To this point, the model presented is mechanistically plausible, but depends on several assumptions, such as the selected binding pose from which to extend the simulation. To validate the mechanistic hypothesis developed by simulation, double mutants of W238 and F241 were constructed and tested using fluorescent assays for their capability to extend the prenyl substrate and bind to lipid bilayers. To measure chain extension, h-cisPT was incubated with MANT-O-GPP, a fluorescent prenyl compound that increases in fluorescent intensity with chain extension, and has been shown to behave analogously to natural prenyl substrates in prior research. Fluorescence was measured in samples with and without lipid nanodiscs (Figure (A,B)). FEP signal by 54%, indicating that aspartic acid mutants have a greater negative effect on membrane association. This data supports our previous FEP results, which also indicated that a W238DF241D double mutant would have a large negative impact on protein-membrane association, and that W238AF241A would have smaller, but still negative effect on association. Regardless, this secondary membrane contact site on DHDDS is clearly important for membrane association.