PMID:9369484

Citation	Wang, Q, Matsushita, K, de Foresta, B, le Maire, M and Kaback, HR (1997) Ligand-induced movement of helix X in the lactose permease from Escherichia coli: a fluorescence quenching study. Biochemistry 36:14120-7
Abstract	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 not change significantly after addition of 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 pKa.
Links	PubMed Online version:10.1021/bi9716433
Keywords	Amino Acid Sequence; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Biological Transport, Active; Escherichia coli; Escherichia coli Proteins; Lactose/analogs & derivatives; Lactose/metabolism; Ligands; Membrane Proteins/genetics; Membrane Proteins/metabolism; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Models, Molecular; Molecular Sequence Data; Monosaccharide Transport Proteins; Motion; Mutagenesis, Site-Directed; Mutation; Protein Structure, Secondary; Sequence Analysis, DNA; Spectrometry, Fluorescence; Symporters; Thiogalactosides/metabolism; Tryptophan/genetics
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