Figure 8

Calculated relative binding free energies for the mutation of residue 211 between the arginine and serine end states reveal discrepancies based on the starting structure. (A) Computed relative binding free energies, analogous to Fig. 5, with the addition of the S211R mutation. The pink markers show the negative of the S211R values to compare on the same scale as R211S. The R211S relative binding free energies ideally should be the same as those for \(- 1 \times\) S211R. The error bars represent the root sum square values of standard errors from the Bennett acceptance ratio for the apo and holo states. (B) Bar plot showing the change in the computed \(\Delta G_3\) (apo protein) or \(\Delta G_4\) (holo protein) of individual processes of the thermodynamic cycle (Fig. 2) for the R211S/S211R mutation. The apo protein value represents \(\Delta G_{3,\> R211S} - (- 1 \times \Delta G_{3,\>S211R})\), and the analogous expression is applied to the \(\Delta G_4\) values of gbi1 and gbi2. These values should be zero since free energy is a state function which is independent of the path taken.