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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要研究在Griffin实验室，我将使用固态NMR技术来研究hVDAC1的传输机制 hVDAC2，线粒体外膜 (MOM) 中的高效门控蛋白。患有许多疾病，包括神经退行性疾病、心脏病和癌症。 hVDAC1 的门控机制涉及 β-桶的构象变化、N- 的运动我将使用允许 13C、15N 和 2H 标记的无细胞标记策略。被整合到 hVDAC1、hVDAC2 和其他蛋白质中它们将被放置在 2D 晶体中。磷脂双层收集高分辨率、1H 检测到的 NMR 谱这些实验将用于分配共振并获得结构约束，以便确定完整的结构 hVDAC1、hVDAC2 以及与其结合的小分子我将使用 1.3 毫米和 0.7 毫米转子来旋转。以高于 100 kHz 的频率进行采样，以消除强 1H-1H 偶极耦合。频率提高了光谱分辨率并延长了均匀弛豫时间。用于共振分配，后者用于在多维中保持可用的高水平灵敏度低温核磁共振将用于冷冻仅与 hVDAC1 和微弱结合的分子。 hVDAC2，例如 ATP 和 NADH，处于结合状态，以便可以获得详细的结构信息。冷冻样品也将有助于动态核极化（DNP）的实施。培训计划我之前有大量执行 DNP 的研究经验，Griffin 实验室是在 Griffin 实验室，我将获得将 DNP 知识应用于生物系统的经验。为了传播我的发现，我将撰写多篇科学文章来确定它们的结构和动态。论文并在国内和国际会议上展示我的原创研究。此外，我将协助准备拨款提案，以帮助我为创办自己的独立实验室做好准备。环境麻省理工学院是一所顶级机构，在成功培养博士后研究员方面拥有悠久的历史。他自己培训了约 75 名博士后，其中许多人已经以独立的身份开始了成功的职业生涯此外，麻省理工学院在磁共振领域拥有强大的影响力。除了格里芬教授的实验室之外，梅红教授还是结构生物学界的主要成员。 John Waugh 教授帮助奠定了固态核磁共振的研究、培训计划和基础。麻省理工学院的格里芬实验室的制度环境成为我接受博士后培训的理想场所。