THE Cus E.coli COPPER EFFLUX SYSTEM – EXPLORING MOLECULAR ASPECTS OF THE INTERACTION BETWEEN CusB AND CusF

Copper plays a key role in all living organisms, serving as a cofactor for a large variety proteins and enzymes involved in electron transfer, oxidase and oxygenase activities, and detoxification of oxygen radicals. Due to its toxicity a homeostasis mechanism is required. In E. coli, the CusCFBA efflux system is one of the copper regulating systems and is responsible for pumping metal ions e.g. Cu(I) out of the periplasm domain into the extracellular domain as well as inserting protons into the cytoplasm in order to detoxify Cu(II) ions. Two of the components of this efflux system, the CusF metallochaperone and the N-terminal domain of CusB have been suggested to have an essential role in triggering the function of this efflux system. Resolving the metal ion transport mechanism through this efflux system is essential for understanding metal and multidrug-resistant microorganism. This work explores one aspect in the E.coli resistance mechanism by observing the interaction between CusB and the CusF protein in-vitro, using Electron Paramagnetic Resonance (EPR) spectroscopy as a factor of Cu(I) and pH titration, Circular Dichroism (CD), and chemical cross-linking.