SDSL-EPR Study of Intermediate Filament Structure

描述（由申请人提供）：中间丝（IF）是在每个细胞中发现的三种主要类型的细胞骨架之一，并且是更丰富的细胞蛋白质之一。IF蛋白的突变导致至少85种不同的人类疾病，包括许多蛋白质聚集疾病。然而，这些蛋白质的不寻常的物理性质已经排除了晶体结构的测定。正因为如此，我们对正常IF功能没有深入的机制性理解，也不知道突变是如何致病的。在这个建议中，我们将使用生物物理学的方法来定义IF结构，目的是阐明IF功能，以及IF结构在IF类中的保守程度。这反过来将打开大门，了解发病机制之间的IF蛋白，特别是那些与蛋白质聚集表型。我们已经组建了一个技术团队：CW SDSL EPR，脉冲场SDSL EPR，计算建模，高分辨率TEM（Cryo和STEM）和X射线晶体学。 公共卫生相关性：中间纤维（IF）是每个脊椎动物细胞的主要组成部分。IFs帮助细胞抵抗机械和代谢压力，并与超过85种人类疾病有关。然而，我们对IF生物学没有机械的理解，因为我们缺乏IF的结构。此外，使IFs稳定的相同性质也有助于其引起蛋白质聚集疾病的潜力。我们将解决正常的IF结构，这样我们就可以理解正常IF功能的机制，从而为理解特定突变是如何致病奠定基础。

DESCRIPTION (provided by applicant): Intermediate Filaments (IFs) are one of three major classes of cytoskeleton found in every cell, and one of the more abundant cellular proteins. Mutations in IF proteins are responsible for at least 85 different human diseases, including many protein aggregation diseases. However, the unusual physical properties of these proteins have precluded determination of crystal structure. Because of this we have no in-depth mechanistic understanding of normal IF function, nor how mutations are pathogenic. In this proposal we will use biophysical approaches to define IF structure with a goal of elucidating IF function, and the degree to which IF structure is conserved across IF classes. This in turn will open the door to understanding pathogenesis among IF proteins, particularly those with protein aggregation phenotypes. We have assembled a team of several technologies: CW SDSL EPR, Pulsed Field SDSL EPR, Computational Modeling, High resolution TEM (Cryo- and STEM) and X Ray Crystallography. PUBLIC HEALTH RELEVANCE: Intermediate Filaments (IFs) are a major component of every vertebrate cell. IFs help cells resist mechanical and metabolic stresses, and have been implicated in more than 85 human diseases. However, we have no mechanistic understanding of IF biology because we lack structure for IFs. Moreover the same properties that make IFs stable, also contribute to their potential to cause proteins aggregation diseases. We will solve normal IF structure, so that we can understand the mechanisms of normal IF function, thus laying the groundwork for understanding how specific mutations are pathogenic.