Structure and Dynamics of Metal-Containing Proteins

描述(申请人提供)：细胞色素P450单加氧酶在许多生理过程中是关键的，包括类固醇生物合成、药物代谢和激活以及异物降解。有关这些酶的结构和动态信息对于了解它们的活性(通常是未活化碳的羟氧基化)至关重要，并将在作为药物设计一部分的预测抑制剂和底物的行为方面发挥重要作用。尽管在生物体中发现了大量的P450酶，但结晶的相对较少，而且缺乏快速表征P450的活性部位及其与底物和抑制剂相互作用的方法。该提案描述了首次将高分辨率多维核磁共振(核磁共振)方法应用于P450酶。细胞色素P450 cam(CYP101)的~1H、~(15)N和~(13)C共振谱已被广泛指认。通过比较酶的顺磁性和抗磁性形式，可以快速区分该酶活性部位的残基。只有在反磁形式下才能观察到活性位共振。任务被用来监测CP101与其生理氧化还原伙伴和效应物Putidaredosin(PDX)的相互作用。在此基础上，提出了PDX效应器活性的新模型。 在本项目的下一阶段，将以多种形式的酶完成对CYP101的顺序分配，局部动力学将被表征为氧化态、底物结合和效应器结合的函数，以进一步阐明“解偶联”的机制，即在不改变底物周转的情况下，还原等价物以超氧化物、过氧化氢和水的形式丢失。将以细胞色素P450 BM-3(细胞色素P450 BM-3)和人类重要的P450酶CYP3A4为测试案例，测试应用核磁共振技术表征其他P450酶活性位点的方法。将进一步完善在当前时期开发的金属蛋白核磁共振结构表征方法。这包括可用于蛋白质顺磁中心附近的二维核磁共振方法，以及从磁各向异性数据中提取结构信息，如残余偶极耦合和偶极移位。

DESCRIPTION (provided by applicant): Cytochrome P450 monooxygenases are critical in many physiological processes, including steroid biosynthesis, drug metabolism and activation, and xenobiotic degradation. Structural and dynamic information concerning these enzymes is critical for understanding their activity (typically hdryoxylation of unactivated carbons), and will play a vital role in predicting the behavior of inhibitors and substrates as a part of drug design. Despite the large numbers of P450 enzymes found in living organisms, relatively few have been crystallized, and methodology for rapid characterization of active sites of P450s and their interactions with substrates and inhibitors is lacking. This proposal describes the first application of high-resolution multidimensional nuclear magnetic resonance (NMR) methods to P450 enzymes. Extensive sequential 1H, 15N and 13C resonance assignments have been made in cytochrome P450cam (CYP101). Residues in the active site of CYP101 can be rapidly distinguished by comparison of paramagnetic and diamagnetic forms of the enzyme. Active site resonances are observed only in the diamagnetic form. Assignments have been used to monitor the interactions of CP101 with its physiological redox partner and effector, putidaredoxin (Pdx). Based on these observations, a new model for effector activity of Pdx has been proposed. During the next period of this project, the sequential assignments of CYP101 will be completed in multiple forms of the enzyme, local dynamics will be characterized as a function of oxidation state, substrate binding and effector binding in order to shed further light on the mechanism of "uncoupling", in which reducing equivalents are lost as superoxide, peroxide and water without turnover of substrate. Methodology for applying NMR to the characterization of active sites of other P450 enzymes will be tested, with CYP102 (cytochrome P450 BM-3) and CYP3A4, an important human P450, as test cases. Methodology developed during the current period for NMR structural characterization of metalloproteins will be further refined. This includes two-dimensional NMR methods that can be used near paramagnetic centers in proteins, and extraction of structural information from magnetic anisotropy data such as residual dipolar couplings and dipolar shifts.