Document Type and Release Option
Thesis (open access)
Dr. Amanda Stewart
Methylated DNA binding (MBD) proteins control transcriptional regulation by binding methylated DNA at CpG islands to modulate transcriptional activation and silencing of specific genes such as tumor suppressors. The goal is to explore the structure/function relationship between MBD proteins and methylated DNA recognition. By synthesizing the methylated DNA binding region of the MBD1 protein, binding studies test the peptide’s affinity and specificity for methylated DNA. Fluorescence, circular dichroism, and thermal denaturation techniques are utilized to determine the binding affinities and structures of peptides. Studies of peptide mutations can monitor the effectiveness of methylated DNA binding compared to the original peptide. The most structured peptide is expected to have the highest affinity and selectivity, leading to a peptide which can compete with the MBD1 protein for methylated DNA binding. The binding region of the MBD1 protein is composed of a two-stranded β-sheet called a 𝛽-hairpin connected by a large loop region. This was not observed in the initial peptide sequence. Instead the CD indicated the structure to be random coil. This was expected, the 𝛽-hairpin portion was synthesized without the full protein limiting favorable interactions to stabilize its structure. However, a fluorescence binding study determined the peptide did bind DNA. Thermal denaturation studies were conducted showing the stability of the peptide. Mutants will be made to increase binding affinity and selectivity in the future. This research could produce a series of peptides which can inhibit methylated DNA binding domains and reduce the silencing of tumor suppressor proteins, providing effective cancer therapeutics.
Scherer, Shelby L., "Exploring the relationship of MBD1 protein binding CpG sites of methylated DNA" (2016). University Honors Program Theses. 214.