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。所有的生物化学教科书都展示了肌动蛋白复合体的模型和它产生力量的atp酶循环，但是这个领域缺少的是肌动蛋白和肌动蛋白如何在原子水平上一起工作的完整图景。问题是f -肌动蛋白形成不同长度的螺旋聚合物，因此它不能用于x射线晶体学和结构测定。我之前的研究主要集中在工程长度的f -肌动蛋白复合物，开发稳定的，具有一个肌球蛋白结合位点的短肌动蛋白聚合物。我现在建议以这个短的f -肌动蛋白结构为基础来确定肌动蛋白聚合物和肌球蛋白之间的相互作用，肌球蛋白负责在细胞中产生力和运动。过去，肌动蛋白被认为是肌凝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起着积极的作用：当肌凝蛋白结合时，f -肌动蛋白的扭曲会发生变化，最近，肌凝蛋白异构体在细胞中有利于不同的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，我们将通过检查肌动蛋白丝的长度如何影响肌凝蛋白活性来确定局部和远程f -肌动蛋白相互作用对肌凝蛋白结合和力产生的贡献。最终，一个目标是确定几十年来一直困扰着研究人员的东西：对肌动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 -肌动蛋白复合物。本研究的目的是：1。确定我们的短f -肌动蛋白结构和肌球蛋白之间的相互作用的要求，反映生理结合和力的产生。我们将进行结合实验，以配合肌球蛋白功能测定。2. 利用我们的短f -肌动蛋白结构和开发更长的结构来确定非进展性和进展性肌动蛋白运动蛋白对肌动蛋白复合体上的远端f -肌动蛋白变构的要求，分析在短f -肌动蛋白结构存在下不同肌动蛋白异构体的肌动蛋白atp酶周期。3. 与当地专家合作，确定包含我们的短f -肌动蛋白结构的配合物的原子分辨率结构。生物运动是生命的基本属性。我们提出的研究处于这一深入领域的前沿，并将为细胞中肌动球蛋白依赖atp收缩的反应机制提供详细的见解。这项研究在更广泛的背景下对肌球蛋白异构体的特异性要求也很重要。此外，通过应用我们从nserc资助的研究中获得的知识，更广泛的生物界将受到影响，因为我们有助于更广泛地了解f -肌动蛋白变构如何在大量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.