Cryo-EM of dynamic protein complexes in protein quality control

蛋白质质量控​​制中动态蛋白质复合物的冷冻电镜

• 批准号: RGPIN-2017-06474
• 负责人: Rubinstein, John
• 金额: $ 5.17万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=663874
• 关键词: Cryo; EM; dynamic; protein; complexes

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.**

背景：蛋白质是可以折叠以折叠以进行精确的3-D结构的长链（氨基酸）的长链，是对活细胞中发生的大部分情况的原因。必须展开不当折叠或不需要的蛋白质，这是由称为AAA+展开的蛋白质进行的活性。增值税是来自单细胞生物热粉嗜酸菌的AAA+展览酶，也是此类蛋白质的简单模型。了解增值税的工作方式需要了解其3D结构，因为它通过展开目标的过程。最近，电子冷冻显微镜（Cryo-EM）的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EM可用于瞬时形成的结构，但是进行这些实验的必要方法尚未完全开发。同样，尽管冷冻EM现在可以常规地达到稳定蛋白的3​​至4Å（3×10-10至4×10-10 m）的溶液，足以构建蛋白质结构中所有原子的模型，但应达到明显更高的分辨率。更重要的是，该技术仍然具有技术限制，可以减少它可以为移动蛋白获得的分辨率，并防止将其应用于不稳定的蛋白质。该研究计划将涵盖生化研究，仪器构建，纳米制作和计算机算法开发。研究计划的具体目的是**** 1：确定增值税最初如何与底物互动，以及其在反应周期中的结构如何变化。**** 2：了解增值税与底物之间形成的化学相互作用。**** 3：阐明VAT如何协作其他蛋白质来进行其活性。 活动。该研究计划中开发的方法将广泛适用于冷冻EM，从而可以使用该技术来回答一系列重要的生物学问题。经过研究计划培训的高素质人员将获得Cero-EM的专业知识，这是加拿大学术界和工业界的感觉，以及仪器设计，计算机编程和纳米制作方面的专业知识，所有这些都在加拿大知识的经济中需求。** ** ** **