Cryo-EM of dynamic protein complexes in protein quality control

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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 (310-10 to 410-10 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.