Folding Cooperativity and Its Influence on pKa Shifting in Nucleic Acids

Open Access
- Author:
- Siegfried, Nathan A.
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 17, 2009
- Committee Members:
- Philip C. Bevilacqua, Dissertation Advisor/Co-Advisor
Philip C. Bevilacqua, Committee Chair/Co-Chair
Squire J Booker, Committee Member
Scott A Showalter, Committee Member
Katsuhiko Murakami, Committee Member - Keywords:
- pKa shift
RNA
DNA
nucleic acids
cooperativity
acid base catalysis - Abstract:
- The ability of RNA to both store genetic information and catalyze chemical reactions has led to the RNA world hypothesis, wherein RNA served as the primary biological molecule. The functional versatility that would, in theory, be required of such a molecule represents a significant challenge to it and to our understanding of the origins of life. In comparison to the variety of chemical side chains available to proteins, RNA (which bears only four, chemically similar side chains) seems to be woefully inadequate, so much so that the discovery of catalytic RNAs in 1982 came as a surprise to many and created a whole new field of study. Since then, a steady stream of RNA-related discoveries have had profound impact on the fields of chemistry, biochemistry, genetics, and molecular biology. Every new discovery furthers our understanding of RNA as a primary biomolecule and expands our understanding of both modern and ancient biology. One particularly powerful catalytic strategy utilized by protein enzymes is general acid-base catalysis. Proteins can utilize amino acid side chains like histidine and lysine which have pKa values at or above physiological pH (~7), to attain efficient proton transfer or electrostatic catalysis, respectively. Nucleic acid bases, on the other hand, have no ionizing groups with pKas in this range and, to make matters worse, routinely experience pKa shifts further from this ideal range upon standard helix formation because the standard protonation state is required for Watson-Crick base pairing. On the other hand, helix formation of non-Watson Crick pairs that require non-standard protonation states causes a pKa shift towards neutrality (or beyond). We hypothesized that coupling many interactions to a protonation event through cooperative structure formation could shift nucleobases pKa values even further. Towards this end, I designed model systems to study folding cooperativity in RNA and DNA hairpins as a function of helical context. Significant cooperativity was observed at the terminus of a helix; in fact, it is demonstrated that a terminal base pair will not form in the absence of a penultimate base pair in both RNA and DNA hairpins. I also note that the thermodynamic impact of mutations at the terminus are small, particularly when compared to the same mutations performed on bases in the middle of the helix. It is at this point where RNA and DNA trends differ: DNA does not exhibit strong cooperativity in the middle of a helix, while RNA does. This difference is attributed to differences in helical structure and flexibility. Experiments were then performed to determine the molecular basis for an unexpectedly large structural disruption observed in the study mentioned above. It was demonstrated that unsatisfied hydrogen bond acceptors can play a surprising and major role in determining the stability (and therefore specificity) of base pair formation in the RNA bases studied. An example of the impact of these interactions is provided in tRNA folding specificity, wherein removing an unsatisfied hydrogen bond acceptor using a base found in the anticodon (2-thiocytosine) increases specificity for canonical Watson Crick base pairing. Having obtained interesting structural information, we turned to pH studies, beginning with a study that details sources of error in experimental pH measurements. This study was instigated by collaboration with Dr. Andrea Cerrone-Szakal on hepatitis delta virus (HDV) ribozyme kinetics in high salt, with an ultimate desire examine pKa shifting under in vivo-like conditions of unusual salt and crowding. A substantial ion- specific effect on pH measurements was found, causing meter readings that are almost 1 unit too low, particularly at high ionic strengths. It was determined that the ionic strength of a solution will affect not only the pKa of the solution species themselves, but also the ability of a pH electrode to produce an accurate reading. A two-step calibration method is presented to correct for this error, and this method is applied to a study of the pKa of C in a single stranded RNA (UUCUU) representing the unfolded state. Lastly, experiments are presented that examine the folded-state pKa of a dsDNA model system. Effects of context, temperature, and ionic strength on the pKa of a non-canonical A+œC wobble pair are presented. This pair requires a protonated A to form, and hence meets the aforementioned criteria for pKa shifting; furthermore, by applying knowledge gained in the cooperativity studies, the effect of cooperativity on pKa shift is studied as well. Conditions under which very large pKa shifts can be observed are identified, and pKa values at and beyond neutrality for this pair are reported, an observation that supports the potential for both general acid-base and electrostatic catalysis in nucleic acids. We present significant pKa shifts in internally located base pairs only, suggesting that the folding free energy, rather than folding cooperativity, is the most important predictor of pKa shifting.