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 α subunit has been examined. The epitopes for each of the mAbs have been localized by using molecular biological approaches to generate fragments of the α subunit. The binding of several of the mAbs has also been examined by cryoelectron microscopy of ECF1 Fab complexes. One of the mAbs, αII, bound in the region Asn 109-Val 153 without affecting ATPase activity. Most of the mAbs bound in the C-terminal third of the α subunit. MAb αI bound between residues Gln 443 and Trp 513. This mAb activated ATPase activity and was visualized in cryoelectron microscopy, superimposed on the α subunit, indicating that the epitope was on the top or bottom of ECF1 in the hexagonal projection. Other mAbs to the C-terminus, including αD 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 α subunit are present, suggesting that the C-terminus of α is a self-folding domain. In cryoelectron microscopy, Fab fragments for αD were seen extending from the sides of the ECF1 complex in hexagonal projection. © 1992.