Single-molecule FRET Reveals Sugar-induced Conformational Dynamics in LacY

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2007 May 16

PMID 17502603

Citations 90

Authors

Devdoot S Majumdar

Irina Smirnova

Vladimir Kasho

Eyal Nir

Xiangxu Kong

Shimon Weiss

H Ronald Kaback

Affiliations

Soon will be listed here.

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.

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References

Sahin-Toth M, Gunawan P, Lawrence M, Toyokuni T, Kaback H . Binding of hydrophobic D-galactopyranosides to the lactose permease of Escherichia coli. Biochemistry. 2002; 41(43):13039-45. DOI: 10.1021/bi0203076. View

Deniz A, Laurence T, Beligere G, Dahan M, Martin A, Chemla D . Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2. Proc Natl Acad Sci U S A. 2000; 97(10):5179-84. PMC: 25802. DOI: 10.1073/pnas.090104997. View

Abramson J, Smirnova I, Kasho V, Verner G, Kaback H, Iwata S . Structure and mechanism of the lactose permease of Escherichia coli. Science. 2003; 301(5633):610-5. DOI: 10.1126/science.1088196. View

Huang Y, Lemieux M, Song J, Auer M, Wang D . Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli. Science. 2003; 301(5633):616-20. DOI: 10.1126/science.1087619. View

Weninger K, Bowen M, Chu S, Brunger A . Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations. Proc Natl Acad Sci U S A. 2003; 100(25):14800-5. PMC: 299806. DOI: 10.1073/pnas.2036428100. View

Margittai M, Widengren J, Schweinberger E, Schroder G, Felekyan S, Haustein E . Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1. Proc Natl Acad Sci U S A. 2003; 100(26):15516-21. PMC: 307599. DOI: 10.1073/pnas.2331232100. View

Diez M, Zimmermann B, Borsch M, Konig M, Schweinberger E, Steigmiller S . Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase. Nat Struct Mol Biol. 2004; 11(2):135-41. DOI: 10.1038/nsmb718. View

Kapanidis A, Lee N, Laurence T, Doose S, Margeat E, Weiss S . Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc Natl Acad Sci U S A. 2004; 101(24):8936-41. PMC: 428450. DOI: 10.1073/pnas.0401690101. View

Rudnick G, Schildiner S, Kaback H . Equilibrium between two forms of the lac carrier protein in energized and nonenergized membrane vesicles from Escherichia coli. Biochemistry. 1976; 15(23):5126-31. DOI: 10.1021/bi00668a028. View

10.

Menick D, Sarkar H, Poonian M, Kaback H . cys154 Is important for lac permease activity in Escherichia coli. Biochem Biophys Res Commun. 1985; 132(1):162-70. DOI: 10.1016/0006-291x(85)91002-2. View

11.

van Iwaarden P, Driessen A, Lolkema J, Kaback H, Konings W . Exchange, efflux, and substrate binding by cysteine mutants of the lactose permease of Escherichia coli. Biochemistry. 1993; 32(20):5419-24. DOI: 10.1021/bi00071a017. View

12.

Sahin-Toth M, Frillingos S, Bibi E, Gonzalez A, Kaback H . The role of transmembrane domain III in the lactose permease of Escherichia coli. Protein Sci. 1994; 3(12):2302-10. PMC: 2142773. DOI: 10.1002/pro.5560031215. View

13.

Frillingos S, Kaback H . Cysteine-scanning mutagenesis of helix VI and the flanking hydrophilic domains on the lactose permease of Escherichia coli. Biochemistry. 1996; 35(16):5333-8. DOI: 10.1021/bi953068d. View

14.

Ha T, Enderle T, Ogletree D, Chemla D, Selvin P, Weiss S . Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc Natl Acad Sci U S A. 1996; 93(13):6264-8. PMC: 39010. DOI: 10.1073/pnas.93.13.6264. View

15.

He M, Sun J, Kaback H . Cysteine-scanning mutagenesis of transmembrane domain XII and the flanking periplasmic loop in the lactose permease of EScherichia coli. Biochemistry. 1996; 35(39):12909-14. DOI: 10.1021/bi960876b. View

16.

Frillingos S, Sahin-Toth M, Wu J, Kaback H . Cys-scanning mutagenesis: a novel approach to structure function relationships in polytopic membrane proteins. FASEB J. 1998; 12(13):1281-99. DOI: 10.1096/fasebj.12.13.1281. View

17.

Weiss S . Fluorescence spectroscopy of single biomolecules. Science. 1999; 283(5408):1676-83. DOI: 10.1126/science.283.5408.1676. View

18.

Lee N, Kapanidis A, Wang Y, Michalet X, Mukhopadhyay J, Ebright R . Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation. Biophys J. 2005; 88(4):2939-53. PMC: 1282518. DOI: 10.1529/biophysj.104.054114. View

19.

Ermolova N, Smirnova I, Kasho V, Kaback H . Interhelical packing modulates conformational flexibility in the lactose permease of Escherichia coli. Biochemistry. 2005; 44(21):7669-77. DOI: 10.1021/bi0502801. View

20.

Kapanidis A, Laurence T, Lee N, Margeat E, Kong X, Weiss S . Alternating-laser excitation of single molecules. Acc Chem Res. 2005; 38(7):523-33. DOI: 10.1021/ar0401348. View