Structural studies of the nucleosome, assembled with different histones, DNA fragments and HMGN proteins, and derivatized with ruthenium anticancer agents

• Main Author: Reyhan Muhammad
• Other Authors: Curtis Alexander Davey
• Format: Theses and Dissertations
• Language: English
• Published: 2013
• Subjects: DRNTU::Science::Biological sciences::Molecular biology; DRNTU::Science::Biological sciences::Biochemistry
• Online Access: https://hdl.handle.net/10356/53656
• Institution: Nanyang Technological University

The nucleosome forms the basic unit of chromatin in eukaryotic cells. This compact structure made of four histone proteins and DNA serves dual interrelated functions for storage and regulation of genomic activities. Nucleosome dynamics, histone variants, histone post-translational modification, nucleosome positioning and occupancy are integral in determining the status of the cell. Structural and biochemical studies of the nucleosome have granted a detailed mechanistic view for understanding the activity of this interesting structure inside the cell. We are interested in studying how nucleosomes interact with other nuclear proteins inside the cell and how we can exploit the nucleosome for anticancer drug development. Xenopus laevis nucleosome core particle has been widely used as a model for nucleosome studies, benefiting from an atomic understanding of its structure. Even though histones and thus nucleosome structure are commonly conserved throughout evolution, we aim to develop a nucleosome platform more relevant for biomedical studies— human nucleosome core structures. We showed that human nucleosome core particle assembled from different DNA fragments can be crystallized to give high resolution structures. Furthermore, we did preliminary studies on the interaction of ruthenium-based compounds with Xenopus laevis and human nucleosomes, and showed that both gave satisfying results. We conducted crystallographic analysis for several derivatives of novel ruthenium-based anticancer drugs, termed RAPTA compounds, with regard to their interaction with the nucleosome. We worked on the principle of optimizing a lead RAPTA-nucleosome structure in order to improve binding affinity and specificity towards the nucleosome. The balance between providing new groups for enhancing RAPTA interaction and avoiding steric clashes with the nucleosome structure is an important determinant in designing new RAPTA compounds. We found that the smallest RAPTA compound (RAPTA-B), with an unsubstituted arene ring, still maintains similar binding selectivity compared to larger RAPTA compounds. Interestingly, the small arene group allows RAPTA-B to access a site on the nucleosome that is sterically restricted due to internucleosome interactions. Nuclear proteins ranging from ATP-dependent chromatin remodelers, histone chaperones, histone modifying enzymes, and structural proteins assist the regulation of chromatin activities. We are interested in studying high mobility group N (HMGN) proteins due to their abundance inside the nucleus, their importance for genomic activities, and their specific interaction with the nucleosome core. Our group showed distinct structural changes on nucleosome structure elicited by different types of HMGN proteins. Several biochemical assays were conducted to investigate the nature of HMGN binding to the nucleosome with the goal of explaining how HMGN proteins may work inside the nucleus. Crystallizations of nucleosome with HMGN proteins have also been conducted, and novel potential crystallization buffers for nucleosomal studies have been discovered.