The T4 lysozyme L99A structure with toluene bound was taken from the 4W53 protein structure from the Protein Data Bank. Hydrogens were added to the protein using tleap from AmberTools14. Hydrogens were added to toluene using Maestro and parameterized using GAFF version 1.7 and AM1-BCC charges. Hydrogens and missing atoms of the protein were added using tleap in AmberTools14, and parameterized using ff99sbildn. A TIP3P rectangular box was added with 10Å padding from the protein to the nearest box edge, and Cl atoms were added to neutralize the charge of the system. The resulting .prmtop and .inpcrd files were converted to the equivalent GROMACS formats using ACPYPE. The system was then minimized using steepest descent running for 2500 steps. The system was then equilibrated in GROMACS 5.1 for 25000 2 fs steps with constant volume, then equilibrated under constant pressure for the same number of steps using a Parrinello-Rahman barostat to maintain a pressure of 1 atm. Long range electrostatics were calculated using Particle Mesh Ewald. Long range dispersion corrections were used for calculating the energy and pressure. These preparatory simulations were performed at 300 K and v-rescale with tau_t set to 0.1 ps was used to perform temperature coupling. v-rescale is the GROMACS option that provides temperature coupling using velocity rescaling with a stochastic term; tau_t specifies the average time between velocity rescalings. Full details of the simulation setup can be found in the .mdp files in the Supporting Information (SI).