Structure, Mechanism and Interactions of Type IA Topoisomerases

IA型拓扑异构酶的结构、机制和相互作用

• 批准号: 10389425
• 负责人: Yuk-Ching Tse-Dinh
• 金额: $ 6.03万
• 依托单位: FLORIDA INTERNATIONAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10389425
• 关键词: Complex; Crystallization; DNA; Equipment; Escherichia coli; Funding; Grant; Human; Ligands; Molecular Conformation; Mycobacterium tuberculosis; National Institute of General Medical Sciences; Proteins; RNA; Recombinants; Request for Applications; Research; Structure; System; Time; Topoisomerase; Topoisomerase III; Type I DNA Topoisomerases; United States National Institutes of Health; X-Ray Crystallography; biophysical analysis; fast protein liquid chromatography; inhibitor/antagonist; milligram; mutant; protein protein interaction; protein purification; repaired; single molecule

Project Summary PI: Yuk-Ching Tse-Dinh This equipment supplement application requests funds to purchase an AKTA FPLC protein purification system for the large scale purification of recombinant topoisomerase proteins that are needed for obtaining new structures of topoisomerases by X-ray crystallography, and biophysical studies to elucidate topoisomerase complex conformations, protein-protein interactions and ligand interaction dynamics under the aims of the PI’s NIGMS active grants R01-GM054226 and R35-GM139817. The AKTA FPLC protein purification system currently available in the PI’s lab requires frequent repairs and needs to be replaced to avoid extensive down times. The replacement of the AKTA FPLC purification system would allow us to produce quickly and efficiently without bottlenecks milligram quantities of wild-type and mutant forms of Escherichia coli and Mycobacterium tuberculosis topoisomerase I, E. coli topoisomerase III, and human topoisomerase IIIB for structural and biophysical studies of their action on both DNA and RNA substrates, as well as inhibitors. The proteins need to be produced in high purity for studies in our lab at FIU, and are also needed by our collaborators at Argonne National Lab and NIH for crystallization screens and single-molecule studies.

项目概要 PI：Yuk-Ching Tse-Dinh 该设备补充申请要求资金购买 AKTA FPLC 蛋白质 用于大规模纯化重组拓扑异构酶蛋白的纯化系统 通过 X 射线晶体学获得拓扑异构酶的新结构是必需的，并且 阐明拓扑异构酶复合物构象、蛋白质-蛋白质的生物物理学研究 PI 的 NIGMS 积极资助目标下的相互作用和配体相互作用动力学 目前的 AKTA FPLC 蛋白质纯化系统为 R01-GM054226 和 R35-GM139817。 PI 实验室中的可用部件需要经常维修并需要更换，以避免大量损坏 更换 AKTA FPLC 纯化系统将使我们能够生产。 快速有效地无瓶颈，毫克量的野生型和突变型 大肠杆菌和结核分枝杆菌拓扑异构酶 I、大肠杆菌拓扑异构酶 III 和 人拓扑异构酶 IIIB，用于结构和生物物理研究其对 DNA 和 RNA 底物以及抑制剂需要以高纯度生产。 我们在佛罗里达国际大学的实验室进行的研究，我们在阿贡国家实验室的合作者也需要 NIH 用于结晶筛选和单分子研究。