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 funct ional lactose permease mutant devoid of Trp residues !Trp-less permeas e). Taking into account expression levels, each single-Trp permease ex cept for Val331-->Trp exhibits significant activity, The intrinsic flu orescence emission of each single-Trp mutant does nor change significa ntly after addition beta-D-galactopyranosyl 1-thio-beta-D-galactopyran oside (TDG), indicating that ligand induces little change in the micro environment of the Trp residues, However, fluorescence quenching studi es with the brominated detergent 7,8-dibromododecyl beta,D-maltoside ( BrDM) demonstrate that a Trp residue in place of Val315, Val326, or Va l331 becomes less accessible to BrDM in the presence of TDG, while a T rp residue in place of Leu318 or Leu329 becomes more accessible. Acryl amide quenching studies with Leu318-->Trp and Val331-->Trp permeases o r 2-(4-maleimidoanilino)-naphthalene-6-sulfonic acid (MIANS)-labeled T hr320-->Cys and Glu325-->Cys permeases indicate that positions 318 and 325 also become more accessible to a hydrophobic environment in the p resence of TDG, while positions 320 and 331 become less accessible. Th e findings are consistent with a recently proposed mechanism for energ y coupling in lactose permease [Kaback, H. R. (1997) Proc. Natl. Acad. Sci, U.S.A. 94, 5539-5543] in which substrate binding causes a confor mational change resulting in movement of Glu325 to a nonpolar environm ent with a dramatic increase in pK(a).