ATP synthase project

FoF1 ATP synthase

FoF1-ATP synthase utilizes the gradient of proton concentration across a membrane to generate ATP in chloroplast and mitochondria. It comprises of the Fo proton-channel (a 10-14 subunit ring, dependent on species and organisms) embedded in a membrane and of the F1 ATPase (a hexameric ring) protruding from Fo by a central stalk and a peripheral stalk (Figure (a)). It is known that the rotational force is generated concomitant with ATP hydrolysis or ATP synthesis, resulting in the rotation of Fo together with the central stalk, while the peripheral stalk fixes the F1 hexameric ring as the stator. The mechanism of coupling between ATP synthesis in F1 and proton channeling in Fo is important and still needs to be investigated. The atomic structures of F1 and F1/Fo-ring have been solved by X-ray crystallography. However, information about the whole structure of FoF1 ATP synthase including the peripheral stalk is limited. Presumably, various conformations, depending on the combination between Fo and F1 due to the continuous rotation, limited the resolution of single particle analysis (Figure (b)), in which the images of the particles are averaged, by existing methodology, including single particle analysis. Electron tomography is likely the suitable technique to visualize these variants. In this study, thylakoid membranes from Chlamydomonas, in which intact FoF1 ATP synthases are abundant, will be used as the specimen. By reconstructing the whole thylakoid membrane, structures of FoF1 ATP synthase will be visualized. The currently unknown structures of d subunit of F1 and b of Fo, which connect Fo and F1 and likely play essential roles for coupling and a subunit of Fo, which is charged for proton channeling will be reconstructed (Figure (b)). In addition, the combination of Fo and F1 at various angles will give deep insight into the mechanism of dynamic coupling between the two domains (Figure (b)), which contribute to proton-pumping and ATP-hydrolysis/synthesis, respectively.

The reconstruction of a whole thylakoid membrane will also give information about the arrangement of ATP synthase together with other key proteins in the chloroplast, that is photosystem I, photosystem II and cytochrome b(6)f complex. Little is known about the architecture of the whole thylakoid membrane, although it is crucial to understand the mechanism of proton- and electron-transfer across the membrane, which is essential reaction in photosynthesis.

Figure (a) FoF1 ATP synthase (modified from Nakamoto, 1999). The structure of the peripheral stalk (in red) is not known yet. (b) Due to the continuous rotation between Fo and F1, there can be variants of the combination between the stator (blue), rotator (green) and the peripheral stalk (red), which causes heterogeneity. This difficulty for current reconstruction will be solved by tomographic reconstruction.