Conformations and Dynamics of Cytochrome P450s via 2D Infrared Spectroscopy

项目总结 细胞色素P450(P450)是一种广泛参与合成和代谢生化的氧化酶 在整个生命王国的有机体中的反应。重要的是，P450是药物的中心酶 新陈代谢。尽管P450同系物的结构和催化机制相似，但它们的特异性有很大的差异 在不同的底物上形成和催化选择性，这些差异可能具有关键的治疗作用 后果。预测任何特定底物上的P450反应性的一个主要障碍是我们的不完整 理解蛋白质动力学的参与--多态种群及其相互转化 --在区域选择性机制中。具体地说，多个束缚态的布居可以定向多个 部分底物相对于活性中间化合物I并导致多个产物。在……里面 此外，活性部位在一个结合状态内的灵活性可以允许底物的多个位置 接近活性化合物I在其生命周期内的中间体。通过实验测试这些可能性 然而，由于蛋白质的复杂、不同的性质和运动的贡献，具有挑战性 在非常快的时间尺度上达到蛋白质的灵活性。红外(IR)光谱可以解析蛋白质构象和 即使是在最快的时间尺度上相互转换的动力学，而且2D技术可以量化 构象异质性，以及它们与之相关的频率波动幅度和时间尺度 都是抽样的。当与红外探针的位置选择性标记提供的空间精度相结合时， 方法应该能够史无前例地描述P450的能源景观。此应用程序是 针对三种柔韧性不同的P450：P450 cam、3A4和2C9及其与底物的络合物 具有不同区域选择性的羟基化产物。机械系统动力学性能的测量与比较 底物复合体将提供信息以评估动力学如何参与其不同的 活动。有关P450的新信息将促进我们对P450生物物理机制的理解 为酶的功能奠定基础，并提高我们预测给定分子上P450活性的能力，因此 开发更好的药物，改善药代动力学。

PROJECT SUMMARY Cytochrome P450s (P450s) are oxidases involved in a wide variety of synthetic and metabolic biochemical reactions in organisms throughout the kingdoms of life. Importantly, P450s are the central enzymes of drug metabolism. Despite similar structures and catalytic mechanisms, P450 homologs vary greatly in their specificity for and catalytic selectivity on different substrates, and these differences can have critical therapeutic consequences. A major obstacle in predicting P450 reactivity on any given substrate is our incomplete understanding of the involvement of protein dynamics - the population of multiple states and their interconversion - in the mechanisms of regioselectivity. Specifically, the population of multiple bound states could orient multiple parts of the substrate with respect to the reactive intermediate compound I and lead to multiple products. In addition, flexibility of the active site within one bound state could permit multiple positions of the substrate to approach the reactive compound I intermediate during its lifetime. Experimentally testing these possibilities is however challenging due to the complex, heterogeneous nature of the proteins and the contribution of motion on very fast timescales to protein flexibility. Infrared (IR) spectroscopy can resolve protein conformations and dynamics that interconvert on even the fastest timescales and, furthermore, 2D techniques can quantify conformational heterogeneity, as well as the frequency fluctuation amplitudes and timescales with which they are sampled. When combined with the spatial precision provided by site-selective labeling with IR probes, the approach should enable unprecedented description of the energy landscapes of P450s. This application is directed at three P450s which vary in flexibility: P450cam, 3A4, and 2C9 and their complexes with substrates that are hydroxylated with differing levels of regioselectivity. Measurement and comparison of the dynamics of the substrate complexes will provide information for evaluating how dynamics are involved in their different activity. The new information about P450s will advance our understanding of the biophysical mechanisms that underlie enzyme function, as well as improve our ability to predict P450 activity on a given molecule, and thus develop better drugs with improved pharmacokinetics.