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bioRxiv · 10.1101/749747

High-throughput living-cell protein crosslinking analysis uncovers the physiological relevance of forming the \"inserted\" state of the ATP synthase ϵ-subunit

Liu, Y.·Yu, J.·Wang, M.·Zeng, Q.·Fu, X.·Chang, Z.
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Abstract

ATP synthase, a highly conserved multi-subunit enzyme complex having a common stoichiometry of 3{beta}3{gamma}{delta}{varepsilon}ab2c8-15, functions to supply ATP as the universal energy currency for cells. It comprises of the peripheral F1 sector (3{beta}3{gamma}{delta}{varepsilon}) and the membrane-integrated Fo sector (ab2c8-15). In vitro structural analyses revealed that the C-terminal domain of the {varepsilon}-subunit could adopt either an \"inserted\" or \"non-inserted\" state (with or without interacting with the /{beta}-subunits), with the former being viewed as inhibitory for the ATP hydrolysis activity of ATP synthase. Nevertheless, as common in current protein researches, the physiological relevance of such an \"inserted\" state for ATP synthase functioning is hardly known. To decipher this, designed an unnatural amino acid-mediated living-cell protein photocrosslinking analysis pipeline by developing the scarless genome-targeted site-directed mutagenesis and the high-throughput gel polyacrylamide gel electrophoresis (HT-PAGE) techniques. Employing this powerful approach, we systematically examined the interactions involving the C-terminal helix of the {varepsilon}-subunit in cells living under a variety of experimental conditions. These studies enabled us to uncover that the \"inserted\" and \"non-inserted\" states of the {varepsilon}-subunit exist as an equilibrium in cells cultured under common experimental conditions, shifting to the former upon the appearance of unfavorable conditions, acting as a low-gear state to strengthen the ATP synthesis function. Such a fine-tuning mechanism allows the ATP synthase to reversibly and instantly switch between two functional states. Further, the two powerful techniques that we developed here might be applied to many aspects of protein researches.

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Liu, Y., Yu, J., Wang, M., Zeng, Q., Fu, X., Chang, Z.. 2019-08-28. High-throughput living-cell protein crosslinking analysis uncovers the physiological relevance of forming the \"inserted\" state of the ATP synthase ϵ-subunit. https://doi.org/10.1101/749747

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