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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。S. pombe激酶样蛋白Klp6p是激酶8家族的成员。对Klp6p的体内分析表明，该酶参与多种过程，包括微管动力学、纺锤体-着丝点相互作用、细胞形态发生和减数分裂。尽管有这些结果，我们对这种微管运动蛋白功能的理解仍然有限。因此，Fiedler的工作主要集中在Klp6p大片段的细菌表达和体外产生的多肽的表征上。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。我们的方法是成像快速冷冻，紫杉醇稳定的mt，在各种条件下用Klp6p运动域加颈（MDN）装饰。结果清楚地表明，细菌表达的Klp6MDN蛋白以atp敏感的方式与微管结合，并且该蛋白在体外主要以二聚体的形式存在。用Klp6MDN修饰的mt直径为36-37nm，而mt本身只有~23nm。邻近的Klp6MDN头的平均间距约为8 nm，表明每个微管蛋白二聚体有一个马达结合。此外，我们已经证明Klp6MDN能够在体外捆绑微管。这些束中相邻mt之间的距离在10-13 nm之间，可以看到相邻mt之间的对角连接物。

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.