IDENTIFICATION OF THE EPITOPE FOR MONOCLONAL-ANTIBODY 4B1 WHICH UNCOUPLES LACTOSE AND PROTON TRANSLOCATION IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI

Monoclonal antibody 4B1 binds to a conformational epitope on the perip lasmic surface of the lactose permease of Escherichia coli, uncoupling lactose and Hf translocation in a manner indicating that it blocks de protonation [Carrasco, N., Viitanen, P., Herzlinger, D., & Kaback, H. R. (1984) Biochemistry 23, 3681; Herzlinger, D., Viitanen, P., Carrasc o, N., & Kaback, H. R. (1984) Biochemistry 23, 3688]. In this paper, 4 B1 binding to purified lactose permease is shown to exhibit a K-D of a bout 5 x 10(-10) M by surface plasmon resonance. Furthermore, the comb ined use of mutants containing 6 contiguous His residues in each perip lasmic loop in the permease and Cys-scanning mutagenesis in conjunctio n with chemical labeling demonstrates that 4B1 binds specifically to t he periplasmic loop between helices VII and VIII and that Phe247 and G ly254 are the primary determinants. Remarkably, although 4B1 binding u ncouples lactose and Hf translocation, none of the amino acid residues in periplasmic loops, particularly Phe247 or Gly254, play an importan t role in the transport mechanism. Moreover, binding of avidin to biot inylated Glu255-->Cys in the loop containing the epitope has no effect on transport activity. Therefore, the uncoupling effect of 4B1 involv es highly specific interactions which in all likelihood exert a torsio nal effect on the loop, resulting in a conformational change in helix VII and/or VIII that alters the pK(a)s of residues involved in lactose -coupled H+ translocation.