SDSL-EPR STUDY OF INTERMEDIATE FILAMENT STRUCTURE

描述（由申请人提供）：中间丝（IPs）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是基于初级序列和有限交联研究的预测。因此，通常提出的中频结构模型的大多数特征都是未经实验测试的，或者是基于可供替代解释的数据。这严重限制了我们理解正常IF功能的能力，以及由人类IF基因中已确定的许多致病突变引起的IF组装和结构的变化。

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.