Date of Award
Doctor of Philosophy (PhD)
Gordon S. Rule
Protein-DNA interactions regulate key biological processes such as gene expression, genetic recombination, DNA synthesis and repair. DNA binding proteins that are enzymes often depend on metal ions (typically Mg2+) for catalysis. Metal ions neutralize the active site electrostatic repulsion generated by a constellation of negatively charged active site residues. They therefore enhance site-specific protein-DNA binding by making the interaction more energetically favorable. Although, specific protein-DNA-metal complexes have been studied extensively by various biophysical and biochemical methods, little is known about the dynamics of these complexes. Many crystal structures of protein-DNA-metal complexes are available but they are very similar to the metal-free structures and show little or no change due to metal-ion binding. Therefore, to understand the altered energetics of protein-DNA recognition complexes due to metal-ion binding, it is essential to study them in solution. In our study, we have used EcoRV-DNA complex, with Lu3+ as the metal ion, to detect metal-induced structural and/or dynamic changes in solution using paramagnetic relaxation enhancement (PRE). Lu3+ inhibits Mg2+ dependent DNA hydrolysis in EcoRV, stimulates EcoRV-DNA affinity, binds in the same site and with the same stoichiometry as Mg2+ and is therefore an excellent substitute for Mg2+. In addition, to verify that Lu3+ binding is confined to the active sites, we have solved crystal structures of the EcoRV-DNALu3+ complex with uncleaved and cleaved DNA. Also, for a comprehensive residue-specific analysis, we have acquired both backbone NH and methyl side-chain (ILV) assignments for EcoRV-DNA complex with and without Lu3+. In order to detect metal-induced changes in the EcoRV-DNA complex, backbone and methyl side-chain PRE rates were measured with spin-labels at S2C, K197C and S234C of EcoRV. The measured PRE rates suggest that the crystal structures of EcoRV-DNA complexes better represent the complex with metal-ion in solution whereas the nometal complex in solution shows differences in structure and/or dynamics when compared to the crystal structure. The C-terminal region of EcoRV is observed to be more flexible in solution in the nometal complex and its conformational flexibility is reduced due to metal-ion binding.
Sangani, Sahil S., "Lanthanides to Probe Metal-induced Structural and Dynamic Changes in EcoRV-DNA Complex" (2016). Dissertations. 685.