Cryo-EM of dynamic protein complexes in protein quality control

背景：蛋白质是化学构件（氨基酸）的长链，可以折叠成精确的 3D 结构，是活细胞中发生的大部分事情的原因。不正确折叠或不需要的蛋白质必须被解折叠，这是由称为 AAA 解折叠酶的蛋白质执行的活动。 VAT 是来自单细胞生物嗜酸热原体的 AAA 解折叠酶，也是此类蛋白质的简单模型。了解增值税如何运作需要了解其展开目标过程的 3D 结构。最近，电子冷冻显微镜（cryo-EM）技术成为确定蛋白质高分辨率结构的可行方法。原则上，冷冻电镜可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出来。此外，虽然冷冻电镜现在通常可以达到稳定蛋白质 3 至 4 Å（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，但应该有可能达到更高的分辨率。更重要的是，该技术仍然存在技术限制，降低了其对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样本和分析冷冻电镜图像，以了解 VAT 如何展开蛋白质。该研究项目将包括生化研究、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如何与其他蛋白质合作来发挥其活性。***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的了解。该研究计划中开发的方法将广泛适用于冷冻电镜，使该技术可用于回答一系列重要的生物学问题。通过该研究项目培训的高素质人才将获得加拿大学术界和工业界所追捧的冷冻电镜专业知识，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.**