Structural and functional characterization of putative Cu importer CopD

假定铜输入者 CopD 的结构和功能表征

• 批准号: 10330998
• 负责人: Rose Currier Hadley
• 金额: $ 2.85万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2022-11-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10330998
• 关键词: Achievement; Affinity; Agriculture; Anti-Bacterial Agents; Antibiotics; Bacteria; Bacterial Genome; Binding; Biochemical; Biogenesis; Biological Assay; Biological Process; Biological Sciences; C-terminal; Carrier Proteins; Cell membrane; Cells; Chemistry; Coenzymes; Collaborations; Complement; Copper; Cryoelectron Microscopy; Crystallography; Cytochrome a; Cytoplasm; Development; Electron Microscopy; Electron Spin Resonance Spectroscopy; Environment; Enzymes; Equipment; Family; Fluorescence; Foundations; Genes; Genetic; Genetic study; Goals; Healthcare; Heme; Homeostasis; Individual; Inductively Coupled Plasma Mass Spectrometry; Innate Immune System; Integral Membrane Protein; Interferometry; Investigation; Ions; Knock-out; Laboratories; Leadership; Length; Liposomes; Mass Spectrum Analysis; Membrane Transport Proteins; Metals; Methane hydroxylase; Methylococcaceae; Methylosinus trichosporium; Molecular; Molecular Biology; Monitor; Nutrient; Operon; Organism; Orthologous Gene; Oxidation-Reduction; Oxidative Stress; Particulate; Pathway interactions; Phenotype; Photons; Play; Positioning Attribute; Prokaryotic Cells; Property; Proteins; Radioactivity; Research; Research Project Grants; Resistance; Resources; Respiration; Role; Series; Source; Spheroplasts; Structure; Technical Expertise; Techniques; Tertiary Protein Structure; Testing; Titrations; Training; Training Programs; Transmembrane Domain; Universities; Work; X-Ray Crystallography; base; beamline; biophysical properties; copper-binding protein; cryogenics; cytochrome c oxidase; delta protein; electronic structure; experimental study; genetic approach; genetic manipulation; in vivo; insight; novel; overexpression; programs; protein function; protein protein interaction; skills; stress management; structural biology; training opportunity

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.

项目总结/文摘