Conformations and Dynamics of Cytochrome P450s via 2D Infrared Spectroscopy

项目摘要 细胞色素P450（Cytochrome P450 s，P450）是参与多种合成和代谢生物化学的氧化酶。 在整个生命王国中的生物反应。重要的是，P450是药物的中心酶， 新陈代谢.尽管结构和催化机制相似，但P450同系物的特异性差异很大 对不同底物的选择性和催化选择性，这些差异可能具有关键的治疗作用。 后果预测P450在任何给定底物上的反应性的主要障碍是我们的不完整性。 了解蛋白质动力学的参与-多种状态的群体及其相互转换 - 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 部分底物相对于反应性中间体化合物I，并导致多种产物。在 此外，活性位点在一个结合状态内的灵活性可以允许底物的多个位置， 在其寿命期间接近反应性化合物I中间体。实验测试这些可能性是 然而，由于蛋白质的复杂性、异质性和运动的贡献， 蛋白质的弹性。红外（IR）光谱可以解析蛋白质构象， 即使在最快的时间尺度上相互转换的动态，而且，2D技术可以量化 构象异质性，以及频率波动幅度和时间尺度，他们 被采样。当与用IR探针进行位点选择性标记所提供的空间精度相结合时， 这种方法应该能够前所未有地描述P450的能源景观。本申请是 针对柔性不同的三种P450：P450 cam、3A 4和2C 9及其与底物的复合物 它们以不同的区域选择性被羟基化。动力学的测量和比较 底物复合物将提供信息，用于评估动力学如何参与它们的不同的 活动关于P450的新信息将促进我们对生物物理机制的理解， 作为酶功能的基础，并提高我们预测给定分子上P450活性的能力， 开发出更好的药物，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.