SDSL-EPR STUDY OF INTERMEDIATE FILAMENT STRUCTURE

中间丝结构的 SDSL-EPR 研究

• 批准号: 7090181
• 负责人: PAUL Gillespie FITZGERALD
• 金额: $ 31.1万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 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/7090181
• 关键词: dimer; gene mutation; head; intermediate filaments; proteins; vimentin

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）：中间细丝（IP）基本上存在于每个人类细胞中。但是，无法实现完整IF蛋白质或IFS完整的晶体结构，这意味着我们对IF结构的理解很大程度上基于由初级序列和有限的交联研究进行的预测。因此，IF结构的常见模型的大多数特征是在实验中未经测试的，或基于对替代解释开放的数据。这对我们理解正常IF功能的能力以及由人类IF基因中已经鉴定出的许多引起疾病的突变引起的IF组装和结构的变化对我们的能力进行了严重限制。 我们已经证明，位点定向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。我们在这里提议对III型蛋白质蛋白进行详尽的研究，并组装完整的中间细丝的第一个综合图。然后，如果蛋白质，脱敏蛋白和由K8和K18制成的细胞角蛋白IFS，我们将对另一种III型进行类似的实验，以确定在三种最常见的中间丝中，如果组装和结构保守的程度。 这些研究将绘制α-螺旋，盘绕螺旋域，接头区域的位置和边界，在所有蛋白质中发现的保守的“口吃”，杆状域的边界，二聚体中单体之间的份量表面，以及完整细丝的晚餐，这些二聚体的注册表和方向以及IF表面可用的IF蛋白的位点，以与其他细胞蛋白/结构相互作用。彼此相互作用的头部和尾部域中的位置也将被映射。最后，我们将探索已知的致病突变对IF组装和结构的影响。