tetrafluoride can act as a Lewis acid. Claims had been presented for the formation of an adduct between SF4 and pyridine, but no conclusive characterization had been performed. In the present study, adducts of SF4 with pyridine, lutidine, 4-picoline and triethylamine were prepared and characterized by low-temperature Raman spectroscopy. Sulfur tetrafluoride also acts as a fluoride-ion donor towards strong Lewis acids, such as AsF5 and SbF5, forming SF3 + salts. Variable-temperature (VT) solid-state 19F NMR spectroscopy showed that SF3 +SbF6 – exists in three phases with phase transitions at ca. –45 and –85°C, while SF3 +AsF6 – exists only as one phase between +20 and –150 °C. The phases of SF3 +AsF6 – were also characterized by VT Raman spectroscopy. / xvi, 170 leaves : ill. (some col.) ; 29 cm

No description available.

The central theme of this thesis is a study of the structural changes accompanying chemical modification and denaturation of eukaryotic cytochrome c as characterised by ¹II nuclear magnetic resonance (n.m.r.) spectroscopy. First, however, it was necessary to obtain and confirm assignments for individual resonances; this was achieved by a novel method of cross assignment between ferricytochrome c and ferrocytochrome c and by double resonance techniques. A variety of perturbations were caused to native cytochromes c, which ranged in degree from the elevation of temperature for ferrocytochrome c to the complete denaturation of the protein with urea or methanol. Modification at single sites both on the surface (e.g. Met 65, Tyr 74) and in the core ( e.g. Tyr 67) of the molecule were found to cause only small local effects to the structure, although the dynamic features of the molecules were altered. One single site modification, the breaking of the iron - sulphur cross linking bond, caused considerable disruption to one side of the molecule, although hydrophobic domains in the other side were preserved; this state of the molecule is analogous to the penultimate state in the refolding pathway. Modification of all the charged lysine residues caused small changes to the surface structure of the molecule, though the complete reversal of the charges in maleyl cytochrome c produced a species which unfolded reversibly from a native configuration with the increase of temperature. The unfolding of the protein is virtually identical with both methanol and urea, but the pathways are shown to differ for the oxidised and reduced proteins.

The human brain consists of a myriad of chemical compounds critical to its functioning. A group of these compounds, collectively known as metabolites, have been a research interest for years because the pathogenesis of neurodegenerative diseases, a tumours classification, the effectiveness of a drug, etc., can be investigated via variations in brain metabolite concentration levels. Nuclear Magnetic Resonance Spectroscopy (NMRS) enables investigators to conduct non-invasive in vivo studies of metabolites in the human brain and the rest of the body. However a number of problems have hindered the usage of NMRS as a clinical diagnostic tool. One is the non-uniqueness of the most widely used analysis methods, i.e. as the parameters and/or prior knowledge data of an analysis method are changed, the results also change. A second problem is the lack of a method that can automatically classify the signal components estimated via signal decomposition based signal analysis methods. Additionally, some of the most widely used analysis methods, by virtue of their algorithms, intrinsically assume the nature of NMRS signals, e.g. stationary, linear, Lorentzian, etc. Hence, this thesis explores a new analysis approach, based on a theoretical and practical understanding of NMRS, that (a) avoids making assumptions about the nature of experimentally acquired NMRS signals, (b) relies on a unique decomposition analysis method, and (c) automatically classifies the estimated peaks of an analysis. Unique decomposition analysis was conducted via the rarely used unique and non-linear signal decomposition method − the Fast Pad´e Transform (FPT). The FPT is compared with the main decomposition based NMRS analysis methods via a detailed mathematical analysis, and a comparative analysis. Automatic classification was conducted via a novel classification method, which is introduced herein, and which is based on quantum mechanical predictions of metabolite NMRS behaviour.

The Application of Magnetic Resonance (MRI) and Computed Tomography Imaging (CT) in the Diagnosis and Management of Maxillofacial Tumours. For decades maxillofacial surgeons over the world have been frustrated by the high and often fatal recurrence of certain advanced jaw tumours. This study conclusively proves that Computed Tomography and especially Magnetic Resonance Imaging significantly decreases recurrence of Odontogenic Keratocyst and Ameloblastoma and allows surgical planning to avoid these recurrences. / Doctor Scientiae (Odontology) - DSc(Odont)

Amyotrophic lateral sclerosis (ALS) is a devastating and progressive disease that results in selective death of motor neurons in the cortex, brain stem and spinal cord. ALS is the most common adult onset motor neuron disease resulting in paralysis and death, commonly within 2 – 5 years of symptom onset, yet there remains no effective treatment for the disease. The majority of ALS cases show no hereditary link (referred to as sporadic ALS or sALS); however, ~10% of cases show a dominant pattern of inheritance (referred to as familial ALS or fALS). Over 170 different mutations in human Cu, Zn superoxide dismutase (SOD1) have been identified to account for ~20% of fALS. SOD1 is a ubiquitously expressed homodimeric antioxidant enzyme. It is widely accepted that mutations in SOD1 result in a gain of toxic function, rather than a loss of native function. A prominent hypothesis for the gain of function is the formation of protein aggregates, which have been shown to be toxic to motor neurons. Protein aggregation is observed in a number of neurodegenerative disorders, including Alzheimer’s, Huntington’s and Parkinson’s disease. Each β-rich monomer of SOD1 binds one catalytic Cu ion and one structural Zn ion. The metallation state of SOD1 significantly influences the structure, dynamics, activity, stability, and aggregation propensity. A similar trend has been observed in a number of metalloenzymes and as such a method to rapidly and accurately quantitate metal ions in proteins is of great importance. Here a review of previous methods using the chromogenic chelator PAR to quantitate metal ions in proteins is presented. Three methods are assessed for their accuracy, precision and ease of use. The methods vary in accuracy, which is highest only under the specific conditions it was designed for. A robust new method is presented here that uses spectral decomposition software to accurately resolve the absorption bands of Cu and Zn with high precision. This method may be successful as a more general method for metal analysis of proteins allowing for the quantitation of additional metal combinations (e.g. Zn/Co, Ni/Cu, Ni/Co). Thermodynamic stability has widely been implicated as playing a major role in the aggregation of globular proteins. Metal loss significantly decreases the global stability of SOD1 and as such metal-depleted (apo) forms of SOD1 have largely been the focus of SOD1 investigations. Recent studies, however, suggest that complete global unfolding is not required for protein aggregation. Local unfolding has been investigated and proposed to be sufficient to induce irreversible protein aggregation in the absence of global destabilization. Enhanced local unfolding has been observed in a number of disease-related proteins. Since SOD1 aggregation may occur from partially unfolded forms, NMR temperature dependence studies have been carried out on the most abundant form of SOD1 in vivo, the fully metallated (holo) dimer, to provide a residue specific picture of subglobal structural changes in SOD1 upon heating. Amide proton (N1H) temperature coefficients report on the hydrogen bonding status of a protein. A curved N1H temperature dependence indicates that the proton populates an alternative conformation generally within 5 kcal/mol of the ground state. NMR temperature dependence studies of pseudoWT indicate that the thermal unfolding process of holo pWT begins with “fraying” of the structure at its periphery. In particular, increased disorder is observed in edge strands β5 and β6, as well as surrounding the zinc binding site. The local stability and conformational heterogeneity of ALS-associated mutants G93A, E100G and V148I was also assessed. All mutants display similar local unfolding patterns to pseudoWT, but also show distinct differences in the hydrogen bonding network surrounding the mutation site. Interestingly, each mutation regardless of its structural context results in altered dynamics at the β-barrel plug, a key stabilizing element in SOD1. A significant proportion of residues (~30%) access alternative states in both pseudoWT and mutants, however, overall mutants appear to be able to access higher free energy alternative states compared to pseudoWT. The implications of these results for the mechanism of protein aggregation and disease are discussed.

The topology and dynamics of biomolecules are intricately linked with their biological function. The focus of this thesis is the NMR-based measurement of topology and dynamics in biomolecular systems, and methods of measuring immersion depth and orientation of membrane-associated molecules. In detergent micelles and lipid bilayers, the local concentrations of hydrophobic and hydrophilic molecules are a function of their bilayer immersion depth. For paramagnetic molecular oxygen or metal cations, the magnitudes of the associated paramagnetic isotropic contact shifts and relaxation rate enhancements (PREs) are therefore depth-dependent. NMR measurements of these effects reveal the immersion depth of bilayer- or detergent-associated molecules. This work first explores transbilayer oxygen solubility and thermodynamics, as measured from contact shifts and PREs of the constituent lipid molecules in the presence of 30 bar oxygen. Contact shifts revealed the transmembrane O2 solubility profile spans a factor of seven across the bilayer, while PREs indicated that oxygen partitioning into bilayers and dodecylphosphocholine (DPC) micelles is entropically driven. Next, this work describes how paramagnetic effects from molecular oxygen and Ni(II) cations may be employed to study the immersion depth and topology of drug and protein molecules in DPC micelles. In one study, the positioning of the amphipathic drug imipramine in micelles was determined from O2- and Ni(II)-induced contact shifts. A second study, relying solely on O2-induced PREs, determined the tilt angles and micelle immersion depths of the two alpha helices in a monomeric mutant of the membrane protein phospholamban. A third study utilized 19F NMR to explore the importance of juxtamembraneous tryptophans on the topology of the membrane protein synaptobrevin, via O2-induced contact shifts and solvent-induced isotope shifts of a juxtamembraneous 19F-phenylalanine. Comparison of synaptobrevin constructs with zero, one, and two juxtamembraneous tryptophans revealed that while one tryptophan is sufficient to ‘anchor’ the protein in micelle, the addition of a second tryptophan dampens local dynamics. These solution state NMR studies demonstrate how paramagnetic effects from dissolved oxygen, complemented with measurements of local water exposure, provide detailed, accurate descriptions of membrane immersion depth and topology. These techniques are readily extended to the study of a wide range of biomolecules.