SDSL-EPR STUDY OF INTERMEDIATE FILAMENT STRUCTURE

描述(申请人提供)：中间纤维(IP)存在于几乎每个人类细胞中。然而，无法获得完整的IF蛋白质或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根据初级序列和有限的交联性研究做出的预测。因此，通常提出的IF结构模型的大多数特征都是未经实验测试的，或者基于可供选择的解释的数据。这严重限制了我们理解正常IF功能的能力，以及由已在人类IF基因中发现的许多致病突变引起的IF组装和结构的变化。 我们已经证明，在生理条件下，定点定向自旋标记和电子顺磁共振可以从完整的IF中提供良好的结构信息。在这里，我们建议对III型IF蛋白波形蛋白进行详尽的研究，组装出第一张完整的中间丝的全面图谱。然后，我们将对另一种类型的IF蛋白质、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纤维形式中的保守程度。 这些研究将绘制出α-螺旋、盘绕结构域、连接区的位置和边界，在所有IF蛋白中发现的保守的“卡顿”，杆状结构域的边界，二聚体中单体之间和完整细丝中的正餐之间的对接表面，这些二聚体的注册和定位，以及IF蛋白表面可用于与其他细胞蛋白质/结构相互作用的位置。头域和尾域中相互作用的站点以及与杆域交互的站点也将被映射。最后，我们将探索已知致病突变对IF组装和结构的影响。

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.