Rl. Dunten et al., CYSTEINE SCANNING MUTAGENESIS OF PUTATIVE HELIX-XI IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI, Biochemistry, 32(47), 1993, pp. 12644-12650
Using a functional lactose permease mutant devoid of Cys residues (C-l
ess permease), each amino acid in putative transmembrane helix XI was
individually replaced with Cys (from Ala347 to Ser366). Fifteen of the
20 mutants are highly functional and accumulate lactose to >60% of th
e level achieved by C-less permease, and an additional three mutants,
all located at the cytoplasmic end of the helix, exhibit lower but sig
nificant lactose accumulation. Cys replacements for Thr348 or Lys358 r
esult in virtually inactive permease. Lys358, however, is not essentia
l for active lactose transport but plays a role in permease folding or
membrane insertion by interacting with Asp237. Immunoblots reveal tha
t all mutant proteins are present in the membrane in amounts comparabl
e to C-less with the exception of Lys358-->Cys which is hardly detecta
ble, as expected. The results highlight Thr348 as a potentially import
ant residue for further analysis. Finally, all active mutants were ass
ayed after treatment with the sulfhydryl reagent N-ethylmaleimide, and
results range from nearly complete inhibition to almost 2-fold stimul
ation. Remarkably, all of the strongly inhibited positions lie on one
face of helix XI. The implications of the findings for packing of tran
smembrane helices in the C-terminal half of the permease are discussed
.