Towards Structural and Dynamic Investigation of Microsomal P450s

微粒体 P450 的结构和动态研究

• 批准号: 7394014
• 负责人: Alana Simorellis
• 金额: $ 4.36万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-02 至 2008-12-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7394014
• 关键词: Active Sites; Agar; Bacteria; Binding; Biochemical; Biological; CYP2B4 gene; Camphor; Camphor 5-Monooxygenase; Catalysis; Chemicals; Collaborations; Complement; Cytochrome P450; Cytochromes; Detection; Deuterium; Electron Transport; Electrons; Enzymes; Equation; Future; Goals; Heme; Human; Hydrogen; Hydroxylation; Integral Membrane Protein; Investigation; Iron-Sulfur Proteins; Laboratories; Localized; Mammals; Mass Spectrum Analysis; Measurement; Membrane Proteins; Methods; Michigan; Mixed Function Oxygenases; Modeling; Molecular; Molecular Conformation; Motion; NADP; Noise; Nuclear Magnetic Resonance; Object Attachment; Organism; Outcome; Oxygen; Play; Protein Dynamics; Protein Overexpression; Proteins; Pseudomonas putida; Public Health; Reaction; Relaxation; Research; Research Personnel; Resources; Role; Signal Transduction; Site; Soil; Solubility; Solutions; Spectrometry; Staging; Standards of Weights and Measures; Structure; Time; Titrations; Toxic effect; Training; Vertebral column; Xenobiotics; cofactor; drug metabolism; inhibitor/antagonist; insight; milligram; oxidation; putidaredoxin; research study; tool

DESCRIPTION (provided by applicant): Overall, the project seeks to investigate the structure and dynamics of two P450 enzymes, P450 cam (CYP101) and the microsomal integral membrane protein P450 2B4 (CYP2B4). The first specific aim of the project is to conduct hydrogen/deuterium exchange experiments using nuclear magnetic resonance and mass spectrometry to investigate the structural dynamics involved in cytochrome P450 catalysis. Collaboration with Prof. Jeffery Agar (Brandeis Univ.) will provide crucial training for the researcher in the field of biological mass spectrometry. Multidimensional NMR methods can be used to complement mass spectral studies, and will be used to further localize in a site-specific manner functionally relevant dynamics. The next specific aim is to conduct backbone resonance assignments for investigation of structure and conformational dynamics of CYP2B4. In collaboration with Prof. Lucy Waskell (Univ. of Michigan) the researcher will have access to milligram quantities of isotopically enriched CYP2B4. Backbone assignment is the first step for NMR relaxation measurements, which are an excellent way to investigate both backbone, sidechain, and global dynamics. To accomplish this goal, the researcher will have on-site access to both 800 and 600 MHz spectrometers. The 800 MHz NMR is equipped with a cryoprobe and cold 1H and 13C preamplifiers. The last specific aim is identification of key backbone resonances involved in xenobiotic turnover by mammalian CYP2B4. Effector binding to other P450s has been hypothesized to cause distinct conformational changes that are necessary for turnover, but there have been no investigations of structural fluctuations of CYP2B4. NMR chemical shift perturbation experiments can identify residues involved in binding in and around the active site, and this evidence will be used to direct future dynamic investigation. The first step will be to use heteronuclear multidimensional solution NMR to investigate both oxidation states of the mammalian protein 2B4 bound to an inhibitor. The biologically relevant effectors will be added and the residues that interact with these proteins will be identified. These initial studies will identify key residues and demonstrate the feasibility of using solution NMR as a tool to further investigate the structure and dynamics of CYP2B4. PUBLIC HEALTH RELEVANCE. CYP2B4 is a critical integral membrane protein involved in xenobiotic and drug metabolism in mammals and a detailed investigation of dynamics would provide valuable insight into the mechanism. The combination of structural and dynamics studies provides the comprehensive analysis required to elucidate the physical origins of the P450 mechanism.

描述（由申请人提供）：总体而言，该项目旨在研究两种P450酶P450 cam（CYP 101）和微粒体整合膜蛋白P450 2B 4（CYP 2B 4）的结构和动力学。该项目的第一个具体目标是利用核磁共振和质谱法进行氢/氘交换实验，以研究细胞色素P450催化作用所涉及的结构动力学。与Jeffery Agar教授（布兰代斯大学）合作将为生物质谱领域的研究人员提供重要的培训。多维NMR方法可用于补充质谱研究，并将用于进一步本地化的网站特定的方式功能相关的动态。下一个具体的目标是进行骨架共振分配的结构和构象动力学的CYP 2B 4的调查。在与Lucy Waskell教授（密歇根大学）的合作中，研究人员将获得毫克量的同位素富集的CYP 2B 4。主链归属是核磁共振弛豫测量的第一步，这是研究主链、侧链和全局动力学的一种很好的方法。为了实现这一目标，研究人员将在现场使用800和600 MHz频谱仪。800 MHz NMR配备有冷冻探针和冷1H和13 C前置放大器。最后一个具体的目标是识别参与哺乳动物CYP 2B 4的异生物质周转的关键骨架共振。已假设效应子与其他P450的结合会引起营业额所需的独特构象变化，但尚未对CYP 2B 4的结构波动进行研究。核磁共振化学位移扰动实验可以确定参与结合的活性位点和周围的残基，这一证据将用于指导未来的动态调查。第一步将是使用异相多维溶液NMR来研究与抑制剂结合的哺乳动物蛋白2B 4的两种氧化态。将添加生物学相关效应物，并鉴定与这些蛋白质相互作用的残基。这些初步研究将确定关键残基，并证明使用溶液NMR作为工具进一步研究CYP 2B 4结构和动力学的可行性。 公共卫生相关性。CYP 2B 4是一种重要的膜蛋白，参与哺乳动物体内的外源性物质和药物代谢，对其动力学的详细研究将为深入了解其机制提供有价值的信息。结构和动力学研究的结合提供了阐明P450机制的物理起源所需的全面分析。