CYSTEINE SCANNING MUTAGENESIS OF HELIX V IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI

Using a functional lactose permease mutant devoid of Cys (C-less perme ase), each amino acid residue in putative transmembrane helix V was re placed individually with Cys (from Met145 to Thr163). Of the 19 mutant s, 13 are highly functional (60-125% of C-less permease activity), and 4 exhibit lower but significant lactose accumulation (15-45% of C-les s permease). Cys replacement of Gly147 or Trp151 essentially inactivat es the permease (<10% of C-less); however, previous studies [Menezes, M. E., Roepe, P. D., and Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U .S.A. 87, 1638; Jung, K., Jung, H., et al. (1995) Biochemistry 34, 103 0] demonstrate that neither of these residues is important for activit y. Immunoblots reveal that all of the mutant proteins are present in t he membrane in amounts comparable to C-less permease with the exceptio n of Trp151-->Cys and single Cys154 permeases which are present in red uced amounts. Finally, only three of the single-Cys mutants are inacti vated significantly by N-ethylmaleimide (Met145-->Cys, native Cys148, and Gly159-->Cys), and the positions of the three mutants fall on the same face of helix V.