No description available.

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 5 uppsatser.

Conventional nuclear magnetic resonance (NMR) spectrometers—the electronic brain that orchestrates and monitors nuclear spin motions—are bulky, expensive, thus, not scalable. In this thesis, we report on scalable 4-mm2 silicon spectrometer chips that perform a broad range of two-dimensional NMR spectroscopy—e.g., correlation spectroscopy, J-resolved spectroscopy, and heteronuclear quantum coherence spectroscopy—as well as one-dimensional spectroscopy and relaxometry. In this way, they examine a wealth of nuclear spin behaviors and interactions in biological, organic, and pharmaceutical compound molecules, elucidating their structures and dynamics. This semiconductor-based NMR spectroscopy opens up new exciting vistas with two prime advantages. First, with size/cost economy and scalability, the spectrometer chips can be parallelized sharing the same bore of a magnet—whether a large superconducting or small permanent magnet—to greatly simplify multi-channel spectroscopy and vastly increase the spectroscopy throughput, overcoming the intrinsic slowness of NMR spectroscopy; such parallelism may enable the much-desired high-throughput NMR paradigm for drug discovery, metabolomics/metabonomics, and structural biology. We demonstrate the concept of this parallelism by 2-channel heteronuclear quantum coherence NMR experiments, where 2 chips run synchronously in an ultra-compact configuration. Second, the chip spectrometers can complement the recent advance in magnet miniaturization to realize bona fide portable NMR spectroscopy systems. To demonstrate this miniaturization benefit (in addition to the orthogonal benefit of parallelism), we perform all our spectroscopy experiments in a platform combining the spectrometer chips with a compact permanent NdFeB magnet. These demonstrations suggest new dimensions to the technology and applications of NMR spectroscopy enabled by the integrated spectrometers. / Engineering and Applied Sciences

The main focus of this thesis is on the ring conformations of carbohydrate molecules; how the conformational equilibria and the rates of the associated interconversions are affected by the molecular constitution and their surroundings. The conformational equilibria of a group of amine linked pseudodisaccharides, designed as potential glycosidase inhibitors, comprising α-D-altrosides are described in Chapter 3. The OS2 conformation was largely populated, and the ring conformation was found to depend on the charge of the amine functionality. The conformations of β-D-xylopyranoside derivatives with naphthyl-based aglycones, which are potential anti-cancer agents, are described in chapter 4. Solvent dependent flexibility was observed. Intramolecular hydrogen bonds were concluded to be involved in the stabilization of 1C4 conformers in non-hydrogen bonding solvents of low polarity. Chapter 5 describes the first measurements of the conformational exchange rates of mannuronic acid ester derivatives between the 4C1 and 1C4 conformations, through DNMR measurements. The relative reactivity of glycosyl triflates as electrophiles in glycosylation reactions were investigated with NMR-based competition experiments. In Chapter 6, investigations of ruthenium-catalyzed epimerizations of the allylic alcohols of glycal derivatives, and stereoselective synthesis of esters through a DYKAT protocol, are described. The kinetics of the epimerizations were elaborated through different NMR-spectroscopic methods. Chapter 7 describes additions of NMR chemical shift data of mono- and oligosaccharides to database of the computer program CASPER, and applications thereof. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Submitted. Paper 5: Manuscript.</p>

Myosins are molecular motor proteins involved in cell movement, vesicle and organelle transport by moving along the cytoskeletal actin filaments. They include a myosin heavy chain and at least one myosin light chain (LC). The latter are typically bilobal proteins like calmodulin, where each lobe comprises a pair of EF-hand Ca2+-binding motifs. The LCs bind to ~25-residue IQ motifs that loosely conform to an IQXXXRGXXXR consensus sequence, and impart rigidity that is crucial for myosin function. The highly motile amoeba Dictyostelium discoideum expresses seven class I myosins, two of which (MyoD and MyoB) recruit the specific LCs MlcD and MlcB, with MlcB being the first observed single-lobe LC. However, the LCs for the remaining D. discoideum class I myosins are unknown. Identifying and characterizing these LCs is one focus of this thesis, with an overall goal of understanding their role in myosin function and regulation. Nuclear magnetic resonance spectroscopy, site-directed mutagenesis, and computational modeling were used to determine the solution structure of apo-MlcB and identify the MyoB IQ motif-binding site. Apo-MlcB differs from the typical closed conformation of an EF-hand Ca2+-binding protein in the apo-state as helix 1 in its structure is splayed from the remaining helices. The MyoB IQ motif-binding surface is not altered by Ca2+, involves residues from helices 1 and 4, and from residues in the N-terminal canonical EF-hand Ca2+-binding loop, and represents a unique mode of IQ recognition by a myosin LC. Calmodulin was identified as the LC for MyoA and MyoE while another single-lobe LC, MlcC, bound to two of three IQ motifs in MyoC. The solution structure of MlcC was more similar to the C-terminal lobe of apo-calmodulin than to apo-MlcB. Chemical shift perturbation studies suggest that like apo-CaM, MlcC undergoes a global MyoC IQ motif-induced conformational change. Computational modeling of the MlcC-MyoC IQ complex indicates that this is a feasible mode of IQ recognition. The structures of MlcB and MlcC, with their different modes of IQ motif binding, provide novel insights into IQ motif binding specificity and begin to illustrate their role in myosin function and regulation. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-01-29 11:42:03.428

Aconitum and Delphinium genera are important rich sources of toxic C19-diterpenoid alkaloids. The alkaloidal content of A. napellus and D. elatum seeds have been investigated in detail. After maceration, crude alkaloidal extracts were obtained and then purified by repeated column chromatography (over silica and alumina gels) to sample homogeneity yielding five known C19-diterpenoid alkaloids from A. napellus, aconitine, neoline, 14-O-acetyltalatisamine, 14-O-benzoylaconine, and taurenine, and two others from D. elatum, delpheline (also including its X-ray single crystal analysis) and methyllycaconitine (MLA). These examples showed that mass spectrometry hyphenated with HPLC or other chromatography can be used as a tool for rapid alkaloid content screening of different extracts. NMR spectroscopic (using a variety of techniques and nuclei) data are presented in support of the first report of iminodelsemine A/B as an imino-alkaloid artefact from D. elatum. A detailed chromatographic study across different pH ranges, and over different solid supports, of aconitine and its main degradation product, 14-O-benzoyl-8-O-methylaconine, together with its semi-synthesis and that of its deuterated analogue are reported within studies to minimize artefact formation during the storage or extraction of A. napellus norditerpenoid alkaloids. Likewise, from D. elatum seeds, as a model source of Delphinium alkaloids, we compared the alkaloid yield using different extraction techniques and conventional chromatographic separations. The structures were confirmed by NMR spectroscopy and mass spectrometry. An NMR spectroscopic approach for the pKa determination of some C19-diterpenoid alkaloids has been developed. A modified calculation method for fatty acid composition quantification has also been developed using 1H-NMR spectroscopic methods.

The conformational preferences of several oligosaccharides are investigated herein using a combination of NMR spectroscopy and molecular dynamics (MD) simulations, focusing on the torsion angles associated with the glycosidic linkages. Strategies for obtaining usable J-HMBC spectra for carbons with an adjacent 13C label are described. By employing a selective pulse or a constant time modification, spectra free from interferences are obtained for site-specifically 13C labeled oligosaccharides. Intermolecular hydrogen bonding in sucrose is investigated using MD simulations performed at different concentrations. One of the most frequent intermolecular hydrogen bonds in the simulations, O3f∙∙∙HO3g, was detected using the HSQC-TOCSY NMR experiment. Based on MD simulations and NMR spectroscopy, the conformational ensemble for a trisaccharide segment of the LeaLex hexasaccharide is proposed to feature conformational exchange between conformations with positive and negative values for the ψ3 torsion angle in the β-D-GlcpNAc-(1→3)-β-D-Galp linkage. Using MD simulations, the conformation of the N-acetyl group is shown to influence the glycosidic conformation at a nearby linkage in two oligosaccharides. Short (1→6)-linked oligosaccharides are shown to exhibit conformational exchange at the ω and ψ torsion angles. Notably, the former torsion angle populates states with ψ ≈ ±90°. Conformationally sensitive homo- and heteronuclear coupling constants are determined using various NMR experiments. The experimental data, including effective distances from NOESY obtained for two of the compounds, is used to improve the representation of the ω torsion angle in the CHARMM36 force field. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 5: Accepted. Paper 6: Manuscript.</p>

In this thesis, we document a comprehensive study of the cation-directed self-assembly of three guanosine derivatives: i) guanosine 5'-monophosphate (5'-GMP), ii) guanosine 5'-thiomonophosphate (5'-GSMP), and iii) 2',3',5'-O-triacetylguanosine (TAG). We discovered that, under the neutral pH condition, Na2(5'-GMP) molecules self-assemble into a right-handed helix structure consisting of alternating all-C2'endo and all-C3'endo planar G-quartets stacking on top of each other with a 30° twist. This self-assembled supramolecular structure uses multiple non-covalent forces (e.g., hydrogen-bond, phosphate-hydroxyl, pi-pi (base-base) stacking, ion-carbonyl, and ion-phosphate) to align individual monomers in a way that resembles RNA and DNA sequences in which covalent bonds are used to link monomers. Na+ ions are located in the channel and surface sites of the G-quadruplex. In contrast, under acidic pH conditions, Na2(5'-GMP) molecules self-assemble into a continuous right-handed helix where guanine bases are hydrogen-bonded in a lock-washer fashion with only C3'-endo monomers. Na+ ions are absent in the channel site due to smaller channel radius and lesser repulsions between phosphate groups (-1 vs. -2 charge under neutral pH) contribute to the stronger stacking mechanism. In Na2(5'-GSMP), a longer phosphate bond compared with Na2(5'-GMP) allows stronger P-O-…Na+…-O-P interactions to occur, thus enhancing self-assembly. Solid-state NMR, FT-IR, powder x-ray diffraction, model building, and calculation showed that Na2(5'-GSMP) forms the same self-assembled structure as Na2(5'-GMP) but with significantly greater tendency. This study proves that single-bond modification can enhance stacking in G self-assemblies, and shows direct evidence that Na+ ions reside at the surface (phosphate) sites. Lastly, using lipophilic TAG, we were able to show for the first time that trivalent lanthanide metal ions can facilitate G-quartet formation. A new mode of metal ion binding in G-quartet structures (i.e., a triple-decker G dodecamer containing a single metal ion in the central G-quartet) is reported. We also report the first 1H and 43Ca NMR characterization of Ca-templated G-quartet formation in a [TAG8-Ca]2+ octamer. / Thesis (Ph.D, Chemistry) -- Queen's University, 2012-06-27 16:53:43.359

No description available.

Solid-state Nuclear Magnetic Resonance (NMR)is one of the premiere biophysical methods that can be applied for addressing the structure and dynamics of biomolecules, including proteins, lipids, and nucleic acids. It illustrates the general problem of determining the average biomolecular structure, including the motional mean-square amplitudes and rates of the fluctuations. Lineshape and relaxtion studies give us a view into the molecular properties under different environments. To help the understanding of NMR theory, both lineshape and relaxation experiments are conducted with hexamethylbezene (HMB). This chemical compound with a simple structure serves as a perfect test molecule. Because of its highly symmetric structure, its motions are not very difficult to understand. The results for HMB set benchmarks for other more complicated systems like membrane proteins. After accumulating a large data set on HMB, we also proceed to develop a completely new method of data analysis, which yields the spectral densities in a body-fixed frame revealing internal motions of the system. Among the possible applications of solid-state NMR spectroscopy, we study the light activation mechanism of visual rhodopsin in lipid membranes. As a prototype of G-protein-coupled receptors, which are a large class of membrane proteins, the cofactor isomerization is triggered by photon absorption, and the local structural change is then propagated to a large-scale conformational change of the protein. Facilitation of the binding of transducin then passes along the visual signal to downstream effector proteins like transducin. To study this process, we introduce 2H labels into the rhodopsin chromophore retinal and the C-terminal peptide of transducin to probe the local structure and dynamics of these two hotspots of the rhodopsin activation process. In addition to the examination of local sites with solid-state 2H NMR spectroscopy, wide angle X-ray scattering (WAXS) provides us the chance of looking at the overall conformational changes through difference scattering profiles. Although the resolution of this method is not as high as NMR spectroscopy, which gives information on atomic scale, the early activation probing is possible because of the short duration of the optical pump and X-ray probe lasers. We can thus visualize the energy dissipation process by observing and comparing the difference scattering profiles at different times after the light activation moments.