Structure and Dynamics of Metal-Containing Proteins

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）对生物体本身是危险的，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，但缺乏方法来监测结构和动力学的变化，作为底物和效应物结合的功能，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。我们将多维核磁共振（NMR）和串联质谱（MS-MS）应用于P450酶来满足这一需求。在细胞色素P450cam （CYP101）中进行了广泛的序列1H， 15N和13C共振分配。当效应物与CYP101结合时，会发生离散的构象变化，从而使底物重新定向，以适应化学反应，这种构象变化的细节已经通过核磁共振、诱变和模拟的结合得到阐明。MS-MS已被用于通过氢/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的核磁共振分配将扩展到慢酰胺交换和顺磁展宽区域。MS-MS和NMR将用于识别对底物结合重要的动态“热点”。效应结合CYP101的结构将使用剩余的偶极偶联来确定。通过比较顺磁和反磁形式，可以很容易地区分p450活性位点的核磁共振。哺乳动物P450酶活性位点的底物和抑制剂与CYP2B4和CYP3A4的结合将被表征。顺磁性金属蛋白的核磁共振结构表征方法将进一步完善。细胞色素P450单加氧酶在许多人体生理过程中至关重要，包括药物代谢和激活、类固醇和花生二酸生物合成以及“有毒废物处理”，即在排泄前降解外来化合物。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。

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.