Tools4Cells:EAGER: CRYO-EM ANALYSIS OF THE CHLOROPLAST CLP PROTEASE SYSTEM THROUGH AFFINITY PURIFICATION OF ENDOGENOUS COMPLEXES

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Protein-protein interactions and protein complexes are critical for cellular viability. Single particle cryogenic electron microscopy (Cryo-EM) has accelerated the pace of high-resolution structure determination of these nano-size protein complexes. Cryo-EM has mostly been used on protein complexes generated in bacterial expression systems to obtain large amount of these proteins for Cryo-EM analysis. However, structural analysis of complexes obtained from their natural environment would be beneficial since these represent the most natural state of the complex. Indeed, the few examples of Cryo-EM of endogenous complexes (i.e. complexes obtained from their physiological environment) is limited to proteins of high relative abundance. Successful tool development and application of Cryo-EM to low abundant endogenous complexes affinity purified from its natural cellular environment will greatly impact biological discoveries, including for plants such as Arabidopsis thaliana. This project is focused on structural analysis of the Clp protease-chaperone system in chloroplasts of Arabidopsis thaliana. This Clp system is central in chloroplast protein homeostasis; insufficient Clp activity is detrimental to the plant. The proposed research will unravel the structural details of this unique plastid Clp chaperone-protease system and help understand its evolutionary adaptation to the chloroplast environment. The Broader Impacts of the work include the intrinsic merit resulting from the biological importance of the protease system, as well as the potential application of the developed methodology for other projects which require structural data from low abundant endogenous complexes in plants. Additionally, the project will provide training opportunities for undergraduate and graduate students.The proposed project aims to determine the structural organization by Cryo-EM of the endogenous Arabidopsis chloroplast ~1000 kDa Clp protease-chaperone complex (ClpPRTC) consisting of a tetradecameric barrel-shaped protease (P,R,T subunits) that dynamically associates with hexameric chaperone rings (C and D subunits). The full complex is stabilized by WalkerB mutations and will be affinity purified from transgenic Arabidopsis plants followed by Cryo-EM. These (sub)complexes cannot be obtained by heterologous (e.g. E. coli) overexpression because more than a dozen subunits need to be assembled in the right order and stoichiometries, involving also post-translational modifications and protein activators. This project is built on extensive prior investments in biological materials (transgenic Arabidopsis plant lines expressing affinity-tagged ClpP3, ClpP5, ClpR4, ClpT1,2 and ClpC1-TRAP) and expertise in chloroplast protein biochemistry, mass spectrometry and Cryo-EM. Since the Clp complex is of very moderate abundance (~100x lower than the Rubisco holocomplex), demonstration that one can determine its 3D structure by Cryo-EM will have a big impact on plant research because it demonstrates that one does not need to use overexpression of protein complexes in heterologous systems. Furthermore, chloroplast proteolysis and protein homeostasis are critical in plant stress response, agriculture and molecular farming and synthetic biology.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.