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1.
FIGURE 8.

FIGURE 8. From: Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium.

Cation activation constants for D2G FRET. On a time trace with λEx 290 nm and λEm 500 nm, Na+ (circle) or Li+ (triangle) at increasing concentrations was added after 10 μm D2G was added. The stimulation in FRET signal was plotted as the concentration of Na+ or Li+ presented in solution, and K0.5Na and K0.5Li were determined by hyperbolic fitting (). A.U., absorbance units.

Lan Guan, et al. J Biol Chem. 2011 February 25;286(8):6367-6374.
2.
FIGURE 7.

FIGURE 7. From: Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium.

EC50 for melibiose displacement of D2G binding. With SLM-8100/DMX fluorescence spectrophotometer, displacements of D2G by melibiose at increasing concentrations were measured on a time trace with λEx 290 nm and λEm 500 nm and expressed as percentages of a total bound D2G. EC50 in the absence (square) or presence of Na+ (circle) or Li+ (triangle) was determined by hyperbolic fitting, and appKd for melibiose was calculated by the equation: appKd = EC50/{1 + [D2G]/Kd for D2G} ().

Lan Guan, et al. J Biol Chem. 2011 February 25;286(8):6367-6374.
3.
FIGURE 6.

FIGURE 6. From: Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium.

appKd for D2G. Titration of D2G was measured by FRET in the presence of H+ (square), Na+ (circle), or Li+ (triangle) with Hitachi F-7000 fluorescence spectrophotometer. diffFRET representing the bound D2G was integrated between 460 and 540 nm for MelB-ST and 440 and 530 nm for MelB-EC, and the hyperbolic function was applied to fit data (). Poor D2G FRET signal in the absence of Na+ and Li+ precluded accurate measurements in MelB-ST. A.U., absorbance units.

Lan Guan, et al. J Biol Chem. 2011 February 25;286(8):6367-6374.
4.
FIGURE 5.

FIGURE 5. From: Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium.

Deduced FRET spectra. A and C, diffFRET emission before and after the addition of excess melibiose was calculated from . Curves filled with light or dark gray represent the deduced diffFRET spectra in the presence of Na+ or H+, and the open blue curves represent diffFRET spectrum in the presence of Li+. B and D, Na+- and Li+-dependent FRET were calculated by subtracting the H+-coupled diffFRET and plotting in filled and open curves, respectively. A.U., absorbance units.

Lan Guan, et al. J Biol Chem. 2011 February 25;286(8):6367-6374.
5.
FIGURE 2.

FIGURE 2. From: Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium.

Effect of ΔΨ on [3H]melibiose uptake. Active transport with RSO vesicles (pH 7.5) containing MelB-ST was carried out in the absence and presence of 20 mm Na+ (circle) or Li+ (triangle). At the 2-min time point of transport time course, 50 μm valinomycin (Vm, dashed lines), 1 μm nigericin (Ng, dotted lines), or both (dashed and dotted lines) were added into RSO vesicles.

Lan Guan, et al. J Biol Chem. 2011 February 25;286(8):6367-6374.
6.
FIGURE 1.

FIGURE 1. From: Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium.

[3H]Melibiose transport. A and B, downhill transport in DW2 intact cells with overexpressed MelB-ST (A) or MelB-EC (B). Calculated intracellular [3H]melibiose concentrations were plotted as a function of time. C, uptake with RSO vesicles containing MelB-ST. Square, circle, and triangle represent data that were obtained in the presence of H+, Na+, and Li+, respectively. Na+ or Li+ (20 mm) was added by premixing with [3H]melibiose. Error bars indicate S.E.

Lan Guan, et al. J Biol Chem. 2011 February 25;286(8):6367-6374.
7.
FIGURE 9.

FIGURE 9. From: Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium.

Cation competition. On a time trace at λEx 290 nm and λEm 500 nm with SLM-8100/DMX fluorescence spectrophotometer, RSO containing MelB-ST was incubated with 5.6 mm Li+ (5-fold K0.5) after the addition of 10 μm D2G and followed by a saturated concentration of either Li+ (40 mm, green curve) or Na+ (44 mm, red curve) as the arrows indicate. As a control, Na+ (44 mm) was directly added without Li+ (blue curve). The FRET signals are displaced with 130 mm melibiose. A.U., absorbance units.

Lan Guan, et al. J Biol Chem. 2011 February 25;286(8):6367-6374.
8.
FIGURE 4.

FIGURE 4. From: Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium.

FRET. RSO vesicles prepared from DW2 cells without or with MelB at a protein concentration of 0.5 mg/ml were excited at 290 nm. Emissions were plotted between 410 and 570 nm. Sugar and cation were added into RSO vesicles sequentially according to the following order: black lines, Trp emission; red lines, D2G emission (10 μm); green lines, Na+, Li+, Rb+, or Cs+ (20 mm); blue lines, Na+ or Li+ (20 mm), Rb+ or Cs+ (100 mm); cyan and magenta lines, melibiose (130 mm). In MelB-ST with Rb+, green and cyan curves were removed for clarity. Data were measured with Hitachi F-7000 fluorescence spectrophotometer. A.U., absorbance units.

Lan Guan, et al. J Biol Chem. 2011 February 25;286(8):6367-6374.
9.
FIGURE 3.

FIGURE 3. From: Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium.

[3H]Melibiose efflux and exchange. Intracellular [3H]melibiose was expressed as a percentage of the zero time point in the presence of external 200 mm Na+ or Li+. Open (Efflux) and filled (exchange) symbols represent equimolar cation concentrations across the membrane. The dashed line represents external 200 mm Na+ or Li+. A, de-energized RSO vesicles were pre-equilibrated with 20 mm [3H]melibiose without Na+ and Li+. Right-filled hexagons and stars represent the presence of external Na+ and Li+, respectively. The dotted line represents the presence of 20 mm external cations. RSO vesicles were also treated with 3.6 mm MIANS for 5 h at 23 °C and diluted into 100 mm potassium Pi, pH 7.5, in the absence of melibiose (crossed square). B and C, de-energized RSO vesicles were pre-equilibrated with 20 mm [3H]melibiose and 20 mm Na+ (B) or Li+ (C). The left-filled symbol represents the dilution buffer without Na+ or Li+ (20/0 mm). The right-filled symbol represents the dilution buffer containing 200 mm Na+ or Li+ (20/200 mm).

Lan Guan, et al. J Biol Chem. 2011 February 25;286(8):6367-6374.

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