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)F(1)) and its subcomplexes CF(O) and CF(1) 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 CF(O)F(1), CF(O) or CF(1) and exchanged with the detergent CHAPS. ATP hydrolysis activity of CF(1) and ATP synthesis activity of reconstituted CF(O)F(1) were determined before and after dye removal. The secondary structure of CF(O) was studied by CD spectroscopy in the presence and the absence of the dye. CBG neither abolishes the catalytic activity of the isolated CF(O)F(1) and CF(1) nor affects the subunit composition and the high alpha-helical content of CF(O). In crystallization attempts, 2D arrays of CF(O)F(1) and of CF(O) before and after dye removal were obtained. In the aggregates of CF(O), 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.
Poetsch
A
A
Institut für Biochemie, Abteilung Physikalische Biochemie, Technische Universität Darmstadt, Germany.
Neff
D
D
Seelert
H
H
Schägger
H
H
Dencher
N A
NA
D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology*; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007202:Indicators and Reagents / Q000737:chemistry; D015394:Molecular Structure; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012394:Rosaniline Dyes / Q000737:chemistry; D018724:Spinacia oleracea / Q000201:enzymology*
D007202:Indicators and Reagents; D012394:Rosaniline Dyes; D006180:Proton-Translocating ATPases; C004692:coomassie Brilliant Blue