Structure and Dynamics of Metal-Containing Proteins

描述(申请人提供)：由细胞色素P450(激活分子氧与有机分子反应)催化的化学对生物体本身是有害的，必须严格控制或在受控环境中进行。在P450酶的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，并有助于确定酶的特异性和效率。虽然许多P450的晶体结构已经被确定，并为理解结构-功能关系提供了一个重要的起点，但缺乏监测作为底物和效应器结合的函数的结构和动力学的变化，或者快速表征P450的活性部位及其与底物和抑制剂的相互作用的方法。我们将多维核磁共振(核磁共振)和串联质谱仪(MS-MS)应用于P450酶来满足这一需求。细胞色素P450 cam(CYP101)的~1H、~(15)N和~(13)C共振谱已被广泛指认。当效应器与CYP101结合时，会发生离散的构象变化，从而适当地重新定位底物以适应化学，这种构象变化的细节已经通过核磁共振、突变和模拟的组合来阐明。MS-MS已被用来通过氢/氢交换来定位CYP101局部蛋白质动力学中氧化还原依赖的变化。在下一个项目期间，CYP101的核磁共振指定将扩展到酰胺交换缓慢和顺磁展宽的区域。MS-MS和核磁共振将被用来识别对底物结合重要的动态“热点”。效应器结合的CYP101的结构将使用剩余偶极耦合来确定。通过顺磁和抗磁形式的比较，可以很容易地区分P450活性中心的核磁共振。将研究哺乳动物P450酶活性部位的底物和抑制剂与细胞色素P2B4和细胞色素P3A4的结合。在当前时期开发的顺磁性金属蛋白的核磁共振结构表征方法将进一步完善。与公共卫生相关：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，包括药物新陈代谢和激活、类固醇和花生四烯酸的生物合成以及“有毒废物处理”，即在排泄之前降解外来化合物。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很重要。这个项目的目的是了解这些酶，以帮助设计更有效的药物。

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.