Conformations and Dynamics of Cytochrome P450s via 2D Infrared Spectroscopy

通过 2D 红外光谱研究细胞色素 P450 的构象和动力学

• 批准号: 9873047
• 负责人: Megan Corrine Thielges
• 金额: $ 22.22万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9873047
• 关键词: Active Sites; Binding; Biochemical Reaction; Biological Assay; Biological Process; Biology; Biophysical Process; Biophysics; Camphor; Complex; Cytochrome P450; Cytochromes; Data; Development; Drug Design; Drug Kinetics; Environment; Enzymes; Evaluation; Family; Frequencies; Generations; Heme; Heterogeneity; Human; Hydrocarbons; Hydroxylation; Label; Lead; Life; Measurement; Measures; Metabolic; Modeling; Molecular; Molecular Conformation; Motion; Mutation; Nature; Organism; Oxidases; Oxides; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacotherapy; Play; Population; Population Dynamics; Positioning Attribute; Protein Conformation; Protein Dynamics; Protein Isoforms; Proteins; Research; Role; Sampling; Series; Site; Specificity; Structure; Techniques; Testing; Therapeutic; Variant; design; drug metabolism; experimental study; flexibility; heme a; human disease; improved; infrared spectroscopy; microbial; mutant; norcamphor; oxidation; two-dimensional; vibration

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.

项目概要 细胞色素 P450 (P450) 是参与多种合成和代谢生化过程的氧化酶 整个生命王国中生物体的反应。重要的是，P450 是药物的核心酶 代谢。尽管结构和催化机制相似，P450 同系物的特异性差异很大 对不同底物的催化选择性，这些差异可能具有关键的治疗作用 结果。预测 P450 在任何给定底物上的反应性的一个主要障碍是我们的不完整 了解蛋白质动力学的参与——多种状态的总体及其相互转换 - 区域选择性机制。具体来说，多个束缚态的总体可以定向多个 部分底物相对于反应性中间体化合物 I 并产生多种产物。在 此外，一种结合态内活性位点的灵活性可以允许底物的多个位置 在其生命周期内接近反应性化合物 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，具有挑战性 在非常快的时间内实现蛋白质的灵活性。红外 (IR) 光谱可以解析蛋白质构象并 即使在最快的时间尺度上也可以相互转换的动力学，此外，2D 技术可以量化 构象异质性，以及它们的频率波动幅度和时间尺度 被抽样。当与红外探针位点选择性标记提供的空间精度相结合时， 该方法应该能够对 P450 的能源景观进行前所未有的描述。这个应用程序是 针对灵活性不同的三种 P450：P450cam、3A4 和 2C9 及其与基材的复合物 具有不同水平的区域选择性羟基化。动力学的测量和比较 底物复合物将提供信息来评估动力学如何参与其不同的过程 活动。有关 P450 的新信息将增进我们对生物物理机制的理解 是酶功能的基础，并提高我们预测给定分子上 P450 活性的能力，从而 开发具有改善药代动力学的更好药物。