Epitope mapping of monoclonal antibodies to the Escherichia coli F1 ATPase alpha subunit in relation to activity effects and location in the enzyme complex based on cryoelectron microscopy.
The interaction of Escherichia coli F1 ATPase (ECF1) with several different monoclonal antibodies (mAbs) specific for the alpha subunit has been examined. The epitopes for each of the mAbs have been localized by using molecular biological approaches to generate fragments of the alpha subunit. The binding of several of the mAbs has also been examined by cryoelectron microscopy of ECF1 Fab complexes. One of the mAbs, alpha II, bound in the region Asn 109-Val 153 without affecting ATPase activity. Most of the mAbs bound in the C-terminal third of the alpha subunit. MAb alpha 1 bound between residues Gln 443 and Trp 513. This mAb activated ATPase activity and was visualized in cryoelectron microscopy, superimposed on the alpha subunit, indicating that the epitope was on the top or bottom of ECF1 in the hexagonal projection. Other mAbs to the C-terminus, including alpha D which also activated the enzyme, reacted between Gly 371 and Trp 513 but failed to bind to small overlapping fragments within this sequence. The epitopes for these mAbs are probably formed by the folded polypeptide which occurs only in Western analysis when long stretches of the alpha subunit are present, suggesting that the C-terminus of alpha is a self-folding domain. In cryoelectron microscopy, Fab fragments for alpha D were seen extending from the sides of the ECF1 complex in hexagonal projection.
Aggeler
R
R
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Capaldi
R A
RA
Dunn
S
S
Gogol
E P
EP
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D004926:Escherichia coli / Q000201:enzymology*; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008854:Microscopy, Electron; D008970:Molecular Weight; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000276:immunology* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins; D006180:Proton-Translocating ATPases