Dissertation - Open Access
Doctor of Philosophy (PhD)
Chemistry and Biochemistry
Bromodomain adjacent to Zn Finger 2A (BAZ2A), Isothermal Titration Colorimeter (ITC), Mutagenesis, PHD finger subtype, Protein expression, Protein purification
Disordered tails of histones are critical information retrieval hub and thus, aberrations in the flow of information through these hubs are associated with a number of pathological consequences in human. Mechanism for retrieval of information from these hubs is achieved by protein-protein interaction, i.e. proteins dock onto histone tails to initiate chromatin signaling. Eukaryotes have a number of small peptide binding domains that have evolved to specifically interact with histone tails, and these domains called histone readers as they read the information encoded on histone tails. Plant homeodomain (hereafter PHD) finger, a binucleated zinc finger, family is one such histone readers. Next-generation sequencing efforts on diagnosed patient’s genomes or cancer tissues show that mutations in PHD finger, particularly a subgroup of PHD fingers, are associated with number of pathological consequences. Therefore, for future understanding of the possible mechanisms for the pathological consequences, as an initial step, detailed characterization of the binding mechanism of the PHD subtype, the PHD_nW_DD, was undertaken here. Starting with human BAZ2A (bromodomain adjacent to zinc-finger 2A), one member of the PHD_nW_DD subtype that is associated with prostate cancer was utilized to probe the effect of mutations on histone tail binding. We experimentally discovered two categories of mutations that disrupt peptide binding: (1) Type-A: positions that are in contact with the peptide and (2) Type-B: positions that are remote from the peptide-binding site (distal site). For my dissertation, I focused on understanding the biochemical basis of the effects of Type-A mutations using recombinant protein chemistry and biophysical chemistry. The peptide-anchoring residue positions of BAZ2A-PHD, interestingly, are enriched in specific type of residues in a subtype specific manner. The energetics revealed that, two non-polar amino acid residues and an Aspartate residue in the treble clef knuckle make significant contributions to the formation of the hBAZ2A-histone peptide complex as mutations at these three positions completely aborted peptide binding. The energetic contributions of the identified positions were further confirmed by mutagenesis in three members of the subtype (UHRF1-PHD, KDM5B-PHD and KAT6A-PHD) that included pairs sharing even less than 40% sequence identity with each other. Despite low sequence similarity, mutations cause similar consequences in histone H3 binding suggesting a strong similarity in the binding mechanism, and thus justifying the subtype classification.
Includes bibliographical references (pages 123-134)
Number of Pages
South Dakota State University
In Copyright - Educational Use Permitted
Boamah, Daniel, "Dissecting the Histone-binding Mechanism of a PHD Finger Subtype" (2017). Theses and Dissertations. 1673.