<p>This thesis describes the use of nuclear magnetic resonance (NMR) spectrometry as an analytical tool. The theory of NMR spectroscopy in general and quantitative NMR spectrometry (qNMR) in particular is described and the instrumental properties and parameter setups for qNMR measurements are discussed. Examples of qNMR are presented by impurity determination of pharmaceutical compounds and analysis of urine samples from rats fed with either water or a drug (metabolic profiling). The instrumental parameter setup of qNMR and traditional data pre-treatments are examined. Spectral smoothing by convolution with a triangular function, which is an unusual application in this context, was shown to be successful regarding the sensitivity and robustness of the method in paper II. In addition, papers III and IV comprise the field of peak alignment, especially designed for ¹H-NMR spectra of urine samples. This is an important preprocessing tool when multivariate analysis is to be applied. A novel peak alignment method was developed and compared to the traditional bucketing approach and a conceptually different alignment method.</p><p>Univariate, multivariate, linear and nonlinear data analyses were applied to qNMR data. In papers I–II, calibration models were created to examine the potential of qNMR for these applications. The data analysis in papers III–VI was mainly explorative. The potential of data fusion and data correlation was examined in order to increase the possibilities of analysing the highly complex samples from metabolic profiling (papers V–VI). Data from LC/MS analysis of the same samples were used with the ¹H-NMR data in different ways. Correlation analyses between the¹H-NMR data and the drug metabolites identified from the LC/MS data were also performed. In this process, data fusion proved to be a valuable tool.</p>

<p>This thesis is based on six research publications concerned with (i) study of the molecular structures and dynamics of modified nucleosides; (ii) investigation of the effect of incorporation of modified nucleosides on the structure of DNA; (iii) examination of the effect of the sugar modifications on the pseudo-aromatic properties (p<i>K</i>_a) of the nucleobases; (iv) analysis of the effect of the CH-π interactions on the relative stability of the DNA-RNA hybrid duplexes. The structural stability of the nucleic acids as well as their behavior in molecular recognition is dominated by hydrogen bonding and stacking interactions beside other non-covalent interactions. Naturally occurring nucleosides are found to have some specific functions. Modifications of nucleic acids, followed by studies of the resulting structural, chemical and functional changes, contribute to an understanding of their role in various biochemical processes, such as catalysis or gene silencing. In papers I-III, analysis of the structures of modified thymidine nucleosides with 1′,2′-(oxetane or azetidine) and 2′,4′-(LNA, 2′-amino LNA, ENA, and Aza-ENA) conformationally constrained sugar moieties, and dynamics of the modified nucleosides by NMR, ab initio, and molecular dynamics simulations are discussed. Based on whether the modification leads to 1′,2′- or 2′,4′- constrained sugar moieties, it is found that they fall into two distinct categories characterized by their respective internal dynamics of the glycosidic and backbone torsions as well as by their characteristic <i>NE</i>-type (P = 37° ± 27°, Φ_m = 25° ± 18°) for 1′,2′-constrained nucleosides, and <i>N</i>-type (P = 19° ± 8°, Φ_m = 48° ± 4°) for 2′,4′-constrained systems, respectively. Moreover, each group has different conformational hyperspace accessible. The effect of the incorporation of 1′,2′-oxetane locked thymidine nucleoside on the structure and dynamics of the Dickerson-Drew dodecamer, d(CGCGAATTCGCG)₂, determined by NMR, is discussed in the paper IV. It shows that the incorporation of oxetane locked T into the dodecamer has made local structural deformations and perturbation in base pairing, where the modification is included. The modulations of physico-chemical properties of the nucleobases in nucleotides by the C2′-modification of the sugar (paper V), 5′-phosphate group, and the effect of constrained pentofuranosyl moiety (sugar, paper III) have been studied. CH-π interactions between the methyl group of thymidine and the neighboring aromatic nucleobase are shown to increase the relative stability of the DNA-RNA hybrid duplexes over the isosequential RNA-DNA duplexes or vice versa (paper VI).</p>

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 143-156).

This thesis covers the process from expression of a heterologous gene in Escherichia coli to structure determination of a protein by nuclear magnetic resonance (NMR) spectroscopy. The first part concerns structural genomics-related parallel screening studies on the effect of fusion tags (in particular the His tag) on protein solubility and the use of fusion tags in fast, parallel purification protocols intended for initial biophysical characterization of human proteins produced in E. coli. It was found that for most proteins the His tag has a negative influence on protein solubility. This influence appears to be more pronounced for our C-terminal His tag than for the N-terminal His tags used in this study. Moreover, high ratios of soluble per total protein do not always guarantee a high yield of soluble protein after purification, as different vector - target protein combinations result in large differences in host cell growth rates. Protein purification protocols for different fusion tags were developed that make it possible to express, purify and study structural properties of low concentration samples of 15N-labeled proteins in one or two days. The second part of this thesis describes the assignment and solution structure determination of ribosomal protein L18 of Thermus thermophilus. The protein is a mixed α/β structure with two α-helices on one side of a four-stranded β-sheet. Comparison to RNA-bound L18 showed that the protein to a large extent adopts identical structures in free and bound states, with exception of the loop regions and the flexible N-terminus. Keywords: protein production, protein solubility, fusion tags, nuclear magnetic resonance, structure determination, ribosomal protein

This thesis concerns different areas of biomolecular nuclearmagnetic resonance spectroscopy (NMR). In the first part of thethesis a new formalism for simulations of NMR pulse sequencesis introduced. The formalism is derived both from classicalmechanics and quantum mechanics and is presented forhomonuclear and heteronuclear spin systems. The formalism hasalso been adapted to systems in chemical exchange. Simulationsof pulse sequences should be more straightforward using the newformalism. In the second part of the thesis the NMR solution structuresof two ribosomal proteins are described. The ribosome isresponsible for protein production in all living cells and tounderstand the mechanism of the ribosome it is important toknow the three dimensional structure. In this thesis thestructures of S16 and S19, two of the proteins in the smallribosomal subunit, are presented. S16 is a mixed α /βprotein with a five-stranded parallel-antiparallel β-sheetand two α -helices. S19 is s mixed α/β proteinwith a three-stranded parallel-antiparallel β -sheet, oneα -helix and a short 310-helix. In the third part of the thesis a program for semiautomaticassignment of NMR-spectra is presented. Assigning resonances inthe NMR spectrum is a labor-intensive process, which can takelong time. In semiautomatic assignment a computer program aidsthe user in finding assignments but leaves all decisions to theuser, thus speeding up the process. The program described inthis thesis is a new version of ANSIG, called Ansig forWindows. The program runs on PCs under Windows and has severaltools for semiautomatic assignment. <b>Keywords:</b>nuclear magnetic resonance, structuredetermination, ribosomal proteins, NMR simulations, NMR theory,NMR assignment software, semiautomatic assignment

This thesis describes the use of nuclear magnetic resonance (NMR) spectrometry as an analytical tool. The theory of NMR spectroscopy in general and quantitative NMR spectrometry (qNMR) in particular is described and the instrumental properties and parameter setups for qNMR measurements are discussed. Examples of qNMR are presented by impurity determination of pharmaceutical compounds and analysis of urine samples from rats fed with either water or a drug (metabolic profiling). The instrumental parameter setup of qNMR and traditional data pre-treatments are examined. Spectral smoothing by convolution with a triangular function, which is an unusual application in this context, was shown to be successful regarding the sensitivity and robustness of the method in paper II. In addition, papers III and IV comprise the field of peak alignment, especially designed for 1H-NMR spectra of urine samples. This is an important preprocessing tool when multivariate analysis is to be applied. A novel peak alignment method was developed and compared to the traditional bucketing approach and a conceptually different alignment method. Univariate, multivariate, linear and nonlinear data analyses were applied to qNMR data. In papers I–II, calibration models were created to examine the potential of qNMR for these applications. The data analysis in papers III–VI was mainly explorative. The potential of data fusion and data correlation was examined in order to increase the possibilities of analysing the highly complex samples from metabolic profiling (papers V–VI). Data from LC/MS analysis of the same samples were used with the 1H-NMR data in different ways. Correlation analyses between the 1H-NMR data and the drug metabolites identified from the LC/MS data were also performed. In this process, data fusion proved to be a valuable tool.

This thesis is based on six research publications concerned with (i) study of the molecular structures and dynamics of modified nucleosides; (ii) investigation of the effect of incorporation of modified nucleosides on the structure of DNA; (iii) examination of the effect of the sugar modifications on the pseudo-aromatic properties (pKa) of the nucleobases; (iv) analysis of the effect of the CH-π interactions on the relative stability of the DNA-RNA hybrid duplexes. The structural stability of the nucleic acids as well as their behavior in molecular recognition is dominated by hydrogen bonding and stacking interactions beside other non-covalent interactions. Naturally occurring nucleosides are found to have some specific functions. Modifications of nucleic acids, followed by studies of the resulting structural, chemical and functional changes, contribute to an understanding of their role in various biochemical processes, such as catalysis or gene silencing. In papers I-III, analysis of the structures of modified thymidine nucleosides with 1′,2′-(oxetane or azetidine) and 2′,4′-(LNA, 2′-amino LNA, ENA, and Aza-ENA) conformationally constrained sugar moieties, and dynamics of the modified nucleosides by NMR, ab initio, and molecular dynamics simulations are discussed. Based on whether the modification leads to 1′,2′- or 2′,4′- constrained sugar moieties, it is found that they fall into two distinct categories characterized by their respective internal dynamics of the glycosidic and backbone torsions as well as by their characteristic NE-type (P = 37° ± 27°, Φm = 25° ± 18°) for 1′,2′-constrained nucleosides, and N-type (P = 19° ± 8°, Φm = 48° ± 4°) for 2′,4′-constrained systems, respectively. Moreover, each group has different conformational hyperspace accessible. The effect of the incorporation of 1′,2′-oxetane locked thymidine nucleoside on the structure and dynamics of the Dickerson-Drew dodecamer, d(CGCGAATTCGCG)2, determined by NMR, is discussed in the paper IV. It shows that the incorporation of oxetane locked T into the dodecamer has made local structural deformations and perturbation in base pairing, where the modification is included. The modulations of physico-chemical properties of the nucleobases in nucleotides by the C2′-modification of the sugar (paper V), 5′-phosphate group, and the effect of constrained pentofuranosyl moiety (sugar, paper III) have been studied. CH-π interactions between the methyl group of thymidine and the neighboring aromatic nucleobase are shown to increase the relative stability of the DNA-RNA hybrid duplexes over the isosequential RNA-DNA duplexes or vice versa (paper VI).

Climate is changing and it is certain that this change is due to human activities. Atmospheric greenhouse gases have been rising in an unprecedented way during the last two centuries, although the land biosphere has dampened their increase by absorbing CO2 emitted by anthropogenic activities. However, it is unclear if this will continue in the future. This uncertainty makes it difficult to predict future climate changes and to determine how much greenhouse gas emissions must be reduced to protect climate. To understand the future role of plants in limiting the atmospheric CO2 level, the effect of increasing CO2 on plant photosynthesis and productivity has been studied. However, studies on trees showed contradictory results, which depended on the duration of the experiment. This revealed that an initial strong CO2 fertilization may be a transient response that disappears after a few years. Because climate changes over centuries, we must explore the response of vegetation to increasing CO2 on this time scale. Studying tree rings is a good alternative to impractical decade-long experiments, because trees have experienced the CO2 increase during the last 200 years and may already have responded to it. This thesis shows that the intramolecular distribution of the stable hydrogen isotope deuterium (deuterium isotopomer distribution, DID) of tree rings is a reliable tool to study long-term plant-climate adaptations. The premise for this is that the deuterium abundance in tree rings depends on environmental as well as physiological factors. Using newly developed methodology for DID measurements, the influences of both factors can be separated. Applied to tree rings, separating both factors opens a strategy for simultaneous reconstruction of climate and of physiological responses. The results presented show that DIDs are influenced by kinetic isotope effects of enzymes, allowing studies of metabolic regulation. We show that the abundances of specific D isotopomers in tree-ring cellulose indeed allow identifying environmental and physiological factors. For example, the D2 isotopomer is mostly influenced by environment, its abundance should allow better reconstruction of past temperature. On the other hand, the abundance ratio of two isotopomers (D6R and D6S) depends on atmospheric CO2, and might serve as a measure of the efficiency of photosynthesis (ratio of photorespiration to assimilation). The presence of this dependence in all species tested and in tree-ring cellulose allows studying adaptations of plants to increasing CO2 on long time scales, using tree-ring series or other remnant plant material. / Klimatet förändras och det är numera allmänt vedertaget att detta beror på människans aktiviteter. Halten av växthusgaser har stigit onormalt mycket under de senaste två århundradena och detta beror i största del på människans användning av fossila bränslen. Landbiosfären har hittills haft en buffrande effekt på klimatförändringen eftersom den tar upp och lagrar mycket av växthusgasen CO2. Det är dock osäkert om, och i så fall hur länge, denna effekt kvarstår. Detta gör det mycket svårt att förutsäga framtida klimatförändringar, och därmed hur mycket utsläppen av växthusgaser måste reduceras för att skydda klimatet. För att förstå växternas framtida förmåga att begränsa halten atmosfäriskt CO2 har man studerat effekten av förhöjda halter av CO2 på växters fotosyntes och produktivitet. Resultaten av dessa försök varierar i stor omfattning. Studier på träd odlade under höga halter CO2 indikerar att den initiala ökningen av en trädets produktivitet kan vara en temporär effekt som försvinner redan efter några år. Eftersom klimatförändringen sker under århundraden, måste även växternas anpassningar på förhöjd CO2 halt utforskas på denna tidsskala, men experiment som skulle ta tiotals år är opraktiska att utföra. Trädringar är ett bra sätt att studera sådana anpassningar, eftersom träd redan har upplevt de senaste två hundra årens ökning av koldioxid och dess trädringar därför kan innehålla information om en respons som redan skett. Denna avhandling visar att den intramolekylära fördelningen av den stabila väteisotopen deuterium i trädringar är en tillförlitlig metod för att studera växters anpassningsförmåga till långsiktiga klimatförändringar. Antagandet bakom denna strategi är att isotopfördelningen i trädringar beror på faktorer både från miljön och växtens fysiologi. Om båda faktorerna skulle kunna utvinnas från trädringar, skulle detta öppna en helt ny väg för parallell rekonstruktion av klimatet och växters anpassning till det. Avhandlingen presenterar den första tekniken för att mäta isotopfördelningen av deuterium i växtglukos. Resultaten visar att deuteriumfördelningen hos växtglukos påverkas av enzymers isotopeffekter, vilket möjliggör att regleringen av växternas metabolism kan kartläggas. I avhandlingen bevisas att halten deuterium i skilda intramolekylära positioner (isotopomerer) av glukos från trädringcellulosa bestäms av miljöfaktorer respektive trädets fysiologi. T.ex. påverkas deuteriumhalten i position 2 (D2 isotopomer) av glukosmolekylen huvudsakligen av miljön, vilket kan användas för att förbättra temperaturrekonstruktioner från trädringar. Å andra sidan är kvoten deuterium mellan två andra positioner (D6R och D6S) relaterat till halten atmosfäriskt koldioxid, och kvoten skulle kunna användas som mått för fotosyntesens effektivitet, dvs. förhållandet mellan fotorespiration och fotosyntes. Närvaron av denna relation i trädringar och annat växtmaterial i alla de växter vi hittills studerat, öppnar en helt ny möjlighet att studera växters anpassning till den ökande mängden CO2 i atmosfären under århundraden.

Cancer is the main cause of death in economically developed countries and the second leading cause of death in developing countries. Along with today’s knowledge that more than two hundred different diseases lie in the category of this prognosis there is an urge for more detailed and case-specific treatments to replace the dramatic actions of available radiation- and chemotherapy, which in many cases do not make a difference between healthy and cancer cells. The transcription factor and onco-protein c-Myc has, after being extensively studied during the past decades, become a prognostic marker for almost all cancer forms known. Still, many questions remain regarding how c-Myc interacts with its many different target proteins involved in cell-cycle regulation, proliferation and apoptosis. Current cell biology states that one of the regulating proteins, hPin1, interacts with c-Myc in a phosphorylation-dependent manner which appears to direct the correct timing of c-Myc activation and degradation through the ubiquitin/proteasome-pathway. The critical phosphorylation sites, T58 and S62, are located in the Myc-Box-I (MBI) region, a highly conserved sequence strongly coupled to aggressive tumourigenesis by hotspot mutations. Interestingly, preliminary results in the Sunnerhagen group suggested that MBI alone did not bind hPin1, suggesting hPin1 targeting a site distal from the residues to be phosphorylated. In this thesis, results from Surface Plasmon Resonance (SPR) and Nuclear Magnetic Resonance (NMR) show that the docking WW-domain of hPin1 binds unphosphorylated c-Myc at a region distal from the phosphorylation site, including residues 13-34. Furthermore, SPR experiments revealed that hPin1 binds unphosphorylated c-Myc with apparently greater affinity and with much slower kinetics than phosphorylated c-Myc. Thus, hPin1 recognition and interaction with c-Myc appears not to be dependent on phosphorylation of c-Myc prior binding. The newly identified binding region of c-Myc, located N-terminal of MBI, may further increase the understanding of protein degradation control and c-Myc function. The studies presented in this thesis provide a brick in the puzzle of c-Myc and hPin1 coupled oncogenesis for further development of new therapeutic strategies.

Dissolved organic matter (DOM) is comprised of a complex array of molecular constituents that are linked to many globally-relevant processes and yet this material is still largely molecularly uncharacterized. Research presented here attempted to probe the molecular complexity of this material from both Arctic and temperate climates via multifaceted and novel approaches. DOM collected from remote Arctic watersheds provided evidence to suggest that permafrost-disturbed systems contain more photochemically- and biologically-labile material than undisturbed systems. These results have large implications for predicted increasing temperatures where widespread permafrost melt would significantly impact stores of organic carbon in polar environments. In attempting to address the complexities and reactivity of DOM within global environments, more information at the molecular-level is necessary. Further research sought to unravel the molecularly uncharacterized fraction via use of nuclear magnetic resonance (NMR) spectroscopy in conjunction with hyphenated and varied analytical techniques. Directly hyphenated high performance size exclusion chromatography (HPSEC) with NMR was explored. This hyphenation was found to separate DOM into structurally distinct fractions but proved limited at reducing DOM heterogeneity. Of the many high performance liquid chromatography (HPLC) techniques tested, hydrophilic interaction chromatography (HILIC) was found the most effective at simplifying DOM. HILIC separations utilizing a sample from Florida resulted in fractions with highly resolved NMR signals and substantial reduction in heterogeneity. Further development with a 2D-HILIC/HILIC system to achieve additional fractionation was employed. This method produced fractions of DOM that were homogenous enough to produce excellent resolution and spectral dispersion, permitting 2D and 3D NMR experiments to be performed. Extensive NMR analyses of these fractions demonstrated strong evidence for the presence of highly oxidized sterols. All fractions, however, provided 2D NMR spectra consistent with oxidized polycyclic structures and support emerging data and hypotheses suggesting that cyclic structures, likely derived from terpenoids, are an abundant, refractory and major component of DOM. Research presented within this thesis demonstrates that HILIC and NMR are excellent co-techniques for the analysis of DOM as well as that oxidized sterols and other cyclic components with significant hydroxyl and carboxyl substituents are major constituents in DOM.