Single-molecule FRET reveals sugar-induced conformational dynamics in LacY.
The N- and C-terminal six-helix bundles of lactose permease (LacY) form a large internal cavity open on the cytoplasmic side and closed on the periplasmic side with a single sugar-binding site at the apex of the cavity near the middle of the molecule. During sugar/H(+) symport, an outward-facing cavity is thought to open with closing of the inward-facing cavity so that the sugar-binding site is alternately accessible to either face of the membrane. In this communication, single-molecule fluorescence (Förster) resonance energy transfer is used to test this model with wild-type LacY and a conformationally restricted mutant. Pairs of Cys residues at the ends of two helices on the cytoplasmic or periplasmic sides of wild-type LacY and the mutant were labeled with appropriate donor and acceptor fluorophores, single-molecule fluorescence resonance energy transfer was determined in the absence and presence of sugar, and distance changes were calculated. With wild-type LacY, binding of a galactopyranoside, but not a glucopyranoside, results in a decrease in distance on the cytoplasmic side and an increase in distance on the periplasmic side. In contrast, with the mutant, a more pronounced decrease in distance and in distance distribution is observed on the cytoplasmic side, but there is no change on the periplasmic side. The results are consistent with the alternating access model and indicate that the defect in the mutant is due to impaired ligand-induced flexibility on the periplasmic side.
Majumdar
Devdoot S
DS
Molecular Biology Institute, Department of Physiology, California NanoSystems Institute, University of California, Los Angeles, CA 90095-1662, USA.
Smirnova
Irina
I
Kasho
Vladimir
V
Nir
Eyal
E
Kong
Xiangxu
X
Weiss
Shimon
S
Kaback
H Ronald
HR
D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D002241:Carbohydrates; D008024:Ligands; D026901:Membrane Transport Proteins; C021499:lactose permease