(A) Molecular systems used to evaluate the change in the free energy of polar/hydrophobic solvation that results from membrane bending, for all-inward GltPh. The solvent-accessible surface area of each residue in the protein was calculated for either case (Materials and methods). (B) Per-residue change in the free-energy of polar/hydrophobic solvation, deduced from two alternative hydrophobicity scales (Materials and methods). Negative values of δGsol indicate the deformed membrane state is favored; positive values favor the flat configuration instead. All residue contributions, in the three protomers, were summed to obtain the total value of ΔGsol. (C) Residues for which the magnitude of δGsol is 1 kBT or greater (with both scales) are highlighted in the context of the proposed membrane deformation for all-inward GltPh. Residues that favor the deformed state are shown in gray; those that favor the flat state are shown in red. The protein structure examined in panels (A, B) is an equilibrated snapshot of the all-atom simulation of all-inward GltPh. An analogous analysis of the X-ray structure of all-inward GltPh is shown in Figure 6—figure supplement 1. (D) Residues involved in H-bonds to lipid head groups (Figure 5C) that are also predicted to have co-evolved with neighboring residues at the protein-lipid interface (Materials and methods). The position of these residues in the outward-facing X-ray structure of GltPh is indicated by their Cα atoms (spheres). On the cytoplasmic side of the protein, there are three main clusters: one on the transport domain (cluster 1, purple) comprising residues E80, K84 and Y88 (TM3), L250, Y254 (TM6), I411, V412, K414, T415, and E416 (TM8); and two mostly on the scaffold: one including A67 (TM3), A164, Y167 (TM4), K196, and G200 (TM5) (cluster 2, gray), and the other including K15 (TM1), Q203 and I207 (TM5) (cluster 3, white). On the periplasmic side, there are two clusters: one on the transport domain (cluster 4, cyan), comprising R105, N108 (TM3), F323 (TM7), V335, and Q338 (HP2a); and one on the scaffold (cluster 5, yellow) containing L30, H32, Y33 (TM1), T41, Y42, and V43 (TM2).