Figure 6. The novel inter-helical hydrogen bonds in HmBRI (green) relative to HsBR (yellow).

In each case, the structure alignment was done locally to emphasize the local effects. (A) The substitution A126_Hs → T124_Hm results in two novel hydrogen bonds connecting the helix D to helices C and E. (B) The coupled substitution M56_Hs → N54_Hm, A84_Hs → T82_Hm results in introduction of the hydrogen bond between the helices B and C. Interestingly, the intra-helical hydrogen bond between the I52 backbone oxygen and T55 is replaced with the hydrogen bond between the homologous I50 backbone oxygen and N54 side-chain amine. (C) Coupled substitution V188_Hs → W192_Hm, L207_Hs → G211_Hm results in introduction of the hydrogen bond between the helices F and G (E208 backbone oxygen and W192 indole nitrogen). Interestingly, the glycine is the only possible amino acid at the position 211, as the C_β atom of any other amino acid would create a steric conflict with W192 side-chain. The hypothetical position of the residue 211 C_β atom is shown by the green sphere, and its van der Waals radius, as well as those of proximal W192 heavy atoms, is shown as a black circle. Introduction of the bulky tryptophan at the position 192 might be another reason for the divergence of the helices F and G in HmBRI, and, as a consequence, the bigger water-accessible volume of the proton release region.