Development of biophysical approaches to investigate high-resolution structure and dynamics of membrane proteins

摘要 膜蛋白的功能重建一直是核磁共振和核磁共振技术应用的主要障碍。 在天然膜上研究其高分辨率动态结构的其他生物物理技术 环境。在这一应用中，我们建议开发能够进行高分辨率结构研究的方法 通过各种生物物理技术对膜蛋白和蛋白质-蛋白质复合体进行研究。我们将发展 免洗涤剂直接提取膜蛋白及膜蛋白功能重构的纳米盘技术 多种膜蛋白的结构研究，包括单程跨膜蛋白(如 哺乳动物细胞色素和血红素加氧酶)和完整的膜蛋白(包括GPCRs和 胍类出口商)。我们实验室开发的合成聚合物具有形成纳米盘的能力 易于控制的尺寸(直径从~8到~60 nm)，对pH和二价金属离子稳定，并能够 直接提取膜蛋白。我们的初步结果表明，这些纳米盘(&lt；20 nm 直径)和宏观纳米盘(直径20 nm)代表了溶液和固态的激发体系 膜蛋白的核磁共振研究。 我们还建议使用最新开发的纳米盘技术和核磁共振方法来研究 哺乳动物细胞色素P450(P450)与其氧化还原伙伴(P450-还原酶(CPR))的结构相互作用 和细胞色素b5(B5))，以更好地了解氧化还原伙伴如何调节P450催化以及P450如何 代谢化学成分多样的底物。与P450催化活性有关的结构方面 由于缺乏全长、活跃形式的高分辨率结构，仍然难以捉摸。目前， P450的结构研究仅限于各种截短的哺乳动物和水溶性细菌的P450 同系物。在这项研究中，我们将研究结构，动力学和跨膜结构域的方向 哺乳动物全长P450(2B4、3A4和3A5亚型)单独以及与其氧化还原伙伴b5和3A5形成复合体 CPR，包含在纳米盘中，使用高分辨率溶液和固态核磁共振技术的组合。 我们还将研究在底物存在的情况下纳米盘中的三元P450-b5-CPR络合物 阐明CPR和b5对P450 2B4催化的显著不同作用的分子来源。这个 P450-氧化还原络合物的拟议研究结果将提供结构和动力学/功能 调节多种底物的P450代谢的原理。从这项研究获得的结果将 这也有助于设计有效的药物，最终治疗和预防包括癌症在内的疾病。

Abstract Functional reconstitution of membrane proteins has been the major roadblock for the application of NMR and other biophysical techniques to investigate their high-resolution dynamic structures in a native membrane environment. In this application, we propose to develop approaches to enable high-resolution structural studies of membrane proteins and protein-protein complexes by a variety of biophysical techniques. We will develop nanodisc technology for detergent-free direct extraction and functional reconstitution of membrane proteins for structural studies of a variety of membrane proteins including single-pass transmembrane proteins (such as mammalian cytochromes and heme oxygenase) and integral membrane proteins (including GPCRs and Guanidine exporter). Synthetic polymers developed in our laboratory exhibit the ability to form nanodiscs with easily controllable sizes (from ~8 to ~60 nm diameter), are stable against pH and divalent metal ions and capable of directly extracting membrane proteins. Our preliminary results demonstrate that these nanodiscs (<20 nm diameter) and macro-nanodiscs (>20 nm diameter) represent an exciting system for solution and solid-state NMR studies of membrane proteins. We also propose to use the newly developed nanodisc technology and NMR approaches to investigate the structural interactions of mammalian cytochrome-P450 (P450) with its redox partners (P450-reductase (CPR) and cytochrome-b5 (b5)) to better understand how redox partners regulate P450 catalysis and how P450s metabolize chemically diverse substrates. The structural aspects pertaining to the catalytic activity of P450s continue to remain elusive due to a lack of high-resolution structures in their full-length, active forms. Presently, structural studies of P450s are restricted to various truncated mammalian and water-soluble bacterial P450 homologs. In this study, we will investigate the structure, dynamics and transmembrane domain orientation of full-length mammalian P450s (2B4, 3A4 and 3A5 isoforms) alone and in complex with its redox partner b5 and CPR, incorporated in nanodiscs, using a combination of high-resolution solution and solid-state NMR techniques. We will also investigate the ternary P450-b5-CPR complex in nanodiscs in the presence of substrates to elucidate the molecular origin of the strikingly different effects CPR and b5 have on P450 2B4 catalysis. The outcome of the proposed studies on P450-redox complexes will provide structure and dynamics/function principles regulating P450 metabolism of a wide variety of substrates. The results obtained from this study will also be useful to design potent drugs to ultimately treat and prevent diseases including cancer.