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.

摘要 膜蛋白的功能重建一直是核磁共振应用的主要障碍， 研究其在天然膜中的高分辨率动态结构的其他生物物理技术 环境在这个应用中，我们提出了开发方法，使高分辨率的结构研究 膜蛋白和蛋白质-蛋白质复合物的各种生物物理技术。我们将开发 纳米盘技术，用于无洗涤剂直接提取和膜蛋白的功能重建， 多种膜蛋白的结构研究，包括单程跨膜蛋白（例如 哺乳动物细胞色素和血红素加氧酶）和整合膜蛋白（包括GPCR和 胍输出者）。我们实验室开发的合成聚合物表现出形成纳米盘的能力， 易于控制的尺寸（直径约8至约60 nm），对pH和二价金属离子稳定， 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）和宏观纳米盘（直径>20 nm）代表溶液和固态的激发系统 膜蛋白的NMR研究。 我们还建议使用新开发的纳米盘技术和NMR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催化和P450如何 代谢化学上不同的底物。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，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 同系物。在这项研究中，我们将研究的结构、动力学和跨膜结构域方向 单独的全长哺乳动物P450（2B 4、3A 4和3A 5同种型）和与其氧化还原配偶体b5的复合物， CPR，结合在纳米盘中，使用高分辨率溶液和固态NMR技术的组合。 我们还将研究在底物存在下纳米盘中的三元P450-b5-CPR复合物， 阐明CPR和b5对P450 2B 4催化作用的显著不同作用的分子起源。的 对P450-氧化还原复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。从这项研究中获得的结果将 还可用于设计有效的药物以最终治疗和预防包括癌症在内的疾病。