Defense Date

2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Integrative Life Sciences

First Advisor

David Jr Williams

Abstract

The nucleosome remodeling and deacetylase (NuRD) complex is an abundant deacetylase complex, which couples histone deacetylation and chromatin remodeling ATPase activities, and has a broad cellular and tissue distribution. Although the working model of how this complex forms and functions is not well known, we have demonstrated that the coiled-coil interaction between two proteins (MBD2 and p66α) is critical for DNA methylation dependent gene silencing in vivo. Chapter one: ‘Unique features of the anti-parallel, heterodimeric coiled-coil interaction between methyl-cytosine binding domain 2 (MBD2) homologues and p66α dictate high affinity binding’ describes this unique coiled coil interaction. Coiled-coils were studied using a variety of biophysical techniques including analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC) and circular dichroism (CD). Results were compared across homologues and mutation studies were carried out to test our hypotheses. The studies reported in this chapter add to our understanding of coiled-coil interaction and thereby facilitate development of small peptide based drugs which target such interactions in nature.A number of proteins have been identified in humans that specifically bind to methylated CpG via a methyl binding domain (MBD). The human genome encodes at least five MBD proteins: MeCP2 and MBD1 through MBD4, which are homologous in their methyl binding domains but not many similarities are seen outside the MBD. Out of the five MBDs, MBD4 has a c-terminal glycosylase domain through which it recognizes mCpG.TpG mismatch and is important for base excision repair system. Chapter two: ‘Dynamic behavior of MBD4 in methylated DNA recognition’ focuses on MBD4 and its preference for DNA methylation mark. Techniques of surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) spectroscopy are used to study binding affinity for variations of methylated DNA mark. Chemical exchange studies are used to demonstrate how MBD4 scans for methylation mark and these studies have added a new dimension to our understanding of how MBD proteins ‘read’ DNA methylation marks. Chapter three: ‘Solving the solution structure of MBD domain of MBD4 on methylated DNA by NMR’ describes a process of structure determination using NMR spectroscopy. The focus of this chapter is not on developing a new technique but rather on using current resources to solve a protein structure, which can be used to further understand our biological system. Here, I have discussed the workflow used to determine a final three-dimensional structure starting from sample preparation, data collection, data analysis to structure calculation.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

August 2013

Included in

Life Sciences Commons

Share

COinS