Cryo-EM of dynamic protein complexes in protein quality control

背景：蛋白质是化学成分(氨基酸)的长链，可以折叠起来形成精确的三维结构，是活细胞中发生的大部分事情的责任。不正确折叠或不需要的蛋白质必须被展开，这一活动由被称为AAA展开酶的蛋白质执行。VAT是一种来自单细胞有机体嗜酸热浆的AAA去折叠酶，是这类蛋白质的一个简单模型。要了解增值税是如何运作的，就需要在其展开目标的过程中了解其3D结构。近年来，电子冷冻显微镜(Cryo-EM)技术成为确定蛋白质高分辨结构的一种可行方法。原则上，低温电磁可以用来捕获瞬时形成的结构，但进行这些实验的必要方法还没有完全开发出来。此外，尽管目前低温电子显微镜对稳定蛋白质的分辨率通常可以达到3到4ä(3×10-10到4×10-10m)，足以建立蛋白质结构中所有原子的模型，但应该有可能达到显著更高的分辨率。更重要的是，该技术仍然存在技术限制，降低了它对可移动蛋白质的分辨率，并阻止其应用于不稳定的蛋白质。*目标：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是如何展开蛋白质的。该研究计划将包括生化研究、仪器建造、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最初如何与其底物接触，以及其结构在其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进行其活性。*意义：这些研究将提供对AAA展开酶这一重要的蛋白质类如何进行其活性的基本了解。在这项研究计划中开发的方法将广泛适用于低温EM，使该技术能够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追捧的冷冻-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型经济所需要的。**

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.**