In addition to residues W238 and F241, a number of potentially significant aromatic residues on the N-terminal domain were identified as potentially significant to protein-membrane binding. From these residues, a series of additional single and double mutants were proposed, in wild type amino acids were replaced with alanine or aspartic acid. To quantify the impact of mutation, free energy perturbation calculations were used to compare the free energy associated with membrane-binding for wild-type and mutant proteins. All mutations tested were found to reduce proteinmembrane association relative to the wild type, with positive ∆∆G (Table ). Among single mutants, mutations F15D and F241D were found to have the most profound negative effects (Table ). The greatest negative effects overall were found in double mutants F15DF241D and W238DF241D, highlighting how critical F241 is for stable membrane association. Negatively charged aspartate mutations were in all cases more harmful to membrane association than alanine mutations that lack a change in charge.