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 在格里芬实验室，我将使用固态核磁共振技术来研究hVDAC1的转运机制 和hVDAC2，线粒体外膜上的高效门控蛋白。它们是相关联的 患有多种疾病，包括神经退行性疾病、心脏疾病和癌症。我们假设 HVDAC1的门控机制涉及β-Barrel的构象变化，N- β-Barrel内的终端，或两者兼而有之。我将使用允许13C、15N和2H标记的无细胞标记策略 整合到hVDAC1、hVDAC2和其他蛋白质中。它们将被放置在2D晶体中 磷脂双层膜采集分辨率高，1H检测到核磁共振波谱。这些实验将被用来 分配共振并获得结构约束，以便确定已完成的结构 HVDAC1、hVDAC2和与它们结合的小分子。我会用1.3毫米和0.7毫米的转子来旋转 以高于100 kHz的频率采样，以消除强1H-1H偶极耦合。这样的纺纱 频率提高了频谱分辨率，延长了均匀弛豫时间。前者很重要 用于共振分配，而后者用于在多维空间中保持有用的高灵敏度水平 实验。低温核磁共振将用于冻结仅与hVDAC1和hVDAC1弱结合的分子 HVDAC2，如ATP和NADH，处于结合状态，从而可以获得详细的结构信息。 冻结样品还有助于实现动态核极化(DNP)。 培训计划 我以前有相当多的执行DNP的研究经验，格里芬实验室是 著名的是。在格里芬实验室，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力学。为了传播我的发现，我将撰写多篇科学论文 发表论文，并在国内和国际会议上展示我的原创研究。此外，我还将帮助 准备拨款提案，帮助我为建立自己的独立实验室做好这方面的准备。 环境 麻省理工学院是一所顶尖学府，在成功培养博士后方面有着悠久的历史。格里芬教授 他自己培养了大约75名博士后，其中许多人开始了独立的成功职业生涯 在各自领域处于领先地位的研究人员。此外，麻省理工学院有很强的磁共振存在 除了格里芬教授的实验室。梅洪教授是结构生物界的领军人物， 约翰·沃教授帮助奠定了固态核磁共振的基础。建议的研究、培训计划和 制度环境使麻省理工学院的格里芬实验室成为我接受博士后培训的完美场所。