Single-molecule FRET reveals sugar-induced conformational dynamics in LacY
Year: 2007
Type: article
Abstract: 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.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Awards: Structural Basis for Mechanism of Secondary Transporters, G222768804, SINGLE MOLECULE METHODS FOR THE LARGE SCALE CHARACTERIZATION OF EXPRESSION LEVELS AND PROTEIN-PROTEIN INTERACTIONS IN SHEWANELLA ONEIDENSIS MR-1, G3006717822, Specialized Center for the Protein Structure Initiative, G3209070901, G3432931296, G3649956556, Center for Biophotonics Science and Technology, Dynamics of the Lactose Permease of Escherichia Coli, G4145698369, Dynamics of the Lactose Permease of Escherichia Coli, G5503935136, FIBR: How Do Proteins Fold Into Their Native and Functional Structures In-Vitro and in The Physiological Milue of The Living cell?, G6642290954, G7205286752, G7562190656, Structure of Cation-Coupled Active Sugar Transporters, G87117393, G9000041628, G9000084337, G9000265271
OpenAlex ID: W2086938888