How things move: Using short F-actin structures to understand the force-generating actomyosin complex.

物体如何运动：使用短 F-肌动蛋白结构来了解产生力的肌动球蛋白复合物。

• 批准号: RGPIN-2014-04566
• 负责人: Dawson, John
• 金额: $ 3.42万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=587885
• 关键词: How; things; move; Using; short

All living things move: to breath, to reproduce, for metabolism. When biological movement goes wrong, cells die or disease develops. For the last decade, my NSERC-funded research program has been aimed at understanding the molecular basis of movement generated by the action of actin and myosin proteins working together in an actomyosin complex. All Biochemistry textbooks show models of the actomyosin complex and its force-generating ATPase cycle, but what is missing in the field is a complete picture of how actin and myosin work together at the atomic level. The problem is that F-actin forms helical polymers of varying lengths and so it cannot be used for X-ray crystallography and structural determination. My previous research focused on engineering F-actin complexes of defined length, developing stable, short actin polymer that possesses one myosin-binding site. I now propose to build upon this short F-actin structure to determine the interactions between actin polymers and myosin proteins responsible for the generation of force and movement in cells. In the past, actin was seen as a passive substrate for myosin and other actin binding proteins; however, evidence has mounted for an active role for actin: the twist of F-actin changes when myosin binds, and more recently, myosin isoforms favour different F-actin structures in cells. Building on our deep experience with engineering defined short F-actin structures, we will answer fundamental questions regarding the role of F-actin allostery that were not testable before. Since we can control the length of the F-actin, we will determine the contribution of local and long-range F-actin interactions to myosin binding and force generation by examining how the length of the actin filament impacts myosin activity. Ultimately, one goal is to determine something that has eluded researchers for decades: an atomic resolution understanding of the actomyosin complex. The work up to this milestone requires optimization of the interactions between our short F-actin structure and myosin, including further engineering of other short F-actin complexes that involve actin binding proteins. The Objectives of this research are: 1. Determine the requirements for interactions between our short F-actin structure and myosin that reflect physiological binding and force generation. We will perform binding experiments in concert with myosin functional assays. 2. Use our short F-actin structure and develop longer structures to determine the requirements for long-range F-actin allostery on the actomyosin complex for both non-processive and processive myosin motor proteins, analyzing the actomyosin ATPase cycle of different myosin isoforms in the presence of short F-actin structures. 3. In collaboration with local experts, determine atomic-resolution structures of complexes containing our short F-actin structure. Biological movement is a fundamental property of life. Our proposed research is at the forefront of this deep field and will provide detailed insights into the reaction mechanisms underwriting the ATP-dependent contraction of actomyosin in cells. This research will also be important in the broader context of the specificity requirements of myosin isoforms. Moreover, by applying the knowledge obtained from our NSERC-sponsored research, the wider biological community will be impacted as we contribute to our broader understanding of how F-actin allostery regulates actin binding proteins in a vast array of physiological processes.

所有的生物都在运动：呼吸，繁殖，新陈代谢。当生物运动出现问题时，细胞死亡或疾病发展。在过去的十年里，我的nserc资助的研究项目一直致力于了解肌动蛋白和肌凝蛋白在肌动球蛋白复合体中共同作用所产生的运动的分子基础。