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The appropriate lipid environment is crucial for the proper function of membrane proteins. There is a tremendous variety of lipid molecules in the membrane and so far it is often unclear which component of the lipid matrix is essential for the function of a respective protein. Lipid molecules and proteins mutually influence each other; parameters such as acyl chain order, membrane thickness, membrane elasticity, permeability, lipid-domain and annulus formation are strongly modulated by proteins. More recent data also indicates that the influence of proteins goes beyond a single annulus of next-neighbor boundary lipids. Therefore, a mesoscopic approach to membrane lipid-protein interactions in terms of elastic membrane deformations has been developed. Solid-state NMR has greatly contributed to the understanding of lipid-protein interactions and the modern view of biological membranes. Methods that detect the influence of proteins on the membrane as well as direct lipid-protein interactions have been developed and are reviewed here. Examples for solid-state NMR studies on the interaction of Ras proteins, the antimicrobial peptide protegrin-1, the G protein-coupled receptor rhodopsin, and the K+ channel KcsA are discussed.

I investigated how land use and human activities affect the sources and cycling of carbon (C) in subtropical rivers. Annually rivers receive a large amount of terrestrial C, process a portion of this C and return it to the atmosphere as CO2. The rest is transported to the ocean. Land use and human activities can affect the sources and fate of terrestrial C in rivers. However, studies on these effects are limited, especially in the humid subtropics. I combined measurements of the partial pressure of dissolved CO2 (pCO2), C isotopes (13C and 14C) and solid-state 13C nuclear magnetic resonance (NMR) to study C cycling in three subtropical rivers in Texas, two small rivers (Buffalo Bayou and Spring Creek) and a midsized river (the Brazos). My pCO2 data show that small humid subtropical rivers are likely a large source of atmospheric CO2 in the global C cycle. My measurements on pCO2, C isotopic and chemical composition of dissolved inorganic C (DIC) and particulate organic C (POC) revealed four types of effects of land use and human activities on river C cycling. First, oyster shells and crushed carbonate minerals used in road construction are being dissolved and slowly drained into Buffalo Bayou and the lower Brazos and may be a source of river CO2 released to the atmosphere. Second, river damming and nutrient input from urban treated wastewater stimulate algal growth and reduce CO2 evasion of the middle Brazos. Third, urban treated wastewater discharge is adding old POC to the middle Brazos and decomposition of the old POC adds to the old riverine DIC pool. Fourth, agricultural activities coupled with high precipitation enhance loss of old organic C (OC) from deep soils to the lower Brazos, and decomposition of the old soil OC contributes to the old CO2 evaded. I document for the first time the river C cycling effects of the use of carbonate minerals in construction and the riverine discharge of urban wastewater. Results presented here indicate the need to study disturbed river systems to better constrain the global C budget.

Concrete, due to its low cost, durability and fire resistance, is one of the world’s most widely used construction materials. Concrete is typically reinforced with steel bars and welded wire mesh. Since the cost of steel is increasing and steel corrosion is a significant contributor to structural failure, it is advantageous to find an alternative replacement reinforcement material which can not only replace the steel, but also resist corrosion. Over the past few decades, polymeric fibres have been used as concrete reinforcement. The chemical bond between the polymeric fibre and the cementitious matrix is an important factor in the fibre’s performance as a concrete reinforcement. Despite the great importance of the chemical bonding at the polymeric fibre/concrete interface, the chemical bonding at the interface is not well understood. To investigate the chemical interactions between polymeric materials and concrete, model systems of polymeric powder/white cement and polymeric fibre/white cement were chosen, where white cement was chosen for its suitability for nuclear magnetic resonance (NMR) experiments. The chemical interactions between poly(ethylenevinyl acetate) (EVA), poly(ether imide) (PEI), and poly(vinylidene fluoride) (PVDF) polymeric powders were studied via 13C NMR spectroscopy. It was found that EVA admixture undergoes hydrolysis in a cementitious matrix and follows a pseudo-second order kinetics model up to 32 days of cement hydration. PEI was also found to undergo hydrolysis at the imide functional group in a cementitious matrix. PVDF powder undergoes dehydrofluorination in the cementitious environment, producing a brown coloured polymer which is a result of conjugation of the polymer backbone. The interfacial transition zone between fluoropolymeric powder/white cement and steel and polymeric fibres (high density polyethylene/polypropylene, poly(vinyl alcohol), PEI, PVDF, and Nylon 6.6) was studied at short range using 19F, 27Al, and 43Ca NMR spectroscopy and at long range using the scanning electron microscopy/energy dispersive spectroscopy method. It was concluded that the chemistry of polymeric fibres themselves can alter the surrounding interfacial transition zone such that the calcium silicate hydrate favours a tobermorite or jennite-like structure, which could contribute to a strong or weak interface.

Nuclear magnetic resonance (NMR) spectroscopy was used to determine the structure of transmembrane (TM) segment IX of the Na+/H+ exchanger isoform 1 (NHE1) in dodecylphosphocholine micelles. Studying isolated TM segments in this fashion constitutes a well-established "divide and conquer" approach to the study of membrane proteins, which are often extremely difficult to produce, purify, and reconstitute in full-length polytopic form. A similar approach was combined with NMR spin relaxation experiments to determine the peptide backbone flexibility of NHE1 TM VII. The combined NMR structural and dynamics studies are consistent with an important role for TM segment flexibility in the function of NHE1, a protein involved in apoptosis and myocardial disease. The study of the rhomboid protease system is also described from two perspectives: 1) I attempted to produce several TM constructs of the substrate spitz or a related construct and the production and purification are described in detail; and 2) I present coarse-grained molecular dynamics simulation results for the E. coli rhomboid ecGlpG and a spitz TM construct. Spitz appears to preferentially associate with rhomboid near TMs 1 and 3 rather than the proposed substrate gate at TM 5. The two proteins primarily interact at the termini of helices rather than within the hydrocarbon core of the bilayer. Finally, I present a detailed analysis of coarse-grained molecular dynamics simulations of the fibroblast growth factor receptor 3 TM domain dimerization. Specifically, algorithms are described for analyzing critical features of wild-type and G380R mutant constructs. The G380R mutation is the cause of achondroplasia, the most common form of human dwarfism. The results suggest that the proximity of a residue to the dimer interface may impact the severity of the mutant phenotype. Strikingly, heterodimer and mutant homodimer constructs exhibit a secondary dimer interface which may explain the increased signaling activity previously reported for the G380R mutation--the helices may rotate with the introduction of G380R. The unifying theme of this work is the 'study of membrane proteins' using complementary techniques from structural biology and computational biochemistry.

In this thesis the synthesis and full characterization of a new bulky diphosphine, tetrakis-(2,6-diisopropylphenyl)diphosphine, are described. This compound displays facile oxidation and a thorough investigation of its redox properties has been studied by combining solution electrochemical techniques such as cyclic voltammetry (CV) and rotating disk electrode (RDE) voltammetry, with spectroscopic methods such as electron paramagnetic resonance (EPR) and Simultaneous Electrochemical Electron Paramagnetic Resonance (SEEPR) spectroscopy over a wide temperature range. Density functional theory (DFT) calculations were carried out to aid in structural characterization of the radical cation that is produced and to provide computed hyperfine splitting (HFS) constants for comparison with experimental results. For comparison to this species with bulky aromatic substituents, similar studies were conducted that have identified the previously unreported radical cation of tetrakis-tert-butyldiphosphine with a bulky aliphatic substituent that provides even higher steric pressure than the 2,6-diisopropylphenyl group. DFT calculations are reported, as is full characterization with fluid and frozen-solution EPR spectroscopy. Further CV and EPR (SEEPR) studies are reported that led to the identification of radical cations of tris(2,6-diisopropylphenyl)arsine and bis(2,4,6-triisopropylphenyl)(2,6-diisopropylphenyl)phosphine. DFT calculations are reported, as is full characterization with fluid and frozen-solution EPR spectroscopy. / xix, 172 leaves : ill (some col.) ; 29 cm

Glass materials surround us, impacting our lives on a daily basis, whether geologically deposited by volcanic activity or synthesized in large volume by industry. These amorphous oxide materials are vastly important due to their variety of applications including solid electrolytes, cookware, and storage of high-level nuclear waste. Although they are used for different applications, one common characteristic of these materials is the absence of long-range periodic order. This makes it difficult to use traditional solid-state characterization methods such as x-ray and neutron diffraction to study glass structure. Nuclear magnetic resonance (NMR), is ideally suited to study materials that exhibit short-range non-periodic order as it probes directly at a nucleus of interest and is sensitive to its local structural environment. This ability of solid-state NMR is illustrated by revealing local structural features in various oxide materials presented in this thesis. Within is a compilation of studies looking at basic borates, followed by borovanadates and complex borosilicate glasses. A multinuclear application of using quantum chemical calculations, single and double resonance methods and charge-balance models are discussed to deconvolute the complex structures of these disordered materials. This is followed by a study of a difficult low-gamma nucleus, 73Ge, (once considered “impossible” for solid-state NMR) which is explored for future material studies by looking at 73Ge NMR of crystalline and glassy germanates. 73Ge chemical shifts were related to coordination environments and quadrupolar coupling constants were related to bond length distortions.

Glass materials surround us, impacting our lives on a daily basis, whether geologically deposited by volcanic activity or synthesized in large volume by industry. These amorphous oxide materials are vastly important due to their variety of applications including solid electrolytes, cookware, and storage of high-level nuclear waste. Although they are used for different applications, one common characteristic of these materials is the absence of long-range periodic order. This makes it difficult to use traditional solid-state characterization methods such as x-ray and neutron diffraction to study glass structure. Nuclear magnetic resonance (NMR), is ideally suited to study materials that exhibit short-range non-periodic order as it probes directly at a nucleus of interest and is sensitive to its local structural environment. This ability of solid-state NMR is illustrated by revealing local structural features in various oxide materials presented in this thesis. Within is a compilation of studies looking at basic borates, followed by borovanadates and complex borosilicate glasses. A multinuclear application of using quantum chemical calculations, single and double resonance methods and charge-balance models are discussed to deconvolute the complex structures of these disordered materials. This is followed by a study of a difficult low-gamma nucleus, 73Ge, (once considered “impossible” for solid-state NMR) which is explored for future material studies by looking at 73Ge NMR of crystalline and glassy germanates. 73Ge chemical shifts were related to coordination environments and quadrupolar coupling constants were related to bond length distortions.

The atomic resolution structure of a low populated and transiently formed on-pathway folding intermediate of the FF domain from human HYPA/FBP11 has recently been reported[1]. The structure was determined on the basis of backbone chemical shift and bond vector orientation restraints measured on the ‘invisible’ intermediate state using relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy that were subsequently input into the data-base structure determination program CS-Rosetta. This thesis focuses on the cross-validation of the structure so produced. We present here the solution NMR structure of a mimic of the folding intermediate that is highly populated in solution, obtained from the wild-type domain by protein mutagenesis. The ensemble of structures generated of the mimic are within 2Å of the relaxation dispersion/CS-Rosetta structures of the intermediate, with the non-native interactions in the intermediate also observed in the mimic. The results presented in this thesis strongly confirm the structure of the FF domain folding intermediate, in particular, and validate the use of relaxation dispersion derived restraints in structural studies of invisible excited states, in general.

The extensive agricultural usage of organohalogenated pesticides has raised many concerns about their potential hazards especially in the soil environment. Environmental metabolomics is an emerging field that investigates the changes in the metabolic profile of native organisms in their environment due to the presence of an environmental stressor. Research presented here explores the potential of Nuclear Magnetic Resonance (NMR)-based metabolomics to examine the sub-lethal exposure of the earthworm, Eisenia fetida to sub-lethal concentrations of organohalogenated pesticides. Various one-dimensional (1-D) and two dimensional (2-D) NMR techniques were compared in a contact filter paper test earthworm metabolomic study using endosulfan, a prevalent pesticide in the environment. The results determined that both the 1H Presaturation Utilizing Gradients and Echos (PURGE) and the 1H-13C Heteronuclear Single Quantum Coherence (HSQC) NMR techniques were most effective in discriminating and identifying significant metabolites in earthworms due to contaminant exposure. These two NMR techniques were further explored in another metabolomic study using various sub-lethal concentrations of endosulfan and an organofluorine pesticide, trifluralin to E. fetida. Principal component analysis (PCA) tests showed increasing separation between the exposed and unexposed earthworms as the concentrations for both contaminants increased. A neurotoxic mode of action (MOA) for endosulfan and a non-polar narcotic MOA for trifluralin were delineated as many significant metabolites, arising from exposure, were identified. The earthworm tissue extract is commonly used as the biological medium for metabolomic studies. However, many overlapping resonances are apparent in an earthworm tissue extract NMR spectrum due to the abundance of metabolites present. To mitigate this spectral overlap, the earthworm’s coelomic fluid (CF) was tested as a complementary biological medium to the tissue extract in an endosulfan exposure metabolomic study to identify additional metabolites of stress. Compared to tests on the tissue extract, a plethora of different metabolites were identified in the earthworm CF using 1-D PURGE and 2-D HSQC NMR techniques. In addition to the neurotoxic MOA identified previously, an apoptotic MOA was also postulated due to endosulfan exposure. This thesis also explored the application of 1-D and 2-D NMR techniques in a soil metabolomic study to understand the exposure of E. fetida to sub-lethal concentrations of endosulfan and its main degradation product, endosulfan sulfate. The earthworm’s CF and tissue extract were both analyzed to maximize the significant metabolites identified due to contaminant exposure. The PCA results identified similar toxicity for both organochlorine contaminants as the same separation, between exposed to the unexposed earthworms, were detected at various concentrations. Both neurotoxic and apopotic MOAs were observed as identical fluctuations of significant metabolites were found. This research demonstrates the potential of NMR-based metabolomics as a powerful environmental monitoring tool to understand sub-lethal organohalogenated pesticide exposure in soil using earthworms as living probes.