Cysteine scanning mutagenesis of the N‐terminal 32 amino acid residues in the lactose permease of Escherichia coli

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Abstract
Using a functional lactose permease mutant devoid of Cys residues (C‐less permease), each amino acid residue in the hydrophilic N‐terminus and the first putative transmembrane helix was systematically replaced with Cys (from Tyr‐2 to Trp‐33). Twenty‐three of 32 mutants exhibit high lactose accumulation (70‐100% or more of C‐less), and an additional 8 mutants accumulate to lower but highly significant levels, Surprisingly, Cys replacement for Gly‐24 or Tyr‐26 yields fully active permease molecules, and permease with Cys in place of Pro‐28 also exhibits significant transport activity, although previous mutagenesis studies on these residues suggested that they may be required for lactose transport. As expected, Cys replacement for Pro‐31 completely inactivates, in agreement with previous findings indicating that “helix‐breaking” propensity at this position is necessary for full activity (Consler TG, Tsolas 0, Kaback HR, 1991, Biochemistry 30:1291‐1297). Twenty‐nine mutants are present in the membrane in amounts comparable to C‐less permease, whereas membrane levels of mutants Tyr‐3 → Cys and Phe‐12 → Cys are slightly reduced, as judged by immunological techniques. Dramatically, mutant Phe‐9 →Cys is hardly detectable when expressed from the lac promoter/operator at a relatively low rate, but is present in the membrane in a stable form when expressed at a high rate from the T7 promoter. Finally, studies with N‐ethylmaleimide show that 6 Cys‐replacement mutants that cluster at the C‐terminal end of putative helix I are inactivated significantly. The results demonstrate that although no residue per se in this region is essential for activity, the structural integrity of the periplasmic half of the first transmembrane helix is important for active lactose transport.