Single-molecule FRET reveals sugar-induced conformational dynamics in LacY
- Devdoot S. Majumdar†,
- Irina Smirnova‡,
- Vladimir Kasho‡,
- Eyal Nir§,
- Xiangxu Kong§,
- Shimon Weiss†,‡,§,¶,‖, and
- H. Ronald Kaback†,‡,‖,††
- †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
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Edited by Robert J. Silbey, Massachusetts Institute of Technology, Cambridge, MA, and approved April 4, 2007 (received for review February 1, 2007)
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Fig. 1.Structural modeling of LacY double-labeled with Alexa fluorophores at Cys pairs introduced at the cytoplasmic ends of helices III and XII (R73C/S401C) (Left) or the periplasmic ends of helices V and XI (I164C/R375C) (Right). Backbone of LacY rendered as ribbons rainbow colored from blue (helix 1) to red (helix 12) with hydrophilic cavity open to cytoplasmic side. Attached fluorophores colored in green (Alexa 488) or magenta (Alexa 647) and shown in space-filling representation. Estimated distances between Cα atoms of the Cys pairs and between centers of the fluorophores are 41 and 48 Å (cytoplasmic), and 32 and 61 Å (periplasmic), respectively.
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Fig. 2.Ligand-induced effects on the FRET distribution E* at the cytoplasmic side of LacY (R73C/S401C, helices III and XII). (A) Two-dimensional S–E* histograms corresponding to wild-type (A1–A3) and C154G mutant (A4–A6) LacY. Measurements for each construct were obtained in the absence of sugar (A1 and A4) and in the presence of 1 mM (saturating) NPG (A2 and A5) or 1 mM NPGlc (A3 and A6). (B and C) Comparison of sugar effects on normalized E* histograms from A for wild-type LacY (B) and C154G LacY (C). Gray bins, no sugar added; red line, 1 mM NPG; blue line, 1 mM NPGlc; broken lines, Gaussian fits to the data with the corrected E values given in Table 2.
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Fig. 3.Concentration dependence of ligand-induced effects on 〈E*〉 and ΔE* at the cytoplasmic side of LacY (R73C/S401C/C154G, helices III and XII). (A) E* distribution without added sugar (red) and with nine NPG concentrations increasing from 1 μM (orange) to 1,000 μM (purple). (B) Fractional change of the fitted Gaussian parameters of histograms from A plotted as a function of NPG concentration. Red, relative 〈E*〉 shift; black, relative ΔE* change. The maximum change in 〈E*〉 and ΔE* at saturating concentration of NPG (1 mM) corresponds to 1. Solid lines, hyperbolic fit to each data set; broken line, theoretical curve showing NPG binding to LacY with K d of 38 μM as measured by stopped-flow (32).
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Fig. 4.Ligand-induced effects on the FRET distribution E* at the periplasmic side of LacY (I164C/S375C, helices V and XI). All details are as described in Fig. 2.
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Fig. 5.Cartoon model illustrating global conformational changes detected by sm-FRET upon sugar binding to wild-type LacY and C154G mutant. Without bound sugar (left) protein is in the protonated state with an inward-facing hydrophilic cavity. Without ligand, both molecules have multiple conformations on the cytoplasmic side (arrows). This conformation is the most energetically stable among multiple unliganded conformers. Binding of sugar induces a global conformational change in both wild-type LacY and C154G mutant resulting in closing of the inward-facing hydrophilic cavity. A cavity opens on the periplasmic in wild-type LacY allowing substrate to be released, and conformational heterogeneity increases after sugar binding. In contrast, the periplasmic cavity in C154G mutant does not form easily after sugar binding, corresponding to a reduced number of conformers in the ligand-bound state and restricted access for the sugar from periplasmic side.
Footnotes
- ‖To whom correspondence may be addressed. E-mail: sweiss{at}chem.ucla.edu or rkaback{at}mednet.ucla.edu
- © 2007 by The National Academy of Sciences of the USA
