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

开发生物物理方法来研究膜蛋白的高分辨率结构和动力学

• 批准号: 10321560
• 负责人: Ayyalusamy Ramamoorthy
• 金额: $ 18.12万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10321560
• 关键词: Caliber; Catalysis; Chemicals; Complex; Cytochrome P450; Cytochromes; Cytochromes b5; Detergents; Development; Disease; Environment; Exhibits; G-Protein-Coupled Receptors; Guanidines; Heme; Human; Integral Membrane Protein; Ions; Laboratories; Length; Malignant Neoplasms; Malignant neoplasm of prostate; Membrane; Membrane Lipids; Membrane Proteins; Metabolism; Molecular; Outcome; Oxidation-Reduction; Oxidoreductase; Oxygenases; Pharmaceutical Preparations; Pharmacologic Substance; Polymers; Protein Isoforms; Resolution; Structure; System; Techniques; Transmembrane Domain; Water; biophysical techniques; design; divalent metal; insight; malignant breast neoplasm; membrane reconstitution; nanodisc technology; nanodisk; prevent; protein complex; reconstitution; solid state nuclear magnetic resonance

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.

抽象的 膜蛋白的功能重构是NMR和 其他生物物理技术研究其在天然膜上的高分辨率动态结构 环境。在此应用中，我们建议开发能够实现高分辨率结构研究的方法 通过多种生物物理技术的膜蛋白和蛋白质 - 蛋白质复合物的构成。我们将发展 纳米轴技术，用于无污染的直接提取和膜蛋白的功能重构 各种膜蛋白（包括单通跨膜蛋白）的结构研究（例如 哺乳动物细胞色素和血红素加氧酶）和整合性膜蛋白（包括GPCR和 鸟根出口商）。在我们的实验室中开发的合成聚合物具有与 易于控制的尺寸（直径约8至〜60 nm），稳定在pH和二价金属离子上，并且能够 直接提取膜蛋白。我们的初步结果表明这些纳米盘（<20 nm 直径）和宏 - 纳米盘（> 20 nm直径）代表一个令人兴奋的溶液和固态系统 膜蛋白的NMR研究。 我们还建议使用新开发的纳米轴技术和NMR方法来调查 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合作伙伴如何调节P450催化以及P450如何调节 代谢化学不同的底物。与P450的催化活性有关的结构方面 由于缺乏全长活性形式的高分辨率结构，因此继续难以捉摸。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P450 同源物。在这项研究中，我们将研究的结构，动力学和跨膜结构域的方向 单独使用全长哺乳动物P450（2B4、3A4和3A5同工型），并与其氧化还原伙伴B5和 CPR，使用高分辨率溶液和固态NMR技术的组合纳入纳米盘。 我们还将在底物存在下研究纳米盘中的三元P450-B5-CPR复合物 阐明了CPR和B5对P450 2B4催化的明显不同效果的分子来源。这 拟议的关于P450-REDOX复合物的研究的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 同样对于设计有效的药物也很有用，以最终治疗和预防包括癌症在内的疾病。