The Structures of hVDAC-1 and hVDAC-2 by High Frequency Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

高频魔角旋转核磁共振波谱分析hVDAC-1和hVDAC-2的结构

• 批准号: 10311025
• 负责人: Edward Paul Saliba
• 金额: $ 6.6万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10311025
• 关键词: Active Sites; Adopted; Affect; Amino Acid Sequence; Amino Acids; Apoptosis; Apoptotic; Applications Grants; B-Cell Lymphomas; BCL-2 Protein; Binding; Binding Sites; Cell Death; Cell Nucleus; Cell Survival; Cell physiology; Cells; Communities; Crystallization; Detection; Disease; Drug Design; Environment; Exhibits; Freezing; Frequencies; Generations; Heart Diseases; Human; Institution; International; Ions; Knowledge; Label; Lipid Bilayers; Location; MEKs; Magic; Magnetic Resonance; Malignant Neoplasms; Measures; Metabolic; Methods; Mitochondria; Molecular Conformation; Movement; Mutation; NADH; NMR Spectroscopy; National Research Service Awards; Neurodegenerative Disorders; Nuclear; Nuclear Magnetic Resonance; Nucleotides; Outer Mitochondrial Membrane; Paper; Pathway interactions; Pharmaceutical Preparations; Phospholipids; Physiologic pulse; Physiological; Physiology; Play; Positioning Attribute; Postdoctoral Fellow; Preparation; Proteins; Protons; Ras/Raf; Recording of previous events; Regulation; Relaxation; Research; Research Personnel; Research Training; Resolution; Rimantadine; Role; Sampling; Signal Transduction; Structure; Techniques; Technology; Temperature; Time; Training; Tumor Suppression; Voltage-Dependent Anion Channel; Writing; biological systems; career; cryogenics; design; erastin; experience; experimental study; fighting; implementation facilitation; improved; member; mitochondrial dysfunction; post-doctoral training; pro-apoptotic protein; protein structure; small molecule; solid state nuclear magnetic resonance; structural biology; symposium; tool; tumor; two-dimensional; voltage-dependent anion channel 2

PROJECT SUMMARY/ABSTRACT Research In the Griffin lab, I will use solid state NMR techniques to study the transport mechanisms of hVDAC1 and hVDAC2, highly efficient gating proteins in the mitochondrial outer membrane (MOM). They are associated with many diseases including neurodegenerative diseases, cardiac diseases, and cancer. We hypothesize that the gating mechanism of hVDAC1 involves a conformational change of the β-barrel, movement of the N- terminus within the β-barrel, or both. I will use cell free labeling strategies that allow for 13C, 15N, and 2H labels to be incorporated into hVDAC1, hVDAC2, and other proteins. They will be placed in 2D crystals of phospholipid bilayers to collect high resolution, 1H detected NMR spectra. These experiments will be used to assign resonances and obtain structural constraints to allow for the determination of completed structures of hVDAC1, hVDAC2, and small molecules that bind to them. I will use 1.3 mm and 0.7 mm rotors to spin the sample at frequencies higher than 100 kHz to remove strong 1H-1H dipolar couplings. Such spinning frequencies improve spectral resolution and lengthen homogenous relaxation times. The former is important for resonance assignments, and the latter for maintaining a usably high level of sensitivity in multidimensional experiments. Cryogenic NMR will be used to freeze molecules that only weakly bind to hVDAC1 and hVDAC2, such as ATP and NADH, in their bound states so that detailed structural information can be obtained. Freezing the sample will also facilitate the implementation of dynamic nuclear polarization (DNP). Training Plan I have a considerable amount of previous research experience performing DNP, which the Griffin lab is renowned for. In the Griffin lab, I will gain experience applying my DNP knowledge to biological systems to determine their structure and dynamics. In order to disseminate my findings, I will write multiple scientific papers and present my original research at national and international conferences. Furthermore, I will aid in the preparation of grant proposals to help prepare me for this aspect of starting my own, independent lab. Environment MIT is a top tier institution with a long history of successfully training postdoctoral fellows. Prof. Griffin has himself trained ~75 postdocs and many of these have gone on to begin successful careers as independent researchers that are leaders in their fields. Furthermore, MIT has a strong magnetic resonance presence besides just Prof. Griffin’s lab. Prof. Mei Hong is a leading member of the structural biology community, and Prof. John Waugh helped to lay the groundwork for solid state NMR. The proposed research, training plan, and institutional environment make the Griffin lab at MIT the perfect place for me to receive postdoctoral training.

项目摘要/摘要 研究 在Griffin实验室中，我将使用固态NMR技术来研究HVDAC1的运输机制 和HVDAC2，线粒体外膜（MOM）中高效的门控蛋白。它们是相关的 许多疾病包括神经退行性疾病，心脏疾病和癌症。我们假设这一点 HVDAC1的门控机制涉及β-桶的构象变化，N-的运动 β-桶内的末端，或两者兼有。我将使用允许13C，15N和2H标签的无细胞标签策略 掺入HVDAC1，HVDAC2和其他蛋白质中。它们将被放置在2D晶体中 磷脂双层以收集高分辨率，1小时检测到NMR光谱。这些实验将用于 分配共振并获得结构性约束，以确定已完成的结构 HVDAC1，HVDAC2和与它们结合的小分子。我将使用1.3毫米和0.7毫米转子旋转 在高于100 kHz的频率下进行样品，以去除强1H-1H偶性耦合。这样的旋转 频率改善了光谱分辨率和长度均匀放松时间。前者很重要 用于共振分配，后者是维持多维的高度敏感性 实验。低温NMR将用于冻结仅弱结合HVDAC1和 HVDAC2（例如ATP和NADH）处于其约束状态，以便可以获得详细的结构信息。 冻结样品还将促进动态核极化（DNP）的实施。 培训计划 我有大量以前的研究经验表现DNP，Griffin Lab是 闻名。在格里芬实验室中，我将获得将我的DNP知识应用于生物系统的经验 确定它们的结构和动态。为了传播我的发现，我将写多个科学 论文并在国家和国际会议上介绍我的原始研究。此外，我将协助 准备赠款建议，以帮助我为启动自己独立实验室的这一方面做好准备。 环境 麻省理工学院是一家顶级机构，拥有成功培训博士后研究员的悠久历史。格里芬教授 他本人曾经训练过〜75个博士后，其中许多已经开始成功地成为独立的职业 研究人员是他们领域的领导者。此外，麻省理工学院具有很强的磁共振 除了格里芬教授的实验室。 Mei Hong教授是结构生物学社区的主要成员， 约翰·沃（John Waugh）教授为固态NMR奠定了基础。拟议的研究，培训计划以及 机构环境使MIT的Griffin Lab成为我接受博士后培训的理想场所。