Structure and Dynamics of Metal-Containing Proteins

含金属蛋白质的结构和动力学

• 批准号: 7924934
• 负责人: Thomas Charles Pochapsky
• 金额: $ 7.72万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924934
• 关键词: Acids; Active Sites; Affect; Agar; Amides; Anabolism; Arts; Attention; Automobile Driving; Behavior; Binding; Biological; Biological Assay; CYP2B4 gene; CYP3A4 gene; Camphor; Camphor 5-Monooxygenase; Carbon Monoxide; Catalysis; Cell Respiration; Chemicals; Chemistry; Collaborations; Complex; Controlled Environment; Coupling; Cytochrome P450; Cytochromes; Data; Detection; Deuterium; Digestion; Distal; Drug Design; Electron Transport; Electrons; Environment; Enzymes; Equilibrium; Excretory function; FDA approved; Ferredoxin; Figs - dietary; Frequencies; Heme; Heme Iron; Hot Spot; Human; Hydrogen; Hydrogen Peroxide; Hydroxylation; Illinois; Individual; Ions; Isotope Labeling; Label; Lead; Letters; Ligation; Link; Liver; Madura; Mass Spectrum Analysis; Metalloproteins; Metals; Methodology; Methods; Michigan; Mixed Function Oxygenases; Modeling; Molecular; Molecular Conformation; Monitor; Motion; Mutagenesis; Mutation; Nuclear Magnetic Resonance; Organism; Oxidation-Reduction; Oxygen; Pathway interactions; Peptides; Peroxides; Pharmaceutical Preparations; Pharmacologic Substance; Physiological; Physiological Processes; Play; Production; Prosthesis; Protein Dynamics; Proteins; Protons; Pseudomonas putida; Reaction; Reactive Oxygen Species; Reducing Agents; Refuse Disposal; Regulation; Relaxation; Residual state; Resistance; Rest; Role; Running; S-1 Antimetabolite agent; Sampling; Scheme; Scientist; Side; Solutions; Specificity; Steroids; Structure; Structure-Activity Relationship; Superoxides; System; Time; Universities; Vertebral column; Work; base; cis trans isomerization; design; drug metabolism; enzyme activity; experience; improved; inhibitor/antagonist; insight; interest; knowledge base; metalloenzyme; molecular dynamics; molecular recognition; monomer; norcamphor; novel; oxidation; oxidative damage; prevent; programs; prolylisoleucine; protonation; public health relevance; putidaredoxin; research study; simulation; tandem mass spectrometry

DESCRIPTION (provided by applicant): The chemistry catalyzed by cytochromes P450 (activation of molecular oxygen to react with organic molecules) is inherently hazardous to the organism, and must be tightly regulated or take place in a controlled environment. Structural and dynamic changes that take place in the course of the catalytic cycle of the P450 enzyme are part of this regulation, and help determine enzyme specificity and efficiency. While crystal structures have been determined for many P450s, and provide an important starting point for understanding structure-function relationships, methodology is lacking for monitoring changes in structure and dynamics as a function of substrate and effector binding or for rapid characterization of active sites of P450s and their interactions with substrates and inhibitors. We apply multidimensional nuclear magnetic resonance (NMR) and tandem mass spectrometry (MS-MS) to P450 enzymes to fill this need. Extensive sequential 1H, 15N and 13C resonance assignments have been made in cytochrome P450cam (CYP101). A discrete conformational change occurs upon binding of effector to CYP101 that reorients the substrate appropriately for chemistry, and details of this conformational change have been elucidated by a combination of NMR, mutagenesis and simulations. MS-MS has been used to localize redox-dependent changes in local protein dynamics in CYP101 by hydrogen/deuterium exchange. During the next project period, NMR assignments of CYP101 will be extended to regions of slow amide exchange and paramagnetic broadening. MS-MS and NMR will be used to identify dynamic "hot-spots" important for substrate binding. The structure of effector-bound CYP101 will be determined using residual dipolar couplings. NMR resonances in the active sites of P450s can be easily distinguished by comparison of paramagnetic and diamagnetic forms. The binding of substrates and inhibitors in the active sites of mammalian P450 enzymes with CYP2B4 and CYP3A4 will be characterized. Methodology developed during the current period for NMR structural characterization of paramagnetic metalloproteins will be further refined. PUBLIC HEALTH RELEVANCE: Project Narrative Cytochrome P450 monooxygenases are critical in many human physiological processes, including drug metabolism and activation, steroid and arachadonic acid biosynthesis, and "toxic waste disposal", that is, degradation of foreign compounds prior to excretion. Understanding the activity of these enzymes is important for predicting the behavior of inhibitors and substrates as a part of drug design. This project is aimed at understanding these enzymes to aid in the design of more effective pharmaceuticals.

描述（由申请人提供）：由细胞色素 P450 催化的化学反应（激活分子氧与有机分子反应）对生物体本身是有害的，必须严格监管或在受控环境中进行。 P450 酶催化循环过程中发生的结构和动态变化是这种调节的一部分，有助于确定酶的特异性和效率。虽然许多 P450 的晶体结构已被确定，并为理解结构-功能关系提供了重要的起点，但缺乏方法来监测结构和动力学随底物和效应子结合的变化，或快速表征 P450 的活性位点及其与底物和抑制剂的相互作用。我们对 P450 酶应用多维核磁共振 (NMR) 和串联质谱 (MS-MS) 来满足这一需求。已在细胞色素 P450cam (CYP101) 中进行了广泛的连续 1H、15N 和 13C 共振分配。效应子与 CYP101 结合后会发生离散的构象变化，从而适当地重新定向底物以进行化学反应，并且这种构象变化的细节已通过 NMR、诱变和模拟的组合得到阐明。 MS-MS 已用于通过氢/氘交换来定位 CYP101 局部蛋白质动力学中氧化还原依赖性变化。在下一个项目期间，CYP101 的 NMR 任务将扩展到缓慢酰胺交换和顺磁展宽的区域。 MS-MS 和 NMR 将用于识别对底物结合很重要的动态“热点”。效应器结合的 CYP101 的结构将使用残余偶极耦合来确定。通过比较顺磁性和抗磁性形式，可以轻松地区分 P450 活性位点的 NMR 共振。将表征哺乳动物 P450 酶活性位点中的底物和抑制剂与 CYP2B4 和 CYP3A4 的结合。目前开发的顺磁性金属蛋白核磁共振结构表征方法将进一步完善。公共健康相关性：项目叙述细胞色素 P450 单加氧酶在许多人类生理过程中至关重要，包括药物代谢和激活、类固醇和花生四烯酸生物合成以及“有毒废物处理”，即排泄前外来化合物的降解。作为药物设计的一部分，了解这些酶的活性对于预测抑制剂和底物的行为非常重要。该项目旨在了解这些酶，以帮助设计更有效的药物。