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. 2007 Feb 9;3(2):e22. doi: 10.1371/journal.pcbi.0030022

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© 2007 Bostick and Brooks.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Free-energy cycle used to determine pK_a of NH₄ ⁺ as a function of the transport axis. is the free energy for the alchemical reaction NH₄ ⁺ → NH₃ in the bulk, far from the membrane, and ΔG ^z _dp is the free energy for the same reaction at a particular position, z, along the transport axis. is the free energy to bring NH₄ ⁺ from the bulk to a particular position, z, and is the analogous quantity for NH₃.

(B) Apparent pK_a (pKa_app) for NH₄ ⁺ as a function of z for the relevant region of the channel. The top panel shows a closeup of the portion of the bottom panel enclosed in the rectangular box drawn with dashed lines. A large dashed black line marks neutral pH in both panels. Vertical dotted lines (bottom panel) mark the average positions of lipid phosphate groups. As in Figures 3 and 4, gray bars represent NH₄ ⁺ binding sites, red bars represent NH₃ binding sites, and green bars represent (de)protonation regions. The periplasmic (de)protonation region is at z = 0.77 ± 0.08 nm, and the cytoplasmic (de)protonation region is at z = −1.13 ± 0.07 nm. See the text for an explanation of the uncertainties (width of the green bars) in these regions.

(C) Synopsis of binding sites and deprotonation regions generated by multiple structural alignment of AmtB channel structures singly bound at each binding site (a total of five structures taken from umbrella sampling simulations) and the X-ray structure [17]. Some transmembrane helices have been removed from the protein (orange) to allow visibility of the binding sites. The positions of the X-ray sites are shown as red spheres, and Am binding sites derived from our analysis are shown as either blue spheres (in the case of NH₃) or blue spheres with white hydrogen atoms (in the case of NH₄ ⁺). Any observed differences in the positions of the sites are within fluctuations. (De)protonation regions are marked in green. The periplasmic and cytoplasmic (de)protonation regions coincide with the phenyl groups of F107 and F31, respectively.

(D) Hydration of NH₃ (diamonds with dashed line) and NH₄ ⁺ (circles with solid line) as a function of the transport axis derived by combining statistics from free MD production runs and umbrella sampling runs. The hydration number is defined as the average number of water molecules falling within the first coordination shell of Am. The first coordination shell of Am was taken to be the position of the first minimum in the N_Am − O_water pair correlation function (R_coord = 0.362 nm, see Figure S4).

Inline graphic — (A) Free-energy cycle used to determine pK_a of NH₄ ⁺ as a function of the transport axis. is the free energy for the alchemical reaction NH₄ ⁺ → NH₃ in the bulk, far from the membrane, and ΔG ^z _dp is the free energy for the same reaction at a particular position, z, along the transport axis. is the free energy to bring NH₄ ⁺ from the bulk to a particular position, z, and is the analogous quantity for NH₃.

(B) Apparent pK_a (pKa_app) for NH₄ ⁺ as a function of z for the relevant region of the channel. The top panel shows a closeup of the portion of the bottom panel enclosed in the rectangular box drawn with dashed lines. A large dashed black line marks neutral pH in both panels. Vertical dotted lines (bottom panel) mark the average positions of lipid phosphate groups. As in Figures 3 and 4, gray bars represent NH₄ ⁺ binding sites, red bars represent NH₃ binding sites, and green bars represent (de)protonation regions. The periplasmic (de)protonation region is at z = 0.77 ± 0.08 nm, and the cytoplasmic (de)protonation region is at z = −1.13 ± 0.07 nm. See the text for an explanation of the uncertainties (width of the green bars) in these regions.

(C) Synopsis of binding sites and deprotonation regions generated by multiple structural alignment of AmtB channel structures singly bound at each binding site (a total of five structures taken from umbrella sampling simulations) and the X-ray structure [17]. Some transmembrane helices have been removed from the protein (orange) to allow visibility of the binding sites. The positions of the X-ray sites are shown as red spheres, and Am binding sites derived from our analysis are shown as either blue spheres (in the case of NH₃) or blue spheres with white hydrogen atoms (in the case of NH₄ ⁺). Any observed differences in the positions of the sites are within fluctuations. (De)protonation regions are marked in green. The periplasmic and cytoplasmic (de)protonation regions coincide with the phenyl groups of F107 and F31, respectively.

(D) Hydration of NH₃ (diamonds with dashed line) and NH₄ ⁺ (circles with solid line) as a function of the transport axis derived by combining statistics from free MD production runs and umbrella sampling runs. The hydration number is defined as the average number of water molecules falling within the first coordination shell of Am. The first coordination shell of Am was taken to be the position of the first minimum in the N_Am − O_water pair correlation function (R_coord = 0.362 nm, see Figure S4).