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 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.
Wang
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Q
Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90024-1570, USA.
Matsushita
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K
de Foresta
B
B
le Maire
M
M
Kaback
H R
HR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001693:Biological Transport, Active; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D007785:Lactose / Q000031:analogs & derivatives / Q000378:metabolism*; D008024:Ligands; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009038:Motion; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017433:Protein Structure, Secondary; D017422:Sequence Analysis, DNA; D013050:Spectrometry, Fluorescence; D027981:Symporters; D013862:Thiogalactosides / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D013862:Thiogalactosides; D014364:Tryptophan; C021499:lactose permease; D007785:Lactose