Atomic force microscopy imaging of the membrane part CF0 and the subunit III complex of chloroplast ATP-synthase

Abstract

ATP-synthases consist of a hydrophilic part F1 containing the nucleotide binding sites and a hydrophobic membrane integrated part F0 which is involved in proton translocation. The structure of F0 is only partly known. We isolated the membrane part F0 from Spinacia oleracea chloroplasts using a new protocol. Starting with the holoenzyme, F1 was removed by treatment with Na-thiocyanate, then F0 was purified by a sucrose density gradient centrifugation followed by native preparative electrophoresis. Due to the mild purification conditions all four F0 subunits I, II, III and IV were found in the resulting complex. Heat treatment of this complex leaves intact only the subunit III complex. Since the protein was obtained in dodecylmaltoside-lipid-micelles, 2D crystallisation was initiated by dialysis in presence of additional lipid. The subunit III complex forms two different kinds of 2D crystals, while F0 builds large 2D sheets of F0 aggregated in an unordered way. These sheets and crystals were imaged on mica surface by contact mode atomic force microscopy. The surface profiles of subunit III crystals are easily interpreted and similar to earlier data [H. Seelert (2000) Nature 405, 418-9], whereas the F0 sheets yield profiles of weak height contrast. Therefore these F0 images were processed electronically to reduce image noise. The resulting images are compared to our earlier data obtained by transmission electron microscopy after negative staining.