Kinetic and Thermodynamic Studies Relevant to Biological Iron Transport

无机化学、生物无机化学和金属有机化学项目以及分子和细胞生物学生物化学项目的这一奖项支持杜克大学Alvin Crumbliss教授的研究，该研究旨在研究铁载体和两种周质蛋白-奈瑟氏菌的铁结合蛋白(FbpA)和假单胞菌的周质结合蛋白(PBP)-介导的生物铁运输机制。具体目的是：探索Fe转运的氧化还原开关假说，Fe/配体交换是由Fe3+还原启动的，研究外膜受体TbpA/B在从Fe2hTf中去除Fe的作用，研究TbpA塞域在FbpA插入Fe中的作用，研究协同阴离子(X)在FbpA插入Fe和调节FeFbpA-X生物物理性质中的作用，用体外方法测定FeFbpA-X随着细胞生长环境的变化而发生的协同阴离子变化，通过FbpA研究GA3+的固存作用，探索细胞周铁向细胞膜铁运输的机制研究FpvAI-Plug结构域、吡喃佛丁和与假单胞菌中铁运输相关的周质结合蛋白之间的分子相互作用，研究Brasilibactin A对铁和锌的隔离与该铁载体的生物活性和信号功能的关系，研究用于启动细胞内Fenton化学的铁载体的配位和氧化还原化学，并将体外结果与在生物合作者实验室进行的体内研究直接相关。将使用体外和体外生物物理方法来实现这些目标。MALDI-TOF谱的SUPREX分析将用于表征金属-蛋白质、蛋白质-蛋白质、蛋白质-铁载体和蛋白质-阴离子之间的相互作用，以及它们对铁/配体交换的影响。重组WT和突变蛋白，包括膜受体的插塞域，将被用来探索运输过程中的各个步骤。该项目能够通过种子项目、高中教师研讨会和为期四年的大学研讨会来指导少数族裔学生。关于铁迁移的研究结果被用来说明普通化学中所教授的原理。这项研究计划涉及与五个美国和国际生物实验室的广泛合作，这将增强PI实验室和合作者实验室学生的跨学科和全球研究经验，从而扩大对教育和科学基础设施的贡献。

This award in the Inorganic, Bioinorganic and Organometallic Chemistry program and the Molecular and Cell Biology Biochemistry program supports research by Provessor Alvin Crumbliss at Duke University to investigate mechanisms for biological Fe transport mediated by siderophores and two periplasmic proteins, ferric binding protein (FbpA) from Neisseria and periplasmic binding protein (PBP) from Pseudomonas. Specific aims are to: explore a redox switch hypothesis for Fe transport whereby Fe/ligand exchange is initiated by Fe3+ reduction, investigate the role of the outer membrane receptor TbpA/B in removing Fe from Fe2hTf , investigate the role of the TbpA plug domain in inserting Fe into FbpA, investigate the role of the synergistic anion (X) in inserting Fe into FbpA and in modulating the biophysical properties of FeFbpA-X, use an ex vivo method to determine the in vivo synergistic anion changes in FeFbpA-X with variations in cell growth environment, investigate Ga3+ sequestration by FbpA to probe the mechanism of periplasm-to-cytosol Fe transport, investigate the molecular interactions between the FpvAI-plug domain, pyoverdine, and a periplasmic binding protein that are relevant to Fe transport in Pseudomonas, investigate the relationship between Fe and Zn sequestration by brasilibactin A and the biological activity and signaling functions of this siderophore, investigate the coordination and redox chemistry of a siderophore conjugate designed to initiate intracellular Fenton chemistry, and directly correlate in vitro results with in vivo studies performed in biological collaborators laboratories. In vitro and ex vivo biophysical methods will be used to achieve the objectives. SUPREX analysis of MALDI-TOF mass spectra will be used to characterize metal-protein, protein-protein, protein-siderophore, and protein-anion interactions, and their influence on Fe/ligand exchange. Recombinant WT and mutant proteins, including plug domains from membrane receptors, will be used to explore individual steps in the transport process. This project enables mentoring minority students through Project SEED, high school teacher workshops, and four-year college seminars. Research results on Fe transport are used to illustrate principles taught in General Chemistry. This research program involves extensive collaboration with five US and international biological laboratories, which will enhance the interdisciplinary and global research experience of students in the PI's and collaborators' laboratories, thereby broadening contributions to education and scientific infrastructure.