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

使用冷冻电镜表征分离的孢子驱动蛋白样蛋白 KLP6P

• 批准号: 7355010
• 负责人: DANIELA NICASTRO
• 金额: $ 0.94万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-26 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355010
• 关键词: dimer; kinesin; microtubules; proteins

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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。粟酒裂殖酵母驱动蛋白样蛋白 Klp6p 是驱动蛋白 8 家族的成员。 Klp6p 的体内分析表明，这种酶在多种过程中发挥作用，包括微管动力学、纺锤体-着丝粒相互作用、细胞形态发生和减数分裂。尽管有这些结果，我们对这种微管运动蛋白功能的理解仍然有限。因此，Fiedler 的工作重点是 Klp6p 大片段的细菌表达以及所得多肽的体外表征。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、紫杉醇稳定的 MT 进行成像，这些 MT 装饰有 Klp6p 运动域加颈 (MDN)。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形式存在。用Klp6MDN修饰的MT的直径为36-37nm，而MT本身只有~23nm。相邻的 Klp6MDN 头的平均间距约为 8 nm，表明每个微管蛋白二聚体结合一个马达。此外，我们还证明 Klp6MDN 能够在体外捆绑微管。这些束内相邻 MT 之间的距离范围为 10-13 nm，相邻 MT 之间桥接的对角连接子是可见的。