S. Frillingos et al., CYSTEINE-SCANNING MUTAGENESIS OF PUTATIVE HELIX-VII IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI, Biochemistry, 33(26), 1994, pp. 8074-8081
Using a functional lactose permease mutant devoid of Cys residues (C-l
ess permease), each amino acid residue in putative transmembrane helix
VII and the flanking cytoplasmic and periplasmic regions (from Leu212
to Glu255) was replaced individually with Cys. Of the 44 single-Cys m
utants, 40 exhibit high transport activity, accumulating lactose to >5
0% of the steady-state observed with C-less permease. In contrast, per
mease with Cys in place of Ala213 or Tyr236 exhibits low but significa
nt activity, and Cys substitution for Asp237 or Asp240 yields permease
molecules with little or no activity due to disruption of charge-neut
ralizing interactions between Asp237 and Lys358 or Asp240 and Lys319,
respectively. Immunological analysis reveals that membrane levels of t
he mutant proteins are comparable to that of C-less permease with the
exception of Tyr228-->Cys, which exhibits reduced but significant leve
ls of permease. Finally, the effect of N-ethylmaleimide (NEM) was test
ed on each mutant, and the results indicate that the transport activit
y of the great majority of the mutants is not affected by the alkylati
ng agent. Remarkably, the six positions where Cys replacements render
the permease highly sensitive to inactivation by NEM are confined to t
he C-terminal half of helix VII, a region that is strongly conserved a
mong transport proteins homologous to lactose permease. The results de
monstrate that although no residue per se in the region scanned is ess
ential, structural features of the C terminus of helix VII may be impo
rtant for transport activity.