Figure 1. Two outward-facing... | Rockefeller University Press

Figure 1.

$Two outward-facing substrate-binding states in P-GltPh(S279E/D405N). (a and b) FRET efficiency population histograms of P-GltPh in the presence of 500 mM sodium salts, in the absence (a) or presence (b) of 1 mM L-Asp. N is the number of molecules analyzed. Data shown are an aggregate of two independent experiments. Population contour plots are color-coded from tan (lowest population) to red (highest). Expected conformations according to EFRET values are indicated by arrows. (c–g) ITC experiments on P-GltPh at 15°C were performed at least twice on independently prepared protein samples with similar results. Insets show the thermal power with the corresponding scales. (c–e) Aspartate binding isotherms derived from the ITC experiments in the presence of different amounts of NaCl (green squares): 50 mM (c); 500 mM (d); and 1 M (e). The 50-mM data were fitted to the single-state model, with fitted Kd = 917 nM, ΔH = −2.3 kcal mol−1, and an apparent number of binding sites, n = 1.18. 500 mM NaCl and 1 M NaCl data were fitted to the two-state binding model. The 500-mM NaCl data gave the following fitted parameters for the two states: Kd, 1.3 and 60.8 nM; ΔH, −3.3 and −7.1 kcal mol−1; n, 0.64 and 0.22. The 1-M NaCl data: Kd, 0.5 and 27.4 nM; ΔH, −3.7 and −8.7 kcal mol−1; n, 0.77 and 0.38. (f and g) Aspartate binding isotherms were obtained in 500 mM Na-gluconate (f, red circles), or NaNO3 (g, blue triangles). All data were fitted to the two-state model. The 500-mM Na-gluconate data: Kd, 4.4 and 138.3 nM; ΔH, −2.3 and −5.5 kcal mol−1; n, 1.00 and 0.28. The 500-mM NaNO3 data: Kd, 0.9 and 34.3 nM; ΔH, −2.1 and −6.5 kcal mol−1; n, 0.62 and 0.37. (h) Comparison of the n2 fraction in 500 mM NaCl or NaNO3. Each point is an independent experiment. (i) Schematic representations of the conformational and binding equilibria obtained experimentally at 10°C and 15°C in 500 mM NaCl (solid lines) or inferred (dashed lines). The thermodynamic parameters were estimated under the assumptions that there are two nonexchanging binding states. Directions of the arrows indicate directions of the free energy changes, ∆G-s, shown. All values are in kilocalories per mole. Binding ∆G-s are from Tables S1 and S2. The free energy differences between sodium-bound OFSs were calculated from equilibrium constants Keq = n1/n2. Thin lines represent steps that are slow on the time scale of ITC experiments.$

Two outward-facing substrate-binding states in P-Glt _Ph (S279E/D405N). (a and b) FRET efficiency population histograms of P-Glt_Ph in the presence of 500 mM sodium salts, in the absence (a) or presence (b) of 1 mM L-Asp. N is the number of molecules analyzed. Data shown are an aggregate of two independent experiments. Population contour plots are color-coded from tan (lowest population) to red (highest). Expected conformations according to E_FRET values are indicated by arrows. (c–g) ITC experiments on P-Glt_Ph at 15°C were performed at least twice on independently prepared protein samples with similar results. Insets show the thermal power with the corresponding scales. (c–e) Aspartate binding isotherms derived from the ITC experiments in the presence of different amounts of NaCl (green squares): 50 mM (c); 500 mM (d); and 1 M (e). The 50-mM data were fitted to the single-state model, with fitted K_d = 917 nM, ΔH = −2.3 kcal mol⁻¹, and an apparent number of binding sites, n = 1.18. 500 mM NaCl and 1 M NaCl data were fitted to the two-state binding model. The 500-mM NaCl data gave the following fitted parameters for the two states: K_d, 1.3 and 60.8 nM; ΔH, −3.3 and −7.1 kcal mol⁻¹; n, 0.64 and 0.22. The 1-M NaCl data: K_d, 0.5 and 27.4 nM; ΔH, −3.7 and −8.7 kcal mol⁻¹; n, 0.77 and 0.38. (f and g) Aspartate binding isotherms were obtained in 500 mM Na-gluconate (f, red circles), or NaNO₃ (g, blue triangles). All data were fitted to the two-state model. The 500-mM Na-gluconate data: K_d, 4.4 and 138.3 nM; ΔH, −2.3 and −5.5 kcal mol⁻¹; n, 1.00 and 0.28. The 500-mM NaNO₃ data: K_d, 0.9 and 34.3 nM; ΔH, −2.1 and −6.5 kcal mol⁻¹; n, 0.62 and 0.37. (h) Comparison of the n₂ fraction in 500 mM NaCl or NaNO₃. Each point is an independent experiment. (i) Schematic representations of the conformational and binding equilibria obtained experimentally at 10°C and 15°C in 500 mM NaCl (solid lines) or inferred (dashed lines). The thermodynamic parameters were estimated under the assumptions that there are two nonexchanging binding states. Directions of the arrows indicate directions of the free energy changes, ∆G-s, shown. All values are in kilocalories per mole. Binding ∆G-s are from Tables S1 and S2. The free energy differences between sodium-bound OFSs were calculated from equilibrium constants K_eq = n₁/n₂. Thin lines represent steps that are slow on the time scale of ITC experiments.

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