Direct sugar binding to LacY measured by resonance energy transfer

Abstract

Trp151 in the lactose permease of Escherichia coli (LacY) is an important component of the sugar-binding site and the only Trp residue out of six that is in close proximity to the galactopyranoside in the structure (1PV7). The short distance between Trp151 and the sugar is favorable for Förster resonance energy transfer (FRET) to nitrophenyl or dansyl derivatives with the fluorophore at the anomeric position of galactose. Modeling of 4-nitrophenyl-alpha-d-galactopyranoside (alpha-NPG) in the binding-site of LacY places the nitrophenyl moiety about 12 A away from Trp151, a distance commensurate with the Förster distance for a Trp-nitrobenzoyl pair. We demonstrate here that alpha-NPG binding to LacY containing all six native Trp residues causes galactopyranoside-specific FRET from Trp151. Moreover, binding of alpha-NPG is sufficiently slow to resolve time-dependent fluorescence changes by stopped-flow. The rate of change in Trp --> alpha-NPG FRET is linearly dependent upon sugar concentration, which allows estimation of kinetic parameters for binding. Furthermore, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) covalently attached to the cytoplasmic end of helix X is sensitive to sugar binding, reflecting a ligand-induced conformational change. Stopped-flow kinetics of Trp --> alpha-NPG FRET and sugar-induced changes in MIANS fluorescence in the same protein reveal a two-step process: a relatively rapid binding step detected by Trp151 --> alpha-NPG FRET followed by a slower conformational change detected by a change in MIANS fluorescence.

Figure 1

Location of Trp residues in the structure of C154G LacY with bound sugar (PDB ID: 1PV7). The structures is displayed with Pymol 0.98 (DeLano Scientific LLC, Inc.), and transmembrane helices are colored from blue (helix I) to red (helix XII). A. Overall structure of LacY with all native Trp residues shown as yellow spheres and bound TDG shown as pink spheres. A cyan sphere highlights the Cα atom of position 331. B. Modeling of bound α-NPG. Amino acid residues involved in substrate binding and/or proton translocation (Glu269 is involved in both) are shown in green and cyan sticks, respectively. Bound TDG is shown as pink sticks and the position of the nitrophenyl moiety in α-NPG is shown as grey sticks.

Figure 2

Binding of α-NPG to C154G LacY as detected by Trp→α-NPG FRET. Measurements were carried out in 50 mM NaP_i (pH 7.5)/0.02% DDM at a protein concentration of 0.4 μM; excitation was at 295 nm. A. Trp emission spectra at different concentrations of α-NPG. Solid lines represent Trp fluorescence at increasing α-NPG concentrations: line 1, protein without α-NPG; line 2, 5 μM α-NPG; line 3, 15 μM α-NPG; line 4, 50 μM α-NPG. Arrows indicate change of fluorescence after displacement of bound α-NPG by addition of 10 mM TDG. Broken lines, spectra after addition of TDG. B. Affinity of C154G LacY for α-NPG measured by displacement with TDG. The fluorescence change after addition of 10 mM TDG (shown as arrows in panel A) plotted as a function of α-NPG concentration. Fluorescence after TDG addition was taken as 100% at each α-NPG concentration. Solid line shows hyperbolic fit to the data with estimated K_D = 19 ± 2.0 μM.

Figure 3

Effect of point mutations in LacY and the nature of the sugar on Trp→sugar FRET. Solid lines represent Trp emission spectra (excitation wavelength 295 nm) recorded after short incubation of purified protein in 50 mM NaP_i (pH 7.5)/0.02% DDM with 100 μM sugar derivative prior to TDG addition. Broken lines are Trp emission spectra after addition of 10 mM TDG. The protein concentration was 0.5 μM except in panel E where it was 2 μM. Trp emission spectra without ligand are not shown. A. C154G LacY mixed with α-NPG. B. Wild-type LacY mixed with α-NPG. C. W151Y/C154G LacY mixed with α-NPG. D. C154G LacY mixed with ortho α-NPG. E. C154G LacY mixed with Dns⁶Gal. F. C154G LacY mixed with α- or β-NPGlc or with the β-anomer of para- or ortho-NPG.

Figure 4

Stopped-flow Trp→ α-NPG FRET. Trp fluorescence changes with time were recorded after rapid mixing of purified C154G LacY with ligand in 50 mM NaP_i (pH 7.5)/0.02% DDM. Excitation and emission wavelengths were 295 and 330 nm, respectively. Stopped-flow traces (average of 5-7 measurements shown as solid lines) were fit using a single exponential equation and corrected for the instrument dead-time of 2.7 ms (broken lines). Final protein (1 μM) and sugar concentrations are given after mixing. A. Trp→ α-NPG FRET after mixing protein with buffer only (1), 10 μM α-NPG (2), 25 μM α-NPG (3) or 50 μM α-NPG (4). Estimated rates (k^obs) are 170 s^-1, 210 s^-1, and 420 s^-1 for 10 μM, 25 μM, and 50 μM α-NPG, respectively. B. Displacement of bound α-NPG by excess TDG. Traces were recorded after mixing a saturating concentration of TDG (15 mM) with protein pre-incubated with α-NPG. α-NPG concentrations were 25 μM (1) and 50 μM (2). Estimated k_obs are 128 and 122 s^-1 for 25 and 50 μM α-NPG, respectively.

Figure 5

Kinetics of α-NPG binding to C154G LacY detected by Trp→ α-NPG FRET. Stopped-flow traces were recorded after rapid mixing of purified protein with given concentrations of α-NPG as described in Figure 4 and were individually fitted with a single exponential equation. Kinetic parameters (k^obs and amplitude of fluorescence changes) were estimated statistically and plotted versus α-NPG concentration. Each point is a result of 7-10 measurements with standard deviations shown as vertical bars. A. Concentration dependence of the rates (k^obs) of α-NPG binding. The open circle represents the rate of displacement of bound α-NPG (25 μM) with 15 mM TDG. The intercept with the Y axis is koff (162 ± 9 s^-1) and the slope is k_on (4.3 ± 0.2 × 10⁶ M^-1 s^-1); the estimated K_D = 38 ± 2 μM. B. Concentration dependence of the amplitude of Trp→α-NPG FRET from the experiments shown on Panel A. The amplitude of the fluorescence changes at each α-NPG concentration were corrected for the instrument dead-time (2.7 ms) and expressed as percentage of final fluorescence level in each experiment. Solid line is a hyperbolic equation fit to data. Estimated K_D = 32 ± 5 μM.

Figure 6

Trp→ α-NPG FRET and α-NPG-induced fluorescence change in MIANS-labeled C154G/V331C LacY. Emission spectra were recorded in 50 mM NaP_i (pH 7.5)/0.02% DDM at 0.6 μM final protein concentration. A. Fluorescence of unlabeled (line 1) or MIANS-labeled (lines 2, 3) LacY without sugar; excitation wavelengths 295 nm (lines 1, 2) or 330 nm (line 3) for Trp→ α-NPG FRET or MIANS fluorescence, respectively. B. TDG effect on the fluorescence of MIANS-labeled C154G/V331C LacY; excitation at 295 nm (lines 1,2) or 330 nm (lines 3,4). Solid lines, no TDG; broken lines, addition of 15 mM TDG. The fluorescence change indicated by arrows is 30% in both cases. C. α-NPG effect on fluorescence of MIANS-labeled LacY with excitation at 295 nm: line 1, no α-NPG; line 2, addition of 50 μM α-NPG.

Figure 7

Time-dependent changes in Trp→ α-NPG FRET (A) or MIANS fluorescence (B) with MIANS-labeled C154G/331C LacY. Stopped-flow traces were recorded after rapid mixing of protein (1 μM, final concentration) with α-NPG at excitation and emission wavelengths of 295 and 330 nm (A) or 330 and 415 nm (B), respectively. α-NPG concentrations were: 20 μM (1), 50 μM (2) or 100 μM (3). The upper trace in panel A represents displacement of bound α-NPG (25 μM) by excess of TDG (15 mM). Other experimental details are described in Figure 4. Estimated rates (k^obs) are 210 s^-1, 230 s^-1 and 380 s^-1 for traces 1, 2 and 3 in panel A, and 40 s^-1, 58 s^-1 and 82 s^-1 for traces 1, 2 and 3 in panel B, respectively.

Figure 8

Concentration dependence of the rates of Trp→ α-NPG FRET (A) and α-NPG-induced changes in MIANS fluorescence (B) with MIANS-labeled C154G/V331C LacY. Two sets of data were collected after mixing of MIANS-labeled protein with α-NPG by stopped-flow: panel A, excitation and emission wavelengths, 295 and 330 nm for Trp→ α-NPG FRET; panel B – excitation and emission wavelengths, 330 and 415 nm for MIANS fluorescence. Other experimental details are described in the legend to Figure 5. The solid line in panel A shows linear regression fit with k_off = 166 ± 10 s^-1 and k_on = 1.4 ± 0.1 × 10⁶ M^-1 s^-1. Estimated K_D = 121 ± 8 μM. The solid line in panel B shows the hyperbolic fit (Eq. 4) to the experimental data with estimated parameters k₂ = 238 ± 12 s^-1, k_-2 = 2 ± 7 s^-1 and K_D = 158 ± 31 mM.