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

所有的生物都在运动：呼吸、繁殖、新陈代谢。当生物运动出错时，细胞死亡或疾病发展。在过去的十年里，我的NSERC资助的研究计划一直旨在了解肌动蛋白和肌球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。**所有的生物化学教科书都展示了肌动肌球蛋白复合体及其产生力量的ATPase循环的模型，但该领域缺少的是肌动蛋白和肌球蛋白如何在原子水平上协同工作的完整图景。问题是，F-肌动蛋白形成不同长度的螺旋聚合物，因此不能用于X射线结晶学和结构测定。**我之前的研究集中在设计固定长度的F-肌动蛋白复合体，开发具有一个肌球蛋白结合位点的稳定、短的肌动蛋白聚合物。我现在建议建立在这个短的F-肌动蛋白结构的基础上来确定肌动蛋白聚合物和肌球蛋白蛋白之间的相互作用，肌球蛋白聚合物负责在细胞中产生力量和运动。**在过去，肌动蛋白被视为肌球蛋白和其他肌动蛋白结合蛋白的被动底物；然而，越来越多的证据表明肌动蛋白具有主动作用：当肌球蛋白结合时，F-肌动蛋白的扭曲发生变化，最近，肌球蛋白亚型有利于细胞中不同的F-肌动蛋白结构。**基于我们在工程定义的短F-肌动蛋白结构方面的丰富经验，我们将回答有关F-肌动蛋白变构作用的基本问题，这些问题以前无法测试。由于我们可以控制F-肌动蛋白的长度，我们将通过检测肌动蛋白细丝的长度如何影响肌球蛋白活性来确定局部和远程F-肌动蛋白相互作用对肌球蛋白结合和作用力的贡献。**最终，一个目标是确定研究人员几十年来一直未能解决的问题：对肌动肌球蛋白复合体的原子分辨率理解。达到这一里程碑的工作需要优化我们的短F-肌动蛋白结构和肌球蛋白之间的相互作用，包括进一步设计其他涉及肌动蛋白结合蛋白的短F-肌动蛋白复合体。**本研究的目标是：**1.确定我们的短F-肌动蛋白结构和肌球蛋白之间相互作用的要求，以反映生理结合和力的产生。我们将结合肌球蛋白功能分析进行结合实验。**2.利用我们的短F-肌动蛋白结构和开发较长的结构来确定肌球蛋白复合体上对非进行性和进展性肌球蛋白马达蛋白的长程F-肌动蛋白变构的要求，分析在存在短F-肌动蛋白结构的情况下不同肌球蛋白亚型的肌球蛋白ATPase周期。**3.与当地专家合作，确定含有我们的短F-肌动蛋白结构的络合物的原子分辨结构。**生物运动是生命的基本属性。我们提出的研究处于这一深层领域的前沿，并将为细胞内肌动球蛋白依赖于ATP的收缩的反应机制提供详细的见解。这项研究在肌球蛋白异构体的特异性要求的更广泛的背景下也将是重要的。此外，通过应用我们从NSERC赞助的研究中获得的知识，更广泛的生物界将受到影响，因为我们将有助于我们更广泛地了解F-肌动蛋白变构如何在大量的生理过程中调节肌动蛋白结合蛋白。

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.