CHARACTERISATION OF ISOLATED SPOMBE KINESIN-LIKE PROTEIN KLP6P USING CRYO EM

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。色葡萄粟酒裂殖酵母驱动蛋白样蛋白Klp 6p是驱动蛋白-8家族的成员。Klp 6p的体内分析表明，这种酶在多种过程中起作用，包括微管动力学、纺锤体-动粒相互作用、细胞形态发生和减数分裂。尽管有这些结果，我们对这种微管马达蛋白功能的了解仍然有限。因此，Fiedler的工作集中在Klp 6p大片段的细菌表达和体外所得多肽的表征上。我们正在使用冷冻电子显微镜和断层扫描来补充他的生化实验，以便更好地了解蛋白质的结构和功能。我们的方法是在各种条件下对快速冷冻的紫杉醇稳定的MT进行成像，这些MT用Klp 6p运动域加颈（MDN）进行装饰。结果清楚地表明，细菌表达的Klp 6 MDN蛋白以ATP敏感的方式与微管结合，并且该蛋白在体外主要作为二聚体存在。用Klp 6 MDN修饰的MT具有36- 37 nm的直径，而MT本身仅为~ 23 nm。相邻Klp 6 MDN头部具有约8 nm的平均间距，表明每个微管蛋白二聚体结合一个马达。此外，我们已经表明Klp 6 MDN能够在体外捆绑微管。这些束内的相邻MT之间的距离在10-13 nm的范围内，并且可见相邻MT之间的对角连接体桥接。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The S. pombe kinesin-like protein Klp6p is a member of the Kinesin-8 Family. In vivo analyses of Klp6p have suggested that this enzyme has rolls in a variety of processes, including microtubule dynamics, spindle-kinetochore interaction, cell morphogenesis, and meiosis. Despite these results our understanding of the function of this microtubule motor protein is still limited. Fiedler¿s work has therefore focused on bacterial expression of large segments of Klp6p and characterization of the resulting polypeptides in vitro. We are using cryo-electron microscopy and tomography to complement his biochemical experiments for a better understanding of the protein¿s structure and function. Our approach has been to image rapidly frozen, taxol-stabilized MTs that are decorated with Klp6p motor domain plus neck (MDN) under a variety of conditions. The results clearly demonstrate that the bacterially expressed Klp6MDN protein binds to microtubules in an ATP-sensitive manner and that the protein exists primarily as a dimer in vitro. MTs decorated with Klp6MDN have a diameter of 36-37nm, while MTs themselves are only ~23nm. Neighboring Klp6MDN heads have a mean spacing of approximately 8 nm, suggesting that one motor binds per tubulin dimer. In addition we have shown that Klp6MDN is able to bundle microtubules in vitro. The distance between neighboring MTs within these bundles ranges from 10-13 nm, and diagonal linkers bridging between neighboring MTs are visible.