Project Summary/Abstract Copper is an essential metal ion for all organisms, including prokaryotes. Strict control of copper homeostasis is maintained using a suite of copper-binding and transporting proteins. The transmembrane protein CopD is implicated in maintaining copper homeostasis by transporting copper into the bacterial cytoplasm. The molecular basis for cytoplasmic copper import is not known, and no ortholog of CopD has been studied on the molecular level. In this project, the CopD protein from Methylosinus trichosporium OB3b will be characterized using a multi-pronged structural, functional, and genetic approach. We will test the hypothesis that this protein is involved in import of copper to the cytoplasm and elucidate the molecular basis for function. Complementary genetic manipulation of the native organism will help determine the role of this protein in vivo. This research project will elucidate a new paradigm in bacterial copper homeostasis and broaden our understanding of membrane transporters by providing the first characterization of a novel family. More broadly, these efforts will lay the foundation for targeting copper import pathways as an antibiotic strategy. This research pursuit will be the main focus of a comprehensive training program aimed at bolstering many technical skills and interpersonal leadership skills through training in the Rosenzweig laboratory. Training will be acquired in biochemical, molecular biology, genetic, and structural biology techniques and will facilitate completion of the research project. Training and resources provided by the Structural Biology Facility will enable structural investigation using crystallography and electron microscopy. Training opportunities outside the laboratory, such as programs at the Life Sciences Collaborative Access Team (LS-CAT) beamlines at the Advanced Photon Source (APS) at Argonne National Laboratory, will also be available. All of the necessary equipment is available in the Departments of Molecular Biosciences and Chemistry at Northwestern University with easy access to the beamlines at the APS. In addition, collaborations with the Hoffman laboratory for electron paramagnetic resonance (EPR) spectroscopy, the He laboratory for cryogenic electron microscopy, and the Lewinson laboratory for metal transport assays will facilitate successful completion of this research. This multi-faceted training program combined with the collaborative and scientific environment at Northwestern University will provide excellent interdisciplinary training for an independent academic position.