Structure and Dynamics of Metal-Containing Proteins

描述（由申请人提供）：细胞色素P450催化的化学反应（活化分子氧与有机分子反应）对生物体具有固有危害，必须严格调节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的晶体结构已被确定，并提供了一个重要的起点，了解结构-功能关系，方法是缺乏监测的结构和动力学的变化作为一个功能的基板和效应器结合或快速表征的活性位点的P450及其与基板和抑制剂的相互作用。我们将多维核磁共振（NMR）和串联质谱（MS-MS）应用于P450酶以满足这一需求。细胞色素P450 cam（CYP 101）的~ 1H、~（15）N和~（13）C共振谱已作了广泛的序列归属。在效应物与CYP 101结合后发生离散构象变化，其使底物适当地重新定向用于化学，并且已经通过NMR、诱变和模拟的组合阐明了这种构象变化的细节。MS-MS已被用于通过氢/氘交换定位CYP 101中局部蛋白动力学的氧化还原依赖性变化。在下一个项目期间，CYP 101的NMR分配将扩展到缓慢酰胺交换和顺磁增宽的区域。MS-MS和NMR将用于识别对底物结合重要的动态“热点”。将使用残余偶极偶联测定效应子结合CYP 101的结构。通过比较顺磁性和反磁性形式，可以很容易地区分P450活性部位的NMR共振。将表征哺乳动物P450酶活性位点中底物和抑制剂与CYP 2B 4和CYP 3A 4的结合。将进一步完善在本报告所述期间开发的顺磁性金属蛋白质核磁共振结构表征方法。公共卫生相关性：细胞色素P450单加氧酶在许多人类生理过程中至关重要，包括药物代谢和活化、类固醇和花生四烯酸生物合成以及“有毒废物处理”，即在排泄前降解外来化合物。了解这些酶的活性对于预测作为药物设计一部分的抑制剂和底物的行为是重要的。该项目旨在了解这些酶，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.