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

20亿年前，在古细菌细胞中形成螺旋聚合物的简单蛋白质产生了祖细胞骨架；同样的早期细胞骨架吞没了古老的细菌，产生了最早的真核细胞。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多的蛋白质。其原始遗产的证据是，它普遍存在于所有真核生物中，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必不可少的参与。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）来控制肌动蛋白的功能。因此，了解ABPs如何与肌动蛋白相互作用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)生产短的f -肌动蛋白复合物（SFACs），克服f -肌动蛋白不形成晶体的原子结构测定，2)鉴定f -肌动蛋白结合蛋白（F-ABPs），然后3)从生物化学和结构上表征这些F-ABPs。******我之前的nserc资助的研究表明，统一修饰的SFAC不结合肌凝蛋白，而其他研究表明，具有多种修饰的SFAC与肌凝蛋白相互作用。为了进一步研究肌球蛋白，我们将证实这种组合方法产生了一个活性的SFAC，然后进行生化和结构表征。******在过去的5年里，我们用冷冻电镜（cro -electron microscopy）测定了F-actin与myosin结合的近原子图像；然而，在肌动球蛋白周期的每个阶段的具体相互作用仍然需要描述。除了肌凝蛋白，不同的ABPs是如何与f -肌动蛋白相互作用的？******基于我们在肌动蛋白方面的丰富经验，并与地方和国家合作，我们将使用现代单分子方法解决这些问题。最终，我们的目标是确定F-actin如何与结合伙伴相互作用，从围绕肌球蛋白结合的未解问题开始，并将研究计划扩展到其他ABP类。***本研究的具体目标是：******产生支持肌凝蛋白力生成的SFAC，并确定肌动蛋白c端在肌动凝蛋白循环中的作用。****** 2。用质谱法鉴定和表征与SFACs相互作用的ABPs。****** 3所示。利用冷冻电镜（cryo-EM）测定与肌动蛋白结合的ABPs的高分辨率结构。****** f -肌动蛋白和abp之间的相互作用是生命的基础。我们的研究将利用最先进的蛋白质组学和结构生物学技术，扩大我们对肌动蛋白和肌凝蛋白之间的分子舞蹈以及ABPs与f-肌动蛋白结合模式的理解。这些知识不仅对理解细胞的功能至关重要，而且可以应用于纳米技术的发展。

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.