Dye removal, catalytic activity and 2D crystallization of chloroplast H+-ATP synthase purified by blue native electrophoresis

The proton-ATP synthase of thylakoid membranes from spinach chloroplasts (CF(O)F1) and its subcomplexes CFO and CF1 were isolated by blue native electrophoresis (BN-PAGE) [Neff, D. and Dencher, N.A. (1999) Biochem. Biophys, Res. Commun. 259, 569-575] and subsequently electroeluted from the gel. A method was developed to remove most of the dye Coomassie G-250 (CBG) using gel filtration, a prerequisite for many biophysical investigations. The dye was removed from the electroeluted CFOF1, CFO or CF1 and exchanged with the detergent CHAPS. ATP hydrolysis activity of CF1 and ATP synthesis activity of reconstituted CFOF1 were determined before and after dye removal. The secondary structure of CFO was studied by CD spectroscopy in the presence and the absence of the dye. CBG neither abolishes the catalytic activity of the isolated CFOF1 and CF1 nor affects the subunit composition and the high alpha-helical content of CFO. In crystallization attempts, 2D arrays of CFOF1 and of CFO before and after dye removal were obtained. In the aggregates of CFO, circular structures with a mean diameter of 6.7 nm were observed. Our results indicate that the combination of BN-PAGE and dye removal by gel filtration is a suitable approach to obtain catalytically active protein complexes for further functional and structural characterization. (C) 2000 Elsevier Science B.V. All rights reserved.