SDSL-EPR STUDY OF INTERMEDIATE FILAMENT STRUCTURE

中间丝（IP）基本上存在于每个人类细胞中。但 不能获得完整的IF蛋白或IF的晶体结构意味着我们对IF的理解 结构在很大程度上是基于从原始序列和有限的交叉序列中做出的预测， 联系研究。因此，通常提出的IF结构模型的大多数特征是 未经实验检验，或基于对其他解释开放的数据。这使 严重限制了我们理解正常IF功能的能力，以及IF组装和 结构是由许多致病突变引起的，这些突变已经在 人IF基因。 我们已经证明，定点自旋标记和电子顺磁共振 可以提供生理条件下完整IF的优异结构信息。我们提出 在这里进行III型IF蛋白波形蛋白的详尽研究，组装第一个 完整的中间灯丝的综合图。然后我们将进行类似的实验， 另一种III型IF蛋白，结蛋白，和由K8和K18制成的细胞角蛋白IF，以确定 IF组装和结构在三种最常见形式中的保守程度 中间丝 这些研究将绘制α-螺旋、卷曲螺旋结构域的位置和边界， 连接区，在所有IF蛋白中发现的保守的“口吃”，杆结构域的边界， 二聚体中单体之间和完整长丝中二聚体之间的并置表面， 这些二聚体的注册和定向，以及在表面上可用的IF蛋白的位点 与其他细胞蛋白质/结构的相互作用。头部和尾部内的部位 相互作用以及与杆结构域相互作用的结构域也将被映射。最后我们 将探讨已知致病突变对IF组装和结构的影响。

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.