DNA based platform for visualization of scarce protein assemblies with electron cryo-microscopy

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Background:***Many clinically relevant protein complexes form transiently in cells at low abundance in response to external stimuli. Additionally, growth signaling proteins undergo heterogeneous phosphorylation regulated via upstream signaling that strictly regulate their conformation, subunit composition, and intra-cellular localization within cells. Many of these ‘regulatory class' of proteins are not amenable to over-expression that is necessary to achieve milligram protein quantities for traditional structure determination techniques such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Therefore, it is critical to target native complexes expressed at endogenous levels. However, another level of complication arises due to chemical and conformational heterogeneity of endogenous proteins. To overcome this challenge, we sought to take advantage of post-translationally modified states and scaffolding subunits that define function, subunit composition and intra-cellular localization. Mammalian target of rapamycin (mTOR) protein is found in association with regulatory scaffold proteins RPTOR or RICTOR that target the kinase to mTORC1 or mTORC2, respectively, with define chemical composition. Indeed, these scaffolding proteins have been used to selectively pull-down endogenous mTOR complexes. On the other hand, tuberous sclerosis complex (TSC) function, complex formation, and sub-cellular localization is strictly controlled via phosphorylation by upstream kinases. In starving cells, TSC is found on the lysosomal surface where it down-regulates mTORC1 activity. Upon insulin stimulation, TSC is rapidly phosphorylated by Akt and translocates from lysosomes to the cytoplasm where it remains in a catalytically active state for up to 1h post insulin stimulation. ***Objectives:***We proposed a research program for strtuctural investigation of protein components of TOR pathway, with emphasis on TORC2 from yeast and human TSC.***1) Development and optimization of polymer-coated holey EM grids. ***2) cryoEM of genetically-tagged TORC2 from yeast Saccharomyces cerevisiae. ***3) Signal-integrated affinity cryoEM of TSC. ***Significance:***Atomic-resolution structural knowledge is acutely lacking for mTORCs and TSC holo-complexes and is instrumental in design of selective agonist and antagonist. A detailed structural knowledge of TORC2 is necessary for generation of selective inhibitors for basic biological research into mTORC2 dependent signaling pathways. Similarly TSC structure, will reveal the mechanistic basis of disease associated mutations and identify interfaces between different TSC subunits and their interaction with upstream kinases. Our cryoEM sample preparation scheme will be adoptable to other scarce complexes.