The h-cisPT and its mutants were expressed and purified as previously described. Briefly, Escherichia coli T7-expressed competent cells were co-transformed with DHDDS and NgBR. The cultures were grown in Terrific Broth medium at 37°C until reaching OD600nm = 0.6. Protein expression was induced at 16°C by adding 0.5 mM isopropyl For each mutant, we determine the binding free energy difference for the horizontal legs connecting bound and solution states (Eq. 1). In this representation, wild type proteins are on the left and are circled in red, while mutants are on the right and circled in blue, so ∆G Bound and ∆G Solution follow the solid arrows as indicated from red to blue. In FEP, the dashed arrows represent different hydration free energies for the wild type and mutant proteins, and are not calculated, but complete the thermodynamic cycle, as the sum for the free energy changes around the cycle must be zero.