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

项目总结/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hVDAC 1的转运机制 和hVDAC 2，线粒体外膜（mtDNA）中的高效门控蛋白。它们相关联 包括神经变性疾病、心脏病和癌症在内的许多疾病。我们假设 hVDAC 1的门控机制涉及β-桶的构象变化，N- 末端在β-桶内，或两者。我将使用允许13 C、15 N和2 H标记的无细胞标记策略 将其掺入hVDAC 1、hVDAC 2和其他蛋白质中。它们将被放置在2D晶体中， 磷脂双层收集高分辨率，1H检测NMR光谱。这些实验将用于 分配共振并获得结构约束，以允许确定 hVDAC 1、hVDAC 2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的频率下采样，以消除强1H-1H偶极耦合。这样的旋转 频率提高了光谱分辨率并延长了均匀弛豫时间。前者很重要 对于共振分配，后者用于在多维环境中保持可用的高灵敏度水平。 实验低温NMR将用于冷冻仅与hVDAC 1弱结合的分子， hVDAC 2，如ATP和NADH，在它们的结合状态，以便可以获得详细的结构信息。 冷冻样品也将促进动态核极化（DNP）的实施。 培训计划 我以前有相当多的研究经验，执行DNP，格里芬实验室是 著名的。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， 决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， 论文，并在国家和国际会议上展示我的原创研究。此外，我将帮助 准备拨款申请，帮助我为建立自己的独立实验室做好准备。 环境 麻省理工学院是一个顶级的机构，具有成功培养博士后研究员的悠久历史。格里芬教授 我自己培养了大约75名博士后，其中许多人已经开始成功的独立职业生涯 在各自领域处于领先地位的研究人员。此外，麻省理工学院有一个强大的磁共振存在 除了格里芬教授的实验室梅宏教授是结构生物学领域的领军人物， 教授约翰·沃帮助奠定了固体核磁共振的基础。拟议的研究、培训计划和 麻省理工学院的格里芬实验室是我接受博士后培训的理想场所。

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.