Structure and Dynamics of Metal-Containing Proteins

含金属蛋白质的结构和动力学

基本信息

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
项目类别：
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

项目摘要

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（分子氧激活与有机分子反应）对生物体固有危害，并且必须在受控的环境中严格调节或发生。在P450酶的催化循环过程中发生的结构和动态变化是该调节的一部分，并有助于确定酶的特异性和效率。尽管已经确定了许多P450的晶体结构，并为理解结构功能关系提供了重要的起点，但是对于监测结构和动力学的变化作为底物和效应子结合的函数或对P450S的活动位点的快速表征以及它们与底物和抑制剂的相互作用的快速表征，方法缺乏方法。我们将多维核磁共振（NMR）和串联质谱（MS-MS）应用于P450酶，以满足这种需求。在细胞色素P450CAM（CYP101）中进行了广泛的顺序1H，15N和13C共振分配。在效应子与CYP101结合后，会发生离散的构象变化，从而适当地重新定义了化学的底物，并且通过NMR，诱变和仿真的组合阐明了这种构象变化的细节。 MS-MS已通过氢/氘交换来定位CYP101中局部蛋白质动力学的氧化还原依赖性变化。在下一个项目期间，CYP101的NMR分配将扩展到缓慢的酰胺交换和顺磁性扩展的区域。 MS-MS和NMR将用于识别对于底物结合至关重要的动态“热点”。将使用残留的偶性耦合确定效应构成的CYP101的结构。 P450的活性位点中的NMR共振可以通过比较顺磁性和磁磁形式的比较来区分。将表征哺乳动物P450酶与CYP2B4和CYP3A4的活性位点的底物和抑制剂的结合。在当前时期开发的，用于进一步完善顺磁性金属蛋白的NMR结构表征。公共卫生相关性：项目叙事细胞色素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，类固醇和蛛网膜酸生物合成以及“有毒废物处理”，即降解前外源化合物的降解。了解这些酶的活性对于预测抑制剂和底物作为药物设计的一部分很重要。该项目旨在理解这些酶，以帮助设计更有效的药物。