Date of Award
Master of Science (MS)
College of Science and Mathematics
Chemistry and Biochemistry
Thesis Sponsor/Dissertation Chair/Project Chair
Carlos A. Molina
A relatively new development in the field of research-based chemistry is to study biomolecules, their interactions, and a biochemical mechanisms by examination of their physical properties and application of laboratory techniques rooted in concepts of physical chemistry. Both projects that are encompassed within this master's thesis indeed fall under the umbrella of biophysical chemistry, as they apply physical chemical techniques study particular biomolecular interactions.
The first of these two projects is the study of a leucine zipper protein, Inducible cAMP Early Repressor [ICER], which is a product of the cAMP Responsive Element Modulator [CREM] gene. ICER functions as a transcriptional repressor by binding to cAMP Responsive Elements [CRE's] found in the promotor sequences of genes involved in cellular growth, and is abnormally expressed in certain forms of cancer in which ICER acts as a tumor repressor. It also binds to the four CRE sites on its own promotor, known as CARE-1 through CARE-4, thereby regulating its own expression. This research is based on the hypothesis that ICER may in fact autoregulate its own expression by cooperative binding to its own promotor.
A technique known as Fluorescence Resonance Energy Transfer [FRET] is used to test this hypothesis and determine dissociation constants of purified ICER with double stranded DNA. Titrations were performed with purified ICER and double stranded DNA labeled with a fluorophore-quencher pair and containing one or more of the CARE sites. Observed dissociation constants were largely inconsistent and traced back to difficulties in producing purified ICER on a regular basis. Therefore, the focus shifted to reproducible purification of ICER. It includes a comparison of three purification protocols, one of which is a urea-based denaturing purification, another being a native purification, and the third a combination of the first two. Qualitative data that will illustrate this comparison includes side-by-side SDS-PAGE gel electrophoresis analysis of samples from each step of both purification procedures, among other things.
The second project that makes up this thesis involves an enzyme that repairs DNA. The enzyme E. coli photolyase utilizes a light-driven electron transfer mechanism for repairing DNA damaged by UV-light exposure. The enzyme may be activated by a proton-coupled electron transfer [PCET] mechanism. PCET mechanisms are of considerable interest due to their prevalence in many physiological processes such as enzyme catalysis, as they provide an alternative reaction pathway that circumvents traditional high-energy transition states. In this case, during the electron transfer mechanism, the neutral radical form of the flavin adenine dinucleotide cofactor [FADH-] is reduced to FADH-, and an amino acid radical intermediate is formed. This amino acid, 306Trp, is of particular significance due to its role in the mechanism.
Using voltammetry, it is possible to measure the reduction potential of the Trp in solution, providing insight into the mechanism involving 306Trp. The 306Trp reduction potential represents the charge recombination energy required for oxidation of that amino acid and formation of FADH-. As the mechanism of electron transfer involves a proton, its kinetics are heavily pH-dependent. Thus data was acquired over a wide range of pH values in order to quantify this relationship. Furthermore, this reaction was studied in distilled water and in D2O in order to examine solvent effects on the reduction potential of Trp. An observed "inverse" kinetic isotope is investigated in detail to explain the observed increase in reaction rate in D2O counterintuitive to the mass-related kinetic isotope effect.
The data from this thesis confirm the hypothesis that D2O significantly affects the reduction potential and the pKa of Trp, the combined effects of which explain the observed "inverse" isotope effect in which the reaction occurs more quickly in D2O. All experiments were repeated with a tryptophan-like molecule, N-acetyl-Ltryptophanamide, the properties of which more accurately represent how the 306Trp residue would act in the electron-transfer mechanism as a member of a peptide chain. N-acetyl-L-tryptophanamide, unlike natural amino acids, contains no Cterminus and two amine groups. Therefore, issues of charge formation from amino acid ionization that limited the pH range for the tryptophan experiments were alleviated, and the use of N-acetyl-L-tryptophanamide allowed for greater experimental freedom and lent itself to a wider pH/pD range to be investigated.
DeMilio, Mark, "Investigation of Autoregulation of ICER and Electrochemical Properties of Tryptophan" (2012). Theses, Dissertations and Culminating Projects. 815.