NMR spectroscopy is not only for protein structure determination, but also for drug screening and studies of dynamics and interactions. In both cases, one of the main bottleneck steps is backbone assignment. When a homologous structure is available, it can accelerate assignment. Such structure-based methods are the focus of this thesis. This thesis aims for fast and robust methods for NMR assignment problems; in particular, structure-based backbone assignment and chemical shift mapping. For speed, we identified situations where the number of 15N-labeled experiments for structure-based assignment can be reduced; in particular, when a homologous assignment or chemical shift mapping information is available. For robustness, we modeled and directly addressed the errors. Binary integer linear programming, a well-studied method in operations research, was used to model the problems and provide practically efficient solutions with optimality guarantees. Our approach improved on the most robust method for structure-based backbone assignment on 15N-labeled data by improving the accuracy by 10% on average on 9 proteins, and then by handling typing errors, which had previously been ignored. We show that such errors can have a large impact on the accuracy; decreasing the accuracy from 95% or greater to between 40% and 75%. On automatically picked peaks, which is much noisier than manually picked peaks, we achieved an accuracy of 97% on ubiquitin. In chemical shift mapping, the peak tracking is often done manually because the problem is inherently visual. We developed a computer vision approach for tracking the peak movements with average accuracy of over 95% on three proteins with less than 1.5 residues predicted per peak. One of the proteins tested is larger than any tested by existing automated methods, and it has more titration peak lists. We then combined peak tracking with backbone assignment to take into account contact information, which resulted in an average accuracy of 94% on one-to-one assignments for these three proteins. Finally, we applied peak tracking and backbone assignment to protein-ligand docking to illustrate the potential for fast 3D complex determination.

Copper is one of the most vital trace elements in ruminant nutrition. It is required for several metabolic activities and it is also an essential component of several physiologically important metalloenzymes. Thus copper deficiency in ruminants results in distinctive pathologies, and hence in significant economic losses to farmers. Copper deficiency results from very low copper in diet (primary copper deficiency) and interference with Cu absorption in the animal due to Mo and S in food or water (secondary copper deficiency). The molybdenum-induced copper deficiency that affects ruminants can be attributed to the formation of thiomolybdates (TMs)from molybdate and sulfide in the rumen. The TMs formed then react irreversibly with copper to form insoluble Cu-TM complex which ultimately end up being excreted, thus reducing copper bioavailability to the ruminant. <p>In this study, an attempt has been made to use computer simulations to model speciation of copper in rumen fluid in the presence of TMs with the aim of understanding the extent to which TMs affects the levels of copper in the rumen. <p>This was done by initially refining the computer model of copper speciation with respect to low molecular mass (LMM) ligands in bovine rumen with the aim of correcting the discrepancy that was observed during experimental validation of the computer model in a previous study. To this end, mass balance equations which describes the distribution of Cu(II) amongst the different ligands were encoded into a spreadsheet to calculate equilibrium concentration of all species. Formation constants obtained from literature as well as those obtained from studies in our group were used as input values in the spreadsheet. Results show that at average ruminal pH, the metal would be present mostly as carbonate and phosphate complexes. The results obtained from the computer model in the present study were validated using 1H NMR experiments on simulated rumen fluid as well as actual rumen fluid containing Cu(II); using acetic acid chemical shift as the probe for monitoring the speciation pattern. Excellent agreement was observed between the computer model and experimental results. Discrepancy was however observed upon introduction of copper lysine as copper source into the model. Incorporation of a mixed ligand complex of Cu(II), acetate and lysine into the computer model gave an excellent agreement between the computer model and experimental results. <p>The study was extended to include glycine, histidine, methionine and EDTA complexes as the copper source in both rumen saliva (McDougalls solution) and rumen fluid. Results show that only the histidine and EDTA complexes persist to any significant extent, in spite of the large number of competing ligands present in these matrices.<p>In this study, success has also been achieved in the integration of the slow (kinetically controlled) formation of TMs and copper-tetrathiomolybdate (TM4) complexation into the previously developed model for the rapidly equilibrating copper-ligand speciation. To simulate the formation of the TMs and Cu-TM4 complex with respect to time, the differential equations representing rate expressions for each chemical species were solved to obtain an analytical solution using the Laplace transform method. The analytical solutions obtained were encoded in a spreadsheet and calculated as function of time to obtain time dependent concentrations of TMs and Cu-TM4 complex. This was then integrated with previously developed model for the rapidly equilibrating copper-ligand speciation in the rumen. The kinetic data used in the simulation of the formation thiomolybdates was obtained fron literature wheras that for Cu-TM4 complexation was obtained from our lab using Cu(II) - Ion Selective Electrode. The results show that that in the presence of TM4 the, Cu(II) bound to low molecular ligands in the rumen is drastically reduced confirming the effect TM4 on Cu(II) observed in several in vitro studies.<p>The study shows that in thiomolybdate contaminated rumen environment, the bioavailability of copper is considerably reduced. Though metal bioavailabilities are hard to predict this approach could help better our understanding of this process.

<p>Although microbes may strongly influence wetland phosphorus (P) cycling, specific microbial communities and P metabolic processes have not been characterized in wetlands, and microbial P cycling is poorly understood across global ecosystems, especially in soils. The goal of this work is to test the effects of stress and growth factors on microbial communities in wetlands, and on microbial P metabolism and P cycling at ecosystem scales in wetland soils and beyond. I conducted field and laboratory research experiments in wetland soils, which by definition lie along gradients between terrestrial and aquatic ecosystems, and I explicitly compared results in wetlands to adjacent ecosystems to improve inference and impact. </p><p> To test relationships between microbial communities, soil stress and resource supply, I compared the distribution and abundance of uncultured bacterial communities to environmental factors across a range of wetland soils including a well-characterized P enrichment gradient, and restoration sequences on organic soils across freshwater wetland types. The strongest predictor of bacterial community composition and diversity was soil pH, which also corresponded with the abundance of some bacterial taxa. Land use and restoration were also strong predictors of bacterial communities, diversity, and the relative abundance of some taxonomic groups. Results from wetland soils in this study were similar to both terrestrial and aquatic ecosystems in the relationship of pH to microbial communities. However, patterns of biogeography I observed in wetlands differed from aquatic systems in their poor relationships to nutrient availability, and from terrestrial ecosystems in the response of microbial diversity to ecosystem restoration.</p><p> Accumulation of inorganic polyphosphate (PolyP) is a critical factor in the survival of multiple environmental stresses by bacteria and fungi. This physiological mechanism is best characterized in pure cultures, wastewater, sediments, and I used 31P-NMR experiments to test whether similar processes influence microbial P cycling in wetland soils. I surveyed PolyP accumulation in soils from different wetland types, and observed PolyP dynamics with flooding and seasonal change in field soils and laboratory microcosms. I found PolyP accumulation only in isolated pocosin peatlands, similar to patterns in the published literature. I observed rapid degradation of PolyP with flooding and anerobic conditions in soils and microcosms, and I characterized the biological and intracellular origin of PolyP with soil cell lysis treatments and bacterial cultures. While degradation of PolyP with flooding and anaerobic conditions appeared consistent with processes in aquatic sediments, some seasonal patterns were inconsistent, and experimental shifts in aerobic and anaerobic conditions did not result in PolyP accumulation in soil slurry microcosms. Similar to patterns in wetlands, I found prior observations of PolyP accumulation in published 31P-NMR studies of terrestrial habitats were limited to acid organic soils, where PolyP accumulation is thought to be fungal in origin. Fungal accumulation of PolyP may be useful as an alternative model for PolyP accumulation in wetlands, although I did not test for fungal activity or PolyP metabolism. </p><p> To evaluate relationships between microbial P metabolism and growth, I compared concentrations of P in soil microbial biomass with the soil metabolic quotient (qCO2) by compiling a large-scale dataset of the carbon (C), nitrogen (N) and P contents of soils and microbial biomass, along with C mineralization rates across global wetland and terrestrial ecosystems (358 observations). The ratios of these elements (stoichiometry) in biomass may reflect nutrient limitation (ecological stoichiometry), or be related to growth rates (Biological Stoichiometry). My results suggest that the growth of microbial biomass pools may be limited by N availability, while microbial metabolism was highly correlated to P availability, which suggests P limitation of microbial metabolism. This pattern may reflect cellular processes described by Biological Stoichiometry, although microbial stoichiometry was only indirectly related to respiration or metabolic rates. I found differences in the N:P ratios of soil microbial biomass among ecosystems and habitats, although high variation within habitats may be related to available inorganic P, season, metabolic states, or P and C rich energy storage compounds. Variation in microbial respiration and metabolic rates with soil pH suggests important influences of microbial communities and their responses to stress on metabolism and P cycling.</p><p> My dissertation research represents early contributions to the understanding of microbial communities and specific processes of microbial P metabolism in wetlands, including PolyP accumulation and Biological Stoichiometry, which underpin microbial cycling of P and C. Together, my research findings broadly indicate differences in microbial P metabolism among habitats in wetlands and other ecosystems, which suggests the prevailing paradigm of uniform P cycling by microbes will be inadequate to characterize the role of microbes in wetland P cycling and retention. While I observed some concomitant shifts in microbial communities, PolyP accumulation, and microbial stoichiometry with soil pH, land use, and habitat factors, relationships between specific microbial groups and their P metabolism is beyond the scope of this work, but represents an exciting frontier for future research studies.</p> / Dissertation

<p>Ssu72 is a protein phosphatase that selectively targets phosphorylated serine residues at the 5th position (pS5) in the heptad repeats of the C-terminal domain (CTD) of RNA polymerase II, in order to regulate the CTD-mediated coupling between eukaryotic transcription and co-transcriptional events. The biological importance of Ssu72 is underscored by (1) the requirement of its activity for viability in yeast, and (2) the numerous phenotypes - affecting all three stages of the transcription cycle - that result from its mutation in yeast. Despite limited homology to the low molecular weight (LMW) subclass of protein tyrosine phosphatases (PTPs), several lines of evidence suggest that Ssu72 represents the founding member of a new class of enzymes, including its unique substrate specificity and an in vivo connection with the activity of proline isomerase Ess1.</p><p>The main focus of this thesis has been to structurally and kinetically characterize Ssu72, in order to define its relation to known enzyme families, to provide biochemical explanations for extant in vivo observations, and to allow future structure-guided investigations of its role in coordinating transcription with co-transcriptional events. To this end, we solved the structure of Ssu72 in complex with its pS5 CTD substrate, revealing an enzyme fold with unique structural features and a surprising substrate conformation with the pS5-P6 motif of the CTD adopting the cis configuration. Together with kinetic assays, the structure provides a new interpretation of the role of proline isomers in regulating the CTD phosphorylation state, with broad implications for CTD biology.</p><p>The second goal of this thesis has been to develop new methods for NMR studies of large proteins, which present unique challenges to conventional methods, including fast signal decay and severe signal degeneracy. The first of these new methods, the `just-in-time' HN(CA)CO, improves the sensitivity of a common backbone assignment experiment. The next two methods, the 4-D diagonal-suppressed TROSY-NOESY-TROSY and the 4-D time-shared NOESY, were designed for use with sparse sampling techniques that allow the acquisition of high-resolution, high-dimensionality datasets. These efforts culminate with global fold calculations for large proteins, including the 23 kDa Ssu72, with accurate and unambiguous automated assignment of NOE crosspeaks. We expect that the methods presented here will be particularly useful as the NMR community continues to push toward higher molecular weight targets.</p> / Dissertation

Multicomponent polymer systems comprised of two or more chemically different polymer moieties provide an effective way to attain the desired properties from a limited palette of commodity polymers. Variations in macromolecular topologies often result in unique and unusual properties leading to novel applications. This dissertation addresses the effect of topology on properties of two multicomponent polymers systems: blends and polyrotaxanes. Blends of cyclic and linear polymers were compared to their topological counterparts, polyrotaxanes, in which cyclic components are threaded onto the linear polymer chains. The first part of the dissertation focuses on the synthesis and purification of cyclic polymers derived from linear (polyoxyethylene) (POE). Cyclic POEs of different cycle sizes were synthesized and then purified from their linear byproducts by inclusion complexation with alpha-cyclodextrin. Polystyrene was threaded through the resulting cycles by in situ free radical polymerization of styrene monomer in the presence of an excess of POE cycles. A bulky free radical initiator was utilized to endcap the polystyrene molecule at the two ends to prevent dethreading of cyclic moieties. In the second part of the dissertation, phase behavior, morphology and dynamics of cyclic POE and polystyrene blends were compared to linear POE and polystyrene blends. Advanced solid-state NMR techniques and differential scanning calorimetry were employed for this purpose. Cyclic POE was found to be much more miscible with polystyrene when compared to linear POE, resulting in nanometer-sized domains and significantly reduced mobilities of the cyclic POE components in the blends. The unusual behavior of cyclic POE in the blends was attributed to topological as well as end-group effects with the topological effects being predominant. Polyrotaxanes composed of polystyrene and cyclic POE components exhibited cyclic POE domain sizes similar to that of physical blends. Cyclic POE dynamics in polyrotaxanes were considerably hindered, however, due to the threaded architecture. Surface segregation studies of cyclic POE/polystyrene blends and polyrotaxanes did not show segregation of POE to the surface because of the improved miscibility and the topological constraints present in these systems.

The most critical problem of NMR spectroscopy and magnetic resonance imaging (MRI) is the relatively low sensitivity compared to other forms of spectroscopy, limiting the applicability of these techniques. Most of the existing research focused on trying to alleviate these problems through hyperpolarization techniques, strong magnetic fields, low temperature experiments (<25K), and pulse sequence development. Going beyond the previously stated methods, researchers found that the implementation of smaller resonant coils is a more convenient and effective way to alleviate the problem of low sensitivity in NMR spectroscopy and magnetic resonance imaging. In this work, a systematic optimization of the parameters for micro-coil probes and magic-angle coil spinning (MACS) probes was carried out with various common NMR nuclear species such as 1H, 31P, 23Na, 79Br on two NMR spectrometers (200 MHz and 500 MHz). The optimized wire diameter, coil diameter, number of turns, the inner and outer diameters of the capillary, the matching capacitors etc have been obtained and demonstrated with a real biological system. In addition, we found that the conventional placement of the sample within the glass tube wrapped by a coil yields a lower signal and sensitivity when compared to coating the sample onto the coil prior to wrapping around the capillary. A new method of performing MACS experiment with micro-coil technology, therefore, was subsequently proposed, namely, micro surface coil magic angle spinning (MISCMAS). The optimized experimental conditions were then determined with both liquid and solid state samples.

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The 5’ untranslated region (UTR) of coronaviral genomes contains cis-acting sequences necessary for replication, transcription and translation. A consensus secondary structural model of the 5' 140 nucleotides of the 5' UTRs of nine coronaviruses (CoVs) derived from all three major CoV groups is presented and characterized by three major stem loops, SL1, SL2 and SL4. SL2 is conserved in all CoVs, typically containing a pentaloop (C47-U48-U49-G50-U51 in MHV) stacked on a 5-bp stem, with some sequences containing an additional U 3' to U51. NMR structural studies of SL2 hairpin reveal that SL2 adopts a U-turn-like conformation. Parallel molecular genetic experiments reveal that SL2 plays an essential role in sgRNA synthesis as does SL1. We observe strong genetic selection against viruses that contain a deletion of A35, an extrahelical nucleotide that destabilizes SL1, in favor of genomes that contain a diverse panel of destabilizing second-site mutations, due to introduction of a collection of non-canonical base pairs near the deleted A35. Viruses containing destabilizing SL1-∆A35 mutations also contain one of two specific single nucleotide mutations in the 3' UTR. Thermal denaturation and imino proton solvent exchange experiments reveal that the lower half of SL1 is unstable and that second-site SL1-∆A35 substitutions recover one or more features of the wild-type SL1. We propose a "dynamic SL1" model that supports viral replication; these characteristics of SL1 appear to be conserved in other coronaviral genomes. The coronaviral nucleocapsid (N) protein contains two or more RNA binding domains. We investigated the RNA-binding properties of the N-terminal (NTD) and Cterminal (CTD) domain of MHV N. Our results reveal that the NTD specifically interacts with the TRS-L3 sequence. The role of conserved residues (Y127, Y129 and R110) for this specific interaction were systematically investigated. In contrast to the NTD, the MHV CTD is homodimeric in solution and binds single-strand RNA nonspecifically in a binding mode of the noncooperative large ligand lattice model. The CTD dimer binds with a site size, n=4 nucleotide and the appending of the NTD enhances the single-strand nucleic acid binding affinity.

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Carbonate reservoirs are a network of pores and connecting pore-throats that contain at least half of the world's oil. Genetic classification of carbonate pores enables one to map the pore types that have greatest influence on reservoir performance. Though NMR logging has been used to estimate pore sizes, it has not been used to identify genetic pore types or to aid in determinations of reservoir quality for different pore assemblages. Five genetic pore types identified in 40 carbonate and 7 sandstone samples were subjected to NMR measurements. Results reveal close correspondence between NMRderived pore volumes and 2-D pore size and shape gleaned from petrographic image analysis. Comparisons of real and synthetic pore shapes showed that shapes of all pore types in the medium size range of 0.02-0.5mm can be reliably compared with synthetic varieties, but such comparisons were unreliable for vuggy pores smaller than 0.5mm. T2 relaxation times for depositional pores exhibit low amplitude, narrow wavelength responses. Moldic pores produced medium amplitude, asymmetrical wavelength responses, and intercrystalline pores show high amplitude, narrow wavelength responses. NMR-derived pore volumes on pores with ferroan dolomite interiors underestimated pore diameter by up to 3 orders of magnitude. Calculated pore-throat sizes from MICP data correlate strongly with measured permeability. Samples with high, intermediate, or low poroperm values displayed characteristic T2 curves confirming that reservoir quality can be estimated from NMR measurements. Future work is expected to show that NMR logging can estimate reservoir quality at field scale and aid in mapping flow units in compartmentalized reservoirs.