Ligand-induced movement of helix X in the lactose permease from Escherichia coli: A fluorescence quenching study

Five single-Trp mutants were constructed by replacing Val315, Leu318, Val326, Leu329, or Val331 with Trp in transmembrane helix X of a functional lactose permease mutant devoid of Trp residues !Trp-less permease). Taking into account expression levels, each single-Trp permease except for Val331-->Trp exhibits significant activity, The intrinsic fluorescence emission of each single-Trp mutant does nor change significantly after addition beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG), indicating that ligand induces little change in the microenvironment of the Trp residues, However, fluorescence quenching studies with the brominated detergent 7,8-dibromododecyl beta,D-maltoside (BrDM) demonstrate that a Trp residue in place of Val315, Val326, or Val331 becomes less accessible to BrDM in the presence of TDG, while a Trp residue in place of Leu318 or Leu329 becomes more accessible. Acrylamide quenching studies with Leu318-->Trp and Val331-->Trp permeases or 2-(4-maleimidoanilino)-naphthalene-6-sulfonic acid (MIANS)-labeled Thr320-->Cys and Glu325-->Cys permeases indicate that positions 318 and 325 also become more accessible to a hydrophobic environment in the presence of TDG, while positions 320 and 331 become less accessible. The findings are consistent with a recently proposed mechanism for energy coupling in lactose permease [Kaback, H. R. (1997) Proc. Natl. Acad. Sci, U.S.A. 94, 5539-5543] in which substrate binding causes a conformational change resulting in movement of Glu325 to a nonpolar environment with a dramatic increase in pK(a).