Thesis advisor: Udayan Mohanty / In chapter one, two simple models are used to estimate the electrostatic contributions to the stiffness of short DNA fragments. The first model views DNA as two strands that are appropriately parameterized and are wrapped helically around a straight cylinder radius equal to the radius of the DNA molecule. The potential energy of the DNA due to phosphate-phosphate electrostatic interactions is evaluated assuming that the charges interact through Debye-Hückle potentials. This potential energy is compared with the potential energy as computed using our second model in which DNA is viewed as two helical strands wrapping around a curved tube whose cross-section is a disk of radius equal to the radius of the DNA. The results are compared with counterion condensation models and experimental data (Guo et al. J. Phys. Chem. B, 2008, 112, 16163-16169). In chapter two, the fidelity of translation selection begins with the base pairing of codon-anticodon complex between the mRNA and tRNAs. Binding of cognate and near-cognate tRNAs induces 30S subunit of the ribosome to wrap around the ternary complex, EF-Tu(GTP)aa-tRNA. We have proposed that large thermal fluctuations play a crucial role in the selection process. The binding energies of over a dozen unique site-bound magnesium structural motifs are investigated and provide insights into the nature of interaction of divalent metal ions with the ribosome (Guo et al. Proc. Nat. Acad. Sci. 2011, 108, 3947-3951). In chapter three, we use extensive molecular dynamics simulations to study a series of stapled alpha helical peptides over a range of temperatures in solution. The peptides are found to exhibit substantial variations in predicted helicities that are in good agreement with the experimental value. In addition, we find significant variation in local structural flexibility of the peptides with the position of the linker, which appears to be more closely related to the observed differences in activity than the absolute alpha helical stability (Guo et al. Chem. Biol. Drug. Des. 2010, 75, 348-359.). In chapter four, the alpha helical conformation and structural stability of single and double stapled all-hydrocarbon cross-linked p53 peptides in solution and when bound to MDM2 is investigated. We determined the effects of the peptide sequence, the stereochemistry of the cross-linker, the conformation of the double bond in the alkene bridge, the length of the bridge, on the relative stability of the alpha helix structure. The conformation population distribution indicates a fully helical state and several partially folded states. The distribution of dihedral pairs of the stapled peptides in the bound state indicates a significant population around the alpha helical region. Sequences over which the linker spans tend to have the highest helical occupancy. Significant helical content is observed for a double stapled p53 peptide at 575 K. The probability to form native contacts is increased when the stapled peptides are bound to MDM2. The distribution of the end-to-end distance of the peptides is bimodal. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Most cellular magnesium is bound, yet it is the concentration of free magnesium, [Mg²⁺]_free, in red blood cells that is vital in the regulation of enzyme activity and ion transport. It is unknown how changes in total blood magnesium affect the [Mg²⁺]_free within red blood cells or in tissue, because the presence of other cations, especially H⁺ and potassium, K⁺ , affects the degree to which Mg²⁺ is bound. Consequently, this Thesis presents a new ³¹P NMR spectroscopic method to measure [Mg²⁺]_free in blood, which analyses the changes in the phosphorus chemical shifts of ATP and 2,3-DPG using theoretical equations expressing the observed chemical shift as a function of pH, K⁺ and [Mg²⁺]_free, over the pH range of 5.75 to 8.5 and [Mg²⁺]_free range 0 to 5 mM. The equations were adjusted for the binding of haemoglobin to ATP and DPG, which required knowledge of the intracellular concentrations of ATP, DPG, K⁺ and Hb. These equations enabled, for the first time, the simultaneous analyses of the chemical shifts of 3P-DPG and β-ATP to measure both intracellular 04- pH and [Mg²⁺]_free in normal and sickle blood. To simulate in vivo 100% oxygenated blood, samples were prepared for analysis by equilibration with a mixture of O₂ and CO₂, adjusted to give a pCO₂ of 40 mmHg and pO₂ > 150 mmHg. Under these conditions, normal whole blood had an intracellular pH of 7.20 ± 0.02 and a [Mg²⁺]_free of 0.41 ± 0.03 mM (n = 33). Further work determined blood pH and [Mg²⁺]_free for several clinical conditions including sickle cell anaemia, pre-eclampsia, hypoxia, patients with sub-arachnoid haemorrhage and chronic fatigue syndrome. This Thesis has demonstrated the potential of this new technique to evaluate the importance of [Mg²⁺]_free in the regulation of metabolite concentration and metabolic function, and to elucidate some of the properties of magnesium transport across the erythrocyte cell membrane.

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Le ribozyme VS de Neurospora catalyse des réactions de clivage et de ligation d’un lien phosphodiester spécifique essentielles à son cycle de réplication. Il est formé de six régions hélicales (I à VI), qui se divisent en deux domaines, soit le substrat (SLI) et le domaine catalytique (tiges II à VI). Ce dernier comprend deux jonctions à trois voies qui permettent de reconnaître le substrat en tige-boucle de façon spécifique. Ce mode de reconnaissance unique pourrait être exploité pour cibler des ARN repliés pour diverses applications. Bien que le ribozyme VS ait été caractérisé biochimiquement de façon exhaustive, aucune structure à haute résolution du ribozyme complet n’a encore été publiée, ce qui limite la compréhension des mécanismes inhérents à son fonctionnement. Précédemment, une approche de divide-and-conquer a été initiée afin d’étudier la structure des sous-domaines importants du ribozyme VS par spectroscopie de résonance magnétique nucléaire (RMN) mais doit être complétée. Dans le cadre de cette thèse, les structures de la boucle A730 et des jonctions III-IV-V et II-III-VI ont été déterminées par spectroscopie RMN hétéronucléaire. De plus, une approche de spectroscopie RMN a été développée pour la localisation des ions divalents, tandis que diverses approches de marquage isotopique ont été implémentées pour l’étude d’ARN de plus grandes tailles. Les structures RMN de la boucle A730 et des deux jonctions à trois voies révèlent que ces sous-domaines sont bien définis, qu’ils sont formés de plusieurs éléments structuraux récurrents (U-turn, S-turn, triplets de bases et empilement coaxial) et qu’ils contiennent plusieurs sites de liaison de métaux. En outre, un modèle du site actif du ribozyme VS a été construit sur la base des similarités identifiées entre les sites actifs des ribozymes VS et hairpin. Dans l’ensemble, ces études contribuent de façon significative à la compréhension de l’architecture globale du ribozyme VS. De plus, elles permettront de construire un modèle à haute résolution du ribozyme VS tout en favorisant de futures études d’ingénierie. / The Neurospora VS ribozyme catalyzes the cleavage and the ligation of a specific phosphodiester bond, which is essential for its replication cycle. It is formed of six helical regions (I to VI) that are divided in two domains: the substrate (SLI) and the catalytic domain (stems II-VI). The latter contains two three-way junctions that allow recognition of the stem-loop substrate in a specific manner. This unique mode of substrate recognition could be exploited to target folded RNAs for diverse applications. Even though the VS ribozyme has been extensively characterized biochemically, no high-resolution structure of the complete ribozyme has been published yet and this limits our mechanistic understanding. A divide-and-conquer approach was previously initiated to study the structure of the important subdomains of the VS ribozyme by nuclear magnetic resonance (NMR), but this approach needs to be completed. In this thesis, the structures of the A730 loop, the III-IV-V junction and the II-III-VI junction were determined by heteronuclear NMR spectroscopy. Moreover, a unique NMR approach was developed for localizing divalent metal ions, whereas several isotope-labeling strategies were implemented to facilitate the study or large RNA molecules. The NMR structures of the A730 loop and the two three-way junctions reveal that these subdomains are well defined, that they are formed by several recurrent structural elements (U-turn and S-turn motifs, base triples and coaxial stacking) and that they contain several metal-binding sites. Interestingly, structural similarities were identified between the VS and hairpin ribozymes, which allowed the modeling of the VS ribozyme active site. In summary, these studies significantly contribute to a better understanding of the global architecture of the VS ribozyme. In addition, they will allow the construction of a high-resolution model of the complete VS ribozyme and facilitate future engineering studies.