S1.29 Exploring rotary ATP synthase by electron tomography of two-dimensional crystals

Abstract

ATPase is the catalytic domain of FoF1-ATP synthase. In this presentation, we will introduce three different topics; one is the temperature-sensitive reaction intermediate state of F1-ATPase. This state was found as an intervening pause in the rotation assay of F1-ATPase at low temperature. The intermediate reaction step occurs at the ATP-binding angle, and that it is not relevant to ATPbinding but to ADP-releasing step. The second topic is the correlation between the rotational substeps of F1-ATPase found in single molecule studies and the chemical state in the crystal structures of F1-ATPase. To directly clarify the question which rotational pausing states the crystal structure of F1-ATPase represent, we crosslink the β and γ subunits of F1-ATPase to fix the conformational state in the crystal structure during the single molecule rotation assay. The last topic is the development of a fluorescent ATP sensor. The recent studies show that the e subunit of F1-ATPase undergoes a large conformational change upon ATP binding. By conjugating CFP and YFP at the Nand Ctermini of the e subunit respectively, we developed the fluorescent ATP sensor which largely enhances the efficiency of FRET from CFP to YFP. We will show some movies of intracellular ATP imaging during several cellular activities such as apoptosis and cell division.