Monomaleimidogold labeling of the gamma subunit of the Escherichia coli F1 ATPase examined by cryoelectron microscopy.
A novel approach for locating sites of interest in a protein complex has been developed using monomaleimidonanogold (MMN). The Escherichia coli F1 ATPase, when prepared without the delta subunit, contains only a single reactive cysteine on one of the three copies of the alpha subunit. This site was reacted with MMN and the gold cluster visualized on the protein complex by cryoelectron microscopy. Additional sites for modification with MMN were added by introducing cysteine residues through site-directed mutagenesis. Labeling of two mutants, gamma S8-C and gamma T106-C, in which Ser8 and Thr106, respectively, had been replaced by a cysteine, placed the gold cluster on the central mass that is seen in the hexagonal projection of the ECF1 complex. The results establish that the central mass contains the N-terminal part of the gamma subunit.
Wilkens
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Institute of Molecular Biology, University of Oregon, Eugene 97403.
Capaldi
R A
RA
D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D005615:Freezing; D006046:Gold; D007140:Immunoglobulin Fab Fragments; D046911:Macromolecular Substances; D008301:Maleimides; D016253:Microscopy, Immunoelectron; D050607:Organogold Compounds; D009942:Organometallic Compounds; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000648:ultrastructure*
D007140:Immunoglobulin Fab Fragments; D046911:Macromolecular Substances; D008301:Maleimides; D050607:Organogold Compounds; D009942:Organometallic Compounds; C077615:monomaleimido gold; D006046:Gold; D006180:Proton-Translocating ATPases