Cryo-EM of dynamic protein complexes in protein quality control

背景技术背景：蛋白质是一种长链的化学构件（氨基酸），可以折叠成精确的三维结构，负责活细胞中发生的大部分事情。折叠不当或不需要的蛋白质必须解折叠，这是由称为AAA+解折叠酶的蛋白质进行的活动。VAT是来自单细胞生物嗜酸热浆菌的AAA+解折叠酶，并且是这种类型蛋白质的简单模型。要理解增值税的工作原理，就需要了解它在展开目标过程中的三维结构。近年来，低温电子显微镜（cryo-EM）技术成为一种可行的方法，用于确定蛋白质的高分辨率结构。原则上，低温EM可以用于捕获瞬时形成的结构，但进行这些实验的必要方法尚未完全开发。此外，虽然cryo-EM现在可以常规地达到稳定蛋白质的3至4 μ m（3×10-10至4×10-10 m）的分辨率，足以建立蛋白质结构中所有原子的模型，但应该可以达到更高的分辨率。更重要的是，该技术仍然存在技术局限性，降低了移动的蛋白质的分辨率，并阻止其应用于不稳定的蛋白质。我们将使用和开发新的方法来制备标本和分析冷冻EM图像，以了解VAT如何展开蛋白质。该研究计划将包括生物化学研究，仪器建设，纳米制造和计算机算法开发。该研究计划的具体目标是 *1：确定VAT最初如何与其底物结合以及其结构在反应周期中如何变化。2：了解VAT与其底物之间形成的化学相互作用。* 3：阐明VAT如何与其他蛋白质协作以执行其活性。**意义：这些研究将为AAA+解折叠酶（一类重要的蛋白质）如何发挥其活性提供基本了解。在这项研究计划中开发的方法将广泛适用于cryo-EM，允许该技术用于回答一系列重要的生物学问题。通过该研究计划培训的高素质人员将获得加拿大学术界和工业界所追求的cryo-EM专业知识，以及仪器设计，计算机编程和纳米制造方面的专业知识，所有这些都是加拿大知识经济的需求。

BACKGROUND: Proteins, which are long chains of chemical building blocks (amino acids) that can fold up to take on precise 3-D structures, are responsible for most of what happens in a living cell. Improperly folded or unwanted proteins must be unfolded, an activity performed by proteins known as AAA+ unfoldases. VAT is a AAA+ unfoldase from the single celled organism Thermoplasma acidophilum and a simple model for this type of protein. Understanding how VAT works requires knowing its 3-D structure as it goes through the process of unfolding its targets. Recently, the technique of electron cryomicroscopy (cryo-EM) became a viable approach for determining the high-resolution structures of proteins. In principle, cryo-EM can be used to trap transiently formed structures but the necessary methods to do these experiments have not been developed fully. Also, while cryo-EM can now routinely reach resolutions of 3 to 4 Å (3×10-10 to 4×10-10 m) for stable proteins, sufficient to build models of all of the atoms in the protein structure it should be possible to reach significantly higher resolution. Even more importantly, the technique still has technical limitations that reduce the resolution it can obtain for mobile proteins and prevent it from being applied to unstable proteins.******OBJECTIVES: We will use and develop new methods for preparing specimens and analyzing cryo-EM images to understand how VAT unfolds proteins. The research program will encompass biochemical studies, instrument construction, nano-fabrication, and computer algorithm development. The specific aims of the research program are to***1: Determine how VAT initially engage with its substrates and how its structure changes during its reaction cycle.***2: Understand the chemical interactions formed between VAT and its substrate.***3: Elucidate how VAT collaborates other proteins to carry out its activity.******SIGNIFICANCE: These studies will provide a fundamental understanding of how AAA+ unfoldases, an important class of protein, carry out their activity. The methods developed in this research program will be widely applicable to cryo-EM, allowing the technique to be used to answer a range of important biological questions. Highly qualified personnel trained by the research program will acquire expertise in cryo-EM, which is sought-after by Canadian academia and industry, as well as expertise in instrument design, computer programing, and nanofabrication, all of which are in demand in the Canadian knowledge-based economy.**