Single-molecule FRET reveals sugar-induced conformational dynamics in LacY

doi:10.1073/pnas.0700969104

Single-molecule FRET reveals sugar-induced conformational dynamics in LacY

^†Molecular Biology Institute,
Departments of ^‡Physiology,
^§Chemistry and Biochemistry, and
^††Microbiology, Immunology, and Molecular Genetics
^¶California NanoSystems Institute, University of California, Los Angeles, CA 90095-1662

Edited by Robert J. Silbey, Massachusetts Institute of Technology, Cambridge, MA, and approved April 4, 2007 (received for review February 1, 2007)

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.

Footnotes

^‖To whom correspondence may be addressed. E-mail: sweiss{at}chem.ucla.edu or rkaback{at}mednet.ucla.edu
Author contributions: S.W. and H.R.K. designed research; D.S.M., I.S., V.K., and X.K. performed research; I.S. and V.K. contributed new reagents/analytic tools; D.S.M., I.S., V.K., and E.N. analyzed data; and D.S.M., I.S., V.K., E.N., S.W., and H.R.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0700969104/DC1.
Abbreviations:

NPG,

4-nitrophenyl-α-d-galactopyranoside;

NPGlc,

4-nitrophenyl-α-d-glucopyranoside;

sm-FRET,

single-molecule fluorescence (Förster) resonance energy transfer.