Titration curves for the two protein systems, T4L99A and MUP1, can be found in the Supplementary Information. Figure shows the calculated affinities versus experimental data and ABFE calculations. For T4L99A, the correlation with experiment (R 2 = 0.661) is comparable to other methods and superior to our FEP protocol (R 2 = 0.255, SI). However, the average error and RMSE with respect to experiment are particularly high, highlighting the added complexity of a protein system compared to a simple host-guest test case. Phenol, which is thought to predominantly bind to the unfolded state of T4L99A, was included as a negative control with GCNCMC predicting a slightly negative binding free energy (K D = 0.21M ). It is possible that this weak binding is masked in the thermal shift assay by preferential binding to the unfolded protein. Crucially, the titration results are in good agreement with those obtained using FEP which implies that the methods are consistent.