Cysteine-scanning mutagenesis of transmembrane domain XII and the flanking periplasmic loop in the lactose permease of Escherichia coli

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain XII and the periplasmic loop between putative helices XI and XII (loop XI/XII) was replaced individually with Cys. Out of 34 mutants, 31 exhibit 60-100% or more of C-less activity, mutants Gly377 --> Cys and Leu385 --> Cys exhibit lower rates of transport but accumulate lactose about 60-70% as well as C-less, and mutant Leu400 --> Cys exhibits <20% of C-less activity. Immunoblots reveal that all of the mutant proteins are present in the membrane in amounts comparable to that of C-less with the exception of mutants Gly377 --> Cys and Leu385 --> Cys which are expressed about 40% as well as C-less and mutant Leu400 --> Cys which is hardly detectable. When transferred to the wild-type background, however, mutant Leu400 --> Cys is expressed normally and exhibits highly significant transport activity, Finally, each active Cys-replacement mutant was assayed for sensitivity to N-ethylmaleimide, and with three exceptions, the mutants are essentially unaffected by the alkylating agent. Mutants Va1367 --> Cys, Gly370 --> Cys, and Tyr373 --> Cys which are predicted to be immediately distal to helix XI in loop XI/XII are significantly inactivated. The periodicity observed suggests that the periplasmic end of transmembrane domain XI may extend to position 373. In the following paper [Voss, J., He, M. M., Hubbell, W. L., & Kaback, H. R, (1996) Biochemistry 35, 12915-12918], site-directed spin labeling of single-Cys mutants at positions 387-402 is used to demonstrate that transmembrane domain XII is in an a-helical conformation.