SDSL-EPR STUDY OF INTERMEDIATE FILAMENT STRUCTURE

中间丝结构的 SDSL-EPR 研究

基本信息

批准号：
7207948
负责人：
PAUL Gillespie FITZGERALD
金额：
$ 29.49万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-04-01 至 2011-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7207948
关键词：
Accounting Affect Amino Acids Architecture Area Cells Cellular Structures Class Coiled-Coil Domain Condition Crystallography Cytokeratin Cytoplasm Data Desmin Dialysis procedure Electron Spin Resonance Spectroscopy Event Exhibits Figs - dietary Filament Genes Head Human In Vitro Intermediate Filaments Interruption K-18 conjugate Knowledge Label Left Link Location Maps Membrane Proteins Modeling Monitor Mutation Output Pathologic Pattern Phenotype Physical Dialysis Physiologic pulse Physiological Point Mutation Positioning Attribute Process Property Proteins Pulse taking Randomized Registries Relative (related person)Reporter Research Design Severities Site Solutions Spin Labels Staging Structure Stuttering Surface Tail Techniques Testing Time Translating Urea Vimentin base crosslink dimer disease-causing mutation human disease monomer mutant protein aggregate protein structure research study retinal rods size ultra high resolution

项目摘要

DESCRIPTION (provided by applicant): Intermediate filaments (IPs) are present in essentially every human cell. However, the inability to achieve crystal structure for intact IF proteins or IFs means that our understanding of IF structure is based in large part on predictions made from primary sequence and from limited cross-linking studies. Thus, most features of commonly presented models of IF structure are experimentally untested, or based on data which is open to alternative interpretation. This places serious limits on our ability to understand normal IF function, and the changes in IF assembly and structure that are caused by the many disease-causing mutations that have been identified in human IF genes. We have demonstrated that site directed spin labeling and electron paramagnetic resonance can provide excellent structural information from intact IFs in physiologic conditions. We propose here to conduct an exhaustive study of the Type III IF protein vimentin, assembling the first comprehensive map of an intact intermediate filament. We will then conduct similar experiments on another Type III IF protein, desmin, and on cytokeratin IFs made from K8 and K18 to determine the degree to which IF assembly and structure are conserved among the three most common forms of intermediate filaments. These studies will map the location and boundaries of alpha-helical, coiled-coil domains, linker regions, the conserved "stutter" found in all IF proteins, the boundaries of the rod domain, the surfaces of apposition between monomers in dimers, and between dinners in intact filaments, the registry and orientation of these dimers, and the sites of IF proteins that are available at the surface of the IF for interactions with other cellular proteins/structures. Sites within the head and tail domains that interact with one another, and with the rod domain will be mapped as well. Finally, we will explore the impact of known disease-causing mutations on IF assembly and structure.

描述（由申请人提供）：中间丝（IP）基本上存在于每个人类细胞中。然而，无法获得完整 IF 蛋白或 IF 的晶体结构意味着我们对 IF 结构的理解在很大程度上基于对一级序列和有限的交联研究所做的预测。因此，常见的 IF 结构模型的大多数特征都未经实验测试，或者基于可供替代解释的数据。这严重限制了我们理解正常 IF 功能以及由人类 IF 基因中已发现的许多致病突变引起的 IF 组装和结构变化的能力。我们已经证明，定点自旋标记和电子顺磁共振可以在生理条件下从完整的 IF 中提供出色的结构信息。我们在此建议对 III 型 IF 蛋白波形蛋白进行详尽的研究，组装第一个完整中间丝的综合图谱。然后，我们将对另一种 III 型 IF 蛋白结蛋白以及由 K8 和 K18 制成的细胞角蛋白 IF 进行类似的实验，以确定 IF 组装和结构在三种最常见的中间丝形式中的保守程度。这些研究将绘制 α 螺旋、卷曲螺旋结构域、接头区域、所有 IF 蛋白中发现的保守“口吃”的位置和边界、杆结构域的边界、二聚体中单体之间以及之间的并置表面。完整细丝中的晚餐、这些二聚体的登记和方向，以及 IF 表面可与其他细胞蛋白质/结构相互作用的 IF 蛋白质位点。头域和尾域内相互相互作用以及与杆域相互作用的位点也将被映射。最后，我们将探讨已知的致病突变对 IF 组装和结构的影响。