The works discussed within this dissertation explore two major considerations. The first of which explores key structural conformations within the branch site helix that are critical for ribonucleic acid (RNA) splicing. The branch site helix is formed by pairing U2 snRNA and intron from S. cerevisiae including a phylogenetically conserved pseudouridine (ψ) in U2 snRNA indicates that the branch site adenosine (A) of the intron strand is extruded from the helix and participates in a base triple (Newby and Greenbaum 2002a). Conformational changes experienced by the branch site adenosine(A), which is responsible for the first step in splicing, were observed by altering base triple formation. The function of the base triple was unknown , yet was thought to be a critical component for proper positioning of the branch site A, given its proximity within the branch site helix. 2-aminopurine fluorescence spectroscopy and nuclear magnetic resonance (NMR) were used to establish qualitative and quantitative structural data, respectively, of the branch site A and the branch site helix (Nelson and Greenbaum 2006). These data show that the introduction of an AU → UA mutation to the base triple were consistent with a stacked intrahelical conformation of the branch site A (Nelson and Greenbaum 2006). In order to obtain atomistic details of these solution structures, molecular dynamics simulations were used. A novel and innovative sampling strategy known as orthogonal space random walk (OSRW) (Zheng, Chen, Yang 2008; Zheng, Chen, Yang 2009) was utilized to efficiently calculate the free energy differences between conformational states of interest by using alchemical transitions to flipping uridine or ψ 180º at the phylogenetically conserved position. These data show that uridine significantly prefers it canonical conformation, where ψ may assume either conformation. Base flipping of the branch site A was quantified by using a new algorithm known as multi-time scale technique (MTST) (Lv, Nelson, Yang 2010). Information about this type of phenomenon are of significant importance, given this is common strategy of enzymes and other binding protein to acquired access to chemical relevant nucleic acids. Data obtained in these studies indicated that ψ encourage base flipping through the major groove and is energetically more favored as opposed to uridine. Uridine favors flipping of the branch site A toward the minor groove and energetically less favored. These data identify an energetically favored structural conformation, in the presence of ψ, that is highly probable to interact with other branch site proteins which affect the efficiency of splicing.
In the second half of these works, an evaluation of metal binding in iron-dependent repressors found in Tuberculosis (TB) and Diphtheria Toxin (DT) was conducted. Interestingly, crystal structures for these repressors have been solved under several divalent metal ionic conditions with similar overall structure and metal ion coordination (Wang and others 1999; White and others 1998; Wisedchaisri, Holmes, Hol 2004). Semavina et al. and Stapleton et al. were able to detect cooperative and non-cooperative behaviors between various divalent metal ions using equilibration dialysis and fluorescence (Semavina, Beckett, Logan 2006; Stapleton and Logan 2010). OSRW was use to calculate the free energy difference upon divalent metal binding in various site in the repressors in order to understand cooperatively and the order of metal binding. These data aid in corroboration of data established in experimental approaches. The repressor found in TB has two possible pathways to bind nickel. The second of which seems to be the most favorable.