| dc.description.abstract | Biological macromolecules can bind a wide range of ligands with high affinity and high specificity. In particular, proteins that bind DNA must recognize a particular DNA sequence within a vast array of non-target sites. In many cases, protein-DNA interactions must also be regulated by the cell and its environment. Hox proteins are transcription factors that interact with DNA via the homeodomain with extraordinarily high affinity (pM). Hox homeodomains are highly conserved and bind similar DNA sequences; therefore, it is unclear how different Hox proteins recognize different genes in vivo. Elucidating the molecular basis for high affinity binding and the mechanisms that enable regulation will allow us to understand how Hox proteins normally function in development and wound repair and how malfunction of Hox proteins leads to developmental malformations and cancer. In addition, this information can be exploited to develop novel therapies and biotechnologies.
This thesis explores the DNA binding interactions by the Drosophila melanogaster Hox transcription factor Ultrabithorax (Ubx). First, most transcription factors contain large intrinsically disordered domains, making these proteins prone to proteolysis and complicating protein purification. Furthermore in Hox proteins, proteolytic products out-compete full-length protein for binding to DNA. We developed an innovative protein purification technique to generate Ubx of sufficient quality and purity for sensitive DNA binding assays. Second, it was previously known that nonhomeodomain regions influence DNA-binding. However the specific amino acids that comprise this intraprotein interface have not been identified. We mapped specific amino acids involved in Ubx-DNA interactions that regulate high affinity binding, providing the first model of a full-length Hox protein structure. Third, materials composed of DNA are easily designed but lack the diverse structural and chemical properties thus limiting the range of structures and activities that can be achieved. Conversely, proteins can form materials with diverse structural and chemical properties, but are difficult to design. Incorporating both DNA and protein into composite materials can maximize the advantages of both types of molecules. We demonstrate that materials composed of Ultrabithorax (Ubx) retain the ability to bind DNA noncovalently in a sequence specific manner, which allows for the optimization and generation of novel composite biomaterials. Taken together, the knowledge reported in this thesis explains many aspects of Hox regulation and provides the basis for the development of novel composite materials. | |
