Executive Summary : | Copper is an essential trace metal found in living organisms belonging to all kingdoms of life. At the same time, due to its redox activity, copper ions when present in excess, pose serious threat to the normal working of a cell, by producing highly reactive free radicals. In order to maintain cellular copper level and ensure delivery of copper to target proteins, Nature has evolved several classes of soluble and membrane proteins to traffic copper ions from outside of the cell to the various intracellular compartments. Abnormal copper trafficking in humans has been linked to Wilson and Menkes diseases. Copper ions are transported as Cu+ inside the cell. This proposal focuses on the pathway from the cell surface to the Golgi body. The main molecular actors in this pathway are the copper transporting proteins called copper chaperones and multidomain membrane proteins, residing in the membrane of the Golgi body, called Cu-ATPases. The copper chaperones are 60-80 residue small proteins with a highly conserved ferredoxin fold structure conserved over most eukaryotic organisms. The Cu-ATPase protein consists of three cytoplasmic domains, a TM domain comprising eight helices and several metal binding domains. Chaperone proteins collect the Cu+ ions from the cell surface, transport them through the cytoplasm and deliver the Cu+ ions to the metal binding domains of the Cu-ATPase. After this, it is believed that MBDs transfer the bound Cu+ ion to the transmembrane (TM) binding sites of the ATPase in an intramolecular metal transfer reaction. Over the last two decades structural and biochemical studies have furnished crucial information about the first process. However, the detail mechanism of the metal transfer between two proteins with similar structures is still unknown. The second step is still poorly understood and there is dearth of information about the metal bound state of the membrane protein, nature of transmembrane metal binding sites and molecular mechanism of intramolecular metal transfer between two domains of the Cu-ATPase. Protein-protein interactions (PPIs) are ubiquitous in biology, however most PPIs involve non-bonded interactions between protein residues. Microscopic pictures of these complex molecular processes are not directly accessible from experiments. Detailed computational investigation can complement experimental findings as well as provide new hypothesis for further experimental work. Metal transfer reaction between two proteins involve significant rearrangement of the local protein structures indicating significant entropic effects as well as additional energetic contribution to the reaction profile from second sphere effects. We propose a multistep novel computational strategy by combining multiple classical and quantum simulations techniques to investigate detailed mechanism of protein mediated copper trafficking. |