Cysteine-scanning mutagenesis of helix VI and the flanking hydrophilic domains on the lactose permease of Escherichia coli

Biochemistry
S Frillingos, H R Kaback

Abstract

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in putative transmembrane helix VI and the flanking hydrophilic loops (residues 164- 211) was replaced individually with Cys. Of the 48 mutants, 43 accumulate lactose at highly significant rates to > 80% of the steady state observed with C-less permease. Three mutants (Phe185--> Cys, Ala187--> Cys, and Phe208--> Cys) exhibit lower but significant levels of accumulation (30-60% of C-less). Cys replacement for Ala177 or Leu184 results in low transport activity (ca. 20%) in the C-less background but much higher activity (60-70%) in the wild type. Immunoblot analysis reveals that all of the mutants are inserted into the membrane at concentrations comparable to that of C-less perrmease. The transport activity of the great majority of the mutants is unaffected by treatment with N-ethylmaleimide (NEM). Relatively modest but significant inactivation (ca. 50%) is observed with mutants Phe170--> Cys, Gly173--> Cys, and Ala187--> Cys, and these positions cluster on the same face of the helix VI. Moreover, the two positions where single Cys replacements result in low activity (Ala177 and Lcu184) are on the same face of helix VI. The...Continue Reading

References

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Citations

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