How do different F-actin binding proteins (F-ABPs) interact with F-actin?

20亿年前，一种在古细菌细胞中形成螺旋聚合物的简单蛋白质产生了祖先细胞骨架；正是这种早期细胞骨架吞噬了古代细菌，形成了第一个真核细胞。这种蛋白质进化成肌动蛋白。肌动蛋白是真核细胞中含量最丰富的蛋白质。它在所有真核生物中的普遍存在、序列的极端保守以及在真核生物生命中几乎每一个主要生理过程中的重要参与都是其原始遗产的证据。在整个生命进化过程中，细胞聚集并发展了数百种不同的肌动蛋白结合蛋白(ABPs)来控制肌动蛋白何时何地发挥作用。因此，了解ABPs是如何与肌动蛋白相互作用的，可以告诉我们真核生物是如何工作的。在过去的20年里，我的研究一直专注于建立这种理解。我的方法是1)制备短的F-肌动蛋白复合体(SFAC)以克服F-肌动蛋白不形成用于原子结构测定的晶体，2)鉴定F-肌动蛋白结合蛋白(F-ABPs)，然后3)对这些F-ABP进行生化和结构表征。*我之前由NSERC资助的研究表明，统一修饰的SFAC不结合肌球蛋白，而其他人则建议具有多种修饰组合的SFAC与肌球蛋白相互作用。为了进一步研究肌球蛋白，我们将确认这种组合方法可以产生活性的SFAC，然后进行生化和结构表征。在过去的5年里，用冷冻电子显微镜测定了几张F-肌动蛋白与肌球蛋白结合的近原子图像；然而，肌动球蛋白周期每个阶段的具体相互作用仍需描述。除了肌球蛋白，不同的ABP如何与F-肌动蛋白相互作用？*基于我们在肌动蛋白方面的深厚经验，并与地方和国家合作，我们将使用现代单分子方法来解决这些问题。最终，我们的目标是确定F-肌动蛋白如何与结合伙伴相互作用，首先是关于肌球蛋白结合的悬而未决的问题，然后将研究计划扩展到其他ABP类别。*本研究的具体目标是：*1.制备一种支持肌球蛋白力量产生的SFAC，并确定肌动蛋白C末端在肌动球蛋白循环中的作用。*2.用质谱仪鉴定和表征与SFAC相互作用的ABPs。确定冷冻-EM结合肌动蛋白的ABPs的高分辨结构。F-肌动蛋白和ABPs之间的相互作用是生命的基础。我们的研究将扩大我们对肌动蛋白和肌球蛋白之间的分子舞蹈以及ABPs与F-肌动蛋白结合方式的理解，使用最先进的蛋白质组学和结构生物学技术。这一知识不仅对理解细胞的功能至关重要，而且还可以应用于纳米技术的开发。

Two billion years ago, a simple protein forming helical polymers in archaeal cells produced a progenitor cytoskeleton; the same early cytoskeleton that engulfed ancient bacteria to give rise to the very first eukaryotic cells. That protein evolved into actin. ******Actin is the most abundant protein in eukaryotic cells. Evidence of its primordial legacy is found in its universal presence in all eukaryotes, extreme conservation of sequence, and essential participation in almost every major physiological process in eukaryotic life. Throughout the evolution of life, cells have gathered and developed hundreds of different actin binding proteins (ABPs) to control when and where actin functions. As a result, understanding how ABPs interact with actin tells us how eukaryotic life works. ******My research has been squarely focused on building that understanding for the past 2 decades. My approach has been to 1) produce short F-actin complexes (SFACs) that overcome F-actin not forming crystals for atomic structure determination, 2) identify F-actin binding proteins (F-ABPs), and then 3) characterize those F-ABPs biochemically and structurally. ******My previous NSERC-funded research showed that uniformly modified SFAC does not bind myosin, while others suggested an SFAC with a combination of modifications interacts with myosin. To further our work with myosin, we will confirm that this combination approach produces an active SFAC and then perform biochemical and structural characterizations. ******Several near-atomic pictures of F-actin with myosin bound were determined in the last 5 years using cryo-electron microscopy; however, specific interactions at each stage of the actomyosin cycle still need to be described. Beyond myosin, how do different ABPs interact with F-actin? ******Building on our deep experience with actin and working with local and national collaborations, we will tackle these questions using modern single molecule approaches. Ultimately, our goal is to determine how F-actin interacts with binding partners, starting with unanswered questions around myosin binding and expanding the research program to other ABP classes. ***The specific Objectives of this research are: ****** 1. To produce an SFAC that supports myosin force generation and determine the role of the C-terminus of actin in the actomyosin cycle. ****** 2. To identify and characterize ABPs that interact with SFACs with mass spectroscopy. ****** 3. To determine the high-resolution structure of ABPs bound to actin with cryo-EM. ******Interactions between F-actin and ABPs is fundamental for life. Our research will expand our understanding of the molecular dance between actin and myosin and the modes of binding to F-actin by ABPs using state-of-the art proteomic and structural biology techniques. This knowledge is not only critical for understanding how cells function but can also be applied to the development of nanotechnologies.