Doctor of Philosophy, The Ohio State University, 2018, Biochemistry Program, Ohio State

Histone proteins are extremely important biological targets that are heavily post-translationally modified to control DNA related processes. To study these proteins in vitro, we have developed a number of chemical techniques that allow for the synthesis of selectively modified histones. These proteins can be refolded into nucleosomes and the stability, dynamics, and structure of the nucleosome can be probed using biochemical and biophysical techniques. Here we present an optimized peptide-synthesis strategy for the generation of peptide thioesters, the biophysical characterization of two modified histones, and explore the use of histones as potent cell-penetrating peptides as a new method to study histone cytoplasmic maturation. In order to generate the reactive peptides to generate fully-synthetic histone proteins, we utilize a masked thioester. We discovered that 3,4-diaminobenzoic acid, our standard thioester precursor, would accumulate number side-products where peptide chain extension occurred on both amines when Gly-rich peptides were synthesized. To remedy this, we developed an alternative protection scheme that allowed for the selective protection and deprotection of both amines using Fmoc and alloxycarbonyl (Alloc) protecting groups. This allowed for the synthesis of peptides containing multiple glycines in a row and a 44-amino acid peptide in high yield. This has since been used for the total synthesis of 4 different core histone proteins ranging in size from 101-214 amino acids in size. While many post-translational modifications of histones exist on the unstructured histone tails, modifications to the folded histone core are also important for control of nucleosome dynamics and stability. Here we present data showing that H4-K91ac and H3-T118ph destabilize nucleosomes, but in different manners. H4-K91ac is a modification that occurs between the tetramer and dimer subunits and weakens dimer interactions in nucleosomes. We find that this modificatio (open full item for complete abstract)

Committee: Swenson Richard Dr. (Committee Member); Freitas Michael Dr. (Committee Member); Parthun Mark Dr. (Committee Member); Ottesen Jennifer Dr. (Advisor) Subjects: Biochemistry; Biology; Chemistry

Master of Science, Miami University, 2018, Cell, Molecular and Structural Biology (CMSB)

Enzymes, while incredibly complex, are limited in functionality to their twenty canonical amino acids. To shortcut this limitation, nature has evolved to utilize post translational modification (PTM) of protein structure to regulate cellular functions. Whether this be as essential cofactors or acting as regulators of enzyme activity, controlling cellular processes by PTMs are absolutely essential to the molecular-level events that coordinate and sustain life. Within a massive population of possible binding sites, enzymes responsible for PTMs manage to find their precise target through highly selective recognition sites. LplA has been shown to be a convenient tool for attaching small molecule precursors to its recognition site. The aim of this study is to modify the enzyme Lipoic acid ligase A (LplA) to effectively carve a tunnel through the backside of the enzyme. By carving a tunnel through the ligase, this work aims to broaden the substrate compatibility range of LplA for post translational modification of target proteins. Additionally in this study, crystal structures of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) will be solved and analyzed for evidence of S-nitrosylation. It is the goal of this study to gain a better understanding of how this PTM regulates heme binding to GAPDH.

Committee: Richard Page (Advisor); Michael Robinson (Committee Chair); Yoshi Tomoyasu (Committee Member) Subjects: Biochemistry