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Structural Biology of Membrane Scaffolds

膜支架的结构生物学

基本信息

批准号：
8527804
负责人：
VINZENZ UNGER
金额：
$ 37.89万
依托单位：
NORTHWESTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8527804
关键词：
Actins Amaze Architecture Binding Binding Proteins Biochemical Biogenesis Biological Biological Process Cell division Cell physiology Cells Complex Cryoelectron Microscopy Cytoskeleton DNA Sequence Rearrangement Data Diabetes Mellitus Disease Dynamin Educational process of instructing Electron Spin Resonance Spectroscopy Epilepsy Foundations Generations Goals Guanosine Triphosphate Phosphohydrolases Imagery In Vitro Kinetics Learning Length Life Lipid Bilayers Macromolecular Complexes Malignant Neoplasms Membrane Modeling Molecular Machines Movement N-WASP protein N-terminal Organelles Outcome Play Positioning Attribute Procedures Process Protein Binding Domain Protein Family Proteins Recruitment Activity Regulation Resolution Role SH3 Domains Set protein Shapes Solutions Specificity Structure Tertiary Protein Structure Testing Thinking Time Vesicle Wiskott-Aldrich Syndrome Work amphiphysin base cdc42 GTP-Binding Protein cell motility design dimer human disease image reconstruction insight member molecular assembly/self assembly molecular scale protein complex public health relevance reconstitution research study scaffold structural biology success trafficking

项目摘要

DESCRIPTION (provided by applicant): A vast number of cellular processes depend on the cell's ability to change the shape of their membranes with astounding spatial and temporal accuracy. At a biochemical level, many of the players that participate in these processes are known. What remains unknown, however, is how ensembles of several proteins with often overlapping functions and interaction specificities reproducibly accomplish defined biological outcomes. The largest hurdle towards resolving the mysteries of membrane remodeling is to obtain structural information about the membrane-associated scaffolds that orchestrate every aspect of these processes from changing membrane curvature to membrane fission, and recruitment of the actin cytoskeleton. Overcoming this limitation, we have demonstrated that electron cryomicroscopy provides access to the architecture of membrane-associated scaffolds at resolutions sufficient for the generation of detailed mechanistic models. Exploiting this advance, the long term goals of this project are to understand how members of the BAR superfamily (bin-amphihpysin-rvs family) of proteins generate/stabilize/sense membrane curvature, and how these molecules can selectively recruit interaction partners from a pool of promiscuous multidomain proteins such as the fission GTPase dynamin and the cytoskeletal activator N-WASP. We will use a combination of electron cryomicroscopy, low angle scattering, electron paramagnetic resonance spectroscopy and in vitro biophysical structure-function experiments to pursue three specific aims: (1) we will expand the number of experimentally determined scaffold structures, which will teach us much about their design principles and how these designs contribute to membrane curvature generation and selection of interaction partners, (2) we will exploit what we already learned to test mechanistic models of early steps in scaffold assembly, which may provide vital clues how scaffold assembly is regulated and (3) we will lay the foundation for structural work on higher order macromolecular complexes that BAR-domain proteins form with two of their most important effectors: dynamin and N-WASP. Taken together, these studies will allow us to greatly advance understanding of one of the most fundamental aspects of life: the ability of cells to change the shape of their membranes with amazing spatial and temporal resolution. Understanding these processes will be essential to appreciate how imbalances and errors in membrane remodeling contribute to a broad spectrum of human diseases ranging from epilepsy to diabetes and cancer. PUBLIC HEALTH RELEVANCE: In order to live, cells must continuously change the shape of their membranes with high precision. How cells accomplish this complex task is poorly understood because we have almost no information about how the molecular machines that are responsible for these processes interact with the membranes they reshape. Overcoming this limitation, we established a procedure to visualize membrane-remodeling molecules as they are engaged to their targets. This - for the first time - allows us to closely examine how these molecules function, and how they interact with additional proteins whose recruitment results in a specific biological effect. Visualizing these interactions is key to understanding how errors in these processes can contribute to diseases as varied as epilepsy, diabetes and cancer.

描述(由申请人提供)：大量的细胞过程依赖于细胞以惊人的空间和时间精度改变其膜的形状的能力。在生化层面上，许多参与这些过程的参与者都是众所周知的。然而，尚不清楚的是，几种功能和相互作用特性经常重叠的蛋白质如何重复性地实现既定的生物学结果。解开膜重建之谜的最大障碍是获得膜相关支架的结构信息，这些支架协调这些过程的各个方面，从改变膜的曲率到膜的分裂，以及肌动蛋白细胞骨架的招募。克服了这一限制，我们已经证明，电子冷冻显微镜提供了进入膜相关支架的结构的途径，分辨率足以生成详细的机械模型。利用这一进展，该项目的长期目标是了解BAR超家族(bin-两亲酶-RVS家族)的成员是如何产生/稳定/感受膜曲率的，以及这些分子如何从混杂的多域蛋白质池中选择性地招募相互作用伙伴，例如裂变GTP酶Dynamin和细胞骨架激活剂N-WASP。我们将结合电子冷冻显微镜、低角散射、电子顺磁共振光谱和体外生物物理结构-功能实验来追求三个具体目标：(1)我们将扩大实验确定的支架结构的数量，这将教我们更多关于它们的设计原理以及这些设计如何有助于膜曲率的产生和相互作用伙伴的选择；(2)我们将利用我们已经学到的知识来测试支架组装早期步骤的机械模型，这可能提供重要的线索如何调控支架组装；(3)我们将为酒吧结构域蛋白质与两个最重要的效应器：Dynamin和N-WASP形成的高阶大分子复合体的结构工作奠定基础。总之，这些研究将使我们能够极大地促进对生命最基本方面之一的理解：细胞以惊人的空间和时间分辨率改变其膜的形状的能力。了解这些过程对于理解膜重塑的失衡和错误如何导致从癫痫到糖尿病和癌症的广泛的人类疾病是至关重要的。与公共健康相关：为了生存，细胞必须以高精度持续改变其膜的形状。细胞如何完成这项复杂的任务知之甚少，因为我们几乎没有关于负责这些过程的分子机器如何与它们重塑的膜相互作用的信息。克服了这一局限，我们建立了一种程序，当膜重塑分子与他们的靶点接触时，可以可视化它们。这使我们第一次能够仔细研究这些分子是如何发挥作用的，以及它们如何与额外的蛋白质相互作用，这些蛋白质的招募会导致特定的生物效应。可视化这些相互作用是理解这些过程中的错误如何导致癫痫、糖尿病和癌症等各种疾病的关键。