Cytochrome P450 Oxidoreductase: Conformational Dynamics and Membrane Interactions

项目摘要 膜蛋白的结构和动力学的分析是非常具有挑战性的，由于存在的蛋白质， 相分离边界-磷脂膜。然而，与脂质双分子层的结合对于这些细胞是至关重要的。 蛋白质在运输、信号转导、生物合成和其他细胞过程中发挥作用。膜 催化电子传递的蛋白质甚至更难研究，因为它们通常包含多个 阻碍光谱分析的发色团和荧光团。在这个项目中，我们正在开发一个 核磁共振（NMR）和瞬态吸收（TA）光谱的强大应用， 能够分析这种富含发色团的氧化还原体系。我们的案例研究是细胞色素P450 一种向位于内质网中的细胞色素P450提供电子的氧化还原酶（POR (ER)肝细胞和其他组织的膜。磷脂膜的存在是严格要求的 对于P450与POR的有效相互作用，其需要呈现“开放”构象以允许 P450结合。根据文献报道和我们自己的实验，分离的可溶性胞质片段 的POR是自动抑制的，并在溶液中采取主要的“封闭”状态，导致可忽略的相互作用 P450我们推测，膜结合触发了POR的“开放”构象，并减轻了其 自我抑制这种开放状态通过细胞色素P450的结合而进一步稳定。我们打算测试一下 假设具有以下具体目的：（1）确定氧化的优先构象状态 和减少的膜结合POR;和（2）创建减少的膜结合POR的结构模型， 和氧化状态的存在和不存在的细胞色素P450。在目标1中，我们将测量 利用FRET和瞬态吸收（TA）光谱在POR中的特定位点之间进行比较， 介绍我们期望在一系列的POR中确定供体-受体距离的分布 样本，这将测试我们的假设，即膜结合POR的开放-闭合平衡转移后， 减少并与P450相互作用，以更开放的群体。在目标2中，我们将建立相对 通过绘制POR与细胞膜的接触， 膜表面将检测膜接触的顺磁弛豫增强（PRE）， 甲基-TROSY信号来自POR。膜接触信息结合开闭数据 过渡将被用作结构约束，以创建POR-纳米盘和POR-纳米盘的分子模型。 纳米盘-P450复合物，其将用分子动力学进一步细化。这项工作意义重大 因为我们的目标是获得膜结合POR的第一个结构模型，以帮助制定可测试的 关于POR功能中特定突变作用的假说。第二个重要的含义是 开发基于TA和NMR光谱组合的创新方法， 将广泛用于富含发色团的柔性膜蛋白的结构分析。

PROJECT SUMMARY Analysis of structure and dynamics of membrane proteins is extremely challenging due to presence of the phase-separation boundary—a phospholipid membrane. Yet, association with lipid bilayer is critical for these proteins to function in transport, signal transduction, biosynthesis, and other cellular processes. Membrane proteins that catalyze electron transport are even more difficult to study because they often include multiple chromophores and fluorophores hindering spectroscopic interrogation. In this project, we are developing a powerful application of Nuclear Magnetic Resonance (NMR) and transient absorption (TA) spectroscopies to enable analysis of such chromophore-rich redox systems. Our case study is the cytochrome P450 oxidoreductase (POR) that supplies electrons to the cytochromes P450 localized in endoplasmic reticulum (ER) membrane of liver cells and other tissues. The presence of a phospholipid membrane is strictly required for a productive interaction of P450 with POR, which needs to assume an "open" conformation to allow for P450 binding. According to literature reports and our own experiments, the isolated soluble cytosolic fragment of POR is auto-inhibited and adopts a predominantly "closed" state in solution leading to negligible interaction with P450. We hypothesize that membrane binding triggers an "open" conformation of POR and relieves its auto-inhibition. This open state is further stabilized by binding of the cytochrome P450. We propose to test this hypothesis with the following Specific Aims: (1) Determine the preferential conformational state of oxidized and reduced membrane-bound POR; and (2) Create a structural model of membrane-bound POR in reduced and oxidized states in the presence and absence of cytochrome P450. In Aim 1, we will measure the distances between specific sites in POR using FRET with transient absorption (TA) spectroscopy—a method we recently introduced. We expect to determine the distribution of the donor-acceptor distances in a series of POR samples, which will test our hypothesis that the open-closed equilibrium of membrane-bound POR shifts upon reduction and interaction with P450 to a more open population. In Aim 2, we will establish the relative orientation of the cytosolic domain of POR near the membrane through mapping its contacts with the membrane surface. The membrane contacts will be detected paramagnetic relaxation enhancement (PRE) of methyl-TROSY signals from POR. The membrane contacts information combined with data on the open-closed transition will be used as structural restraints to create a molecular model of POR-nanodisc and POR- nanodisc-P450 complexes which will be further refined with molecular dynamics. This work is significant because we aim to obtain the first structural model of membrane-bound POR to help formulate testable hypotheses on the role of specific mutations in the POR function. The second significant implication is development of the innovative approach based on a combination of the TA and NMR spectroscopies, which will be widely useful for structural analysis of flexible chromophore-rich membrane proteins.