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）：大量的细胞过程取决于细胞以惊人的空间和时间准确性改变膜形状的能力。在生化层面上，许多参与这些过程的参与者都是已知的。然而，未知的是，几种蛋白质的集合通常会重叠函数和相互作用特异性，可重复地完成定义的生物学结果。解决膜重塑的奥秘的最大障碍是获取有关膜相关的脚手架的结构信息，这些信息从变化的膜曲率到膜裂变，并募集肌动蛋白细胞骨架。克服了这一限制，我们已经证明了电子冷冻显微镜可以在足以生成详细机械模型的分辨率下访问与膜相关支架的结构。 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我们已经学会了测试脚手架组装中早期步骤的机械模型，这可能提供了如何调节支架组件的重要线索，并且（3）我们将在高阶大分子分子复合物上为结构性工作奠定基础，这些结构型蛋白质与其两个最重要的效果：dynalin和n-wasp。综上所述，这些研究将使我们能够极大地了解生活中最基本的方面之一：细胞以惊人的空间和时间分辨率改变其膜形状的能力。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等广泛的人类疾病。公共卫生相关性：为了生活，细胞必须以高精度不断地改变其膜的形状。细胞如何完成这项复杂的任务的理解很少，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。克服了这一局限性，我们建立了一个程序，以可视化膜变形分子，因为它们与目标分子合作。这是第一次，这使我们能够仔细研究这些分子的功能，以及它们如何与募集的其他蛋白质相互作用会导致特定的生物学作用。可视化这些相互作用是了解这些过程中的错误如何导致癫痫，糖尿病和癌症等多样化的疾病。