Computational and Vibrational Probes of CYP3A4 Solution Dynamics

 描述(申请人提供)：毫无疑问，细胞色素P450细胞色素P3A4是人类异种代谢中最重要的单一蛋白质。CYP3A4在药物代谢中的突出地位导致每年对作为该酶底物的数千个分子的活性进行常规调查。已经开发了许多计算方法来预测CYP3A4的新陈代谢。基于配体的方法考虑了一系列令人困惑的潜在底物的结构-活性关系，以及一系列相当不可靠的预测。关键问题是，CYP3A4的构象灵活性或可塑性尚未被提供，因此，这些预测中没有考虑到这一因素。这项研究计划的长期目标是开发和应用新的方法，极大地扩大我们对P450酶机制的理解。这项应用的目标是结合计算化学、化学生物学和分子光谱学的最新工具，深入了解血红素的反应性和蛋白质动力学的耦合，以及与氧化还原伙伴的相互作用对CYP3A4对配体的适应的影响。为了达到这一目标，我们将有三个具体的目标，我们将：1)描绘溶液中CYP3A4构象的范围和这些构象之间的过渡途径。能够采样低频率蛋白质运动的分子动力学方法将提供迄今为止通过其他方法难以捉摸的CYP3A4溶液构象的描述。2)确定配体和氧化还原伙伴相互作用对CYP3A4血红素动力学的功能影响。共振拉曼光谱将被用来探测由于细胞色素P450还原酶和细胞色素b5之间的相互作用而导致的构象移动和电子结构变化，这些相互作用预先组织了该活性部位以促进电子转移。3)选择性掺入拉曼活性振动探针对细胞色素P3A4静电图谱的影响。选择性地将氨基酸类似物与振动探针结合，可以通过配体结合引起的溶剂变色位移、蛋白质与氧化还原伙伴的相互作用以及由此产生的构象交换来直接观察局部静电变化。为了对细胞色素P3A4代谢提供准确的代谢预测，必须了解构象可塑性以及构象与血红素动力学之间的相互作用。我们建议使用一套交互式实验设计来解决我们目前理解中的这些漏洞。这组研究的意义在于，有望更清楚地了解配体和氧化还原伙伴诱导的P450动力学和血红素反应活性的变化，以及这些迄今未被研究的因素对CYP3A4适应新底物结构的影响。

 DESCRIPTION (provided by applicant): There is little doubt that cytochrome P450 CYP3A4 is the single most important protein in human xenobiotic metabolism. The prominence of CYP3A4 in drug metabolism results in routine investigation of the activity of thousands of molecules annually as substrates for this enzyme. Numerous computational methods have been developed to predict CYP3A4 metabolism. Ligand-based methods have considered structure-activity relationships for a baffling array of potential substrates, with a complex set of rather unreliable predictions. The key problem is that the conformational flexibility, or plasticity, of CYP3A4 has not been available and hence, has not been factored into these predictions. The long-term goal of this research program is to develop and apply novel approaches that dramatically expand our understanding of the P450 enzyme mechanisms. The objective of this application is to combine state-of-the-art tools of computational chemistry, chemical biology, and molecular spectroscopy to gain insight into the coupling of heme reactivity and protein dynamics, and the influence of interactions with redox partners with the adaptation of CYP3A4 to ligands. To meet this objective, there are three Specific Aims, we will: 1) Delineate the range of CYP3A4 conformational states in solution and the transition pathways between these conformers. Molecular dynamics methods capable of sampling low frequency protein motions will afford a description of CYP3A4 solution conformations, that to date, have been elusive by other means. 2) Define the functional consequences of ligand and redox partner interactions on CYP3A4 heme dynamics. Resonance Raman spectroscopy will be used to probe the conformational shifts and electronic structure changes resulting from interactions between CYP3A4, cytochrome P450 reductase, and cytochrome b5 that pre-organize that active site to facilitate electron transfer. 3) Map changes in CYP3A4 electrostatics by selective incorporation of Raman-active vibrational probes. The selective incorporation of amino acid analogs with vibrational probes will permit direct observation of local electrostatic changes through solvatochromic shifts induced by ligand binding, protein-protein interactions with redox partners, and resultant conformational interchanges. To afford accurate metabolic predictions for CYP3A4 metabolism, the conformational plasticity and the interactions between conformer and heme dynamics must be understood. We propose to approach these holes in our current understanding using a suite of interactive experimental designs. The significance of this set of studies is the promise of a clearer insight into ligand- and redox-partner induced changes in P450 dynamics and heme reactivity and the impact of these heretofore understudied factors in the adaptation of CYP3A4 to new substrate structures.