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 of this research is to explore the structure/function relationship between MBD proteins and methylated DNA recognition by synthesizing and studying the methylated DNA binding region of the MBD1 protein. Fluorescence, circular dichroism (CD), and thermal denaturation techniques are utilized to determine the structures of peptides, binding affinities and specificities of peptides for methylated and unmethylated DNA. Studies of peptide mutations can monitor the effectiveness of methylated DNA binding compared to the original peptide. The same studies will be conducted using unmethylated DNA to visualize affinity and specificity of the peptides for methylated versus unmethylated DNA. 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 beta-sheet connected by a large loop region. In studying the structure of the initial peptide sequence via CD, a beta sheet structure was not observed. Instead the peptide alone displays a random coil structure, as expected, because the beta-hairpin portion was synthesized without the full protein, limiting favorable interactions to stabilize its structure. A fluorescence binding study determined that the peptide does bind DNA. Thermal denaturation studies were conducted showing the stability of the peptide. In an attempt to increase the structure of the mutant peptides, the beta-sheet portion of the binding sequence will be extended. To enhance the specificity for DNA, amino acids will be added to hydrogen bond with the DNA. Further studies will be conducted and compared to the initial findings. The analysis of the initial peptide and the mutant data will determine the impact of structure on the binding affinity and specificity for methylated versus unmethylated DNA. This research could produce a series of peptides to inhibit methylated DNA binding domains and reduce the silencing of tumor suppressor proteins, providing effective cancer therapeutics.

Investigating the specificity of methylated DNA binding by MBD1 and mutant peptides for transcriptional regulation

253rd National Meeting (2017)

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