In line with our ultimate goal of improving accuracy of binding free energy calculations in pharmaceutically relevant systems, we are interested in applying NCMC sidechain rotations to the binding sites of receptors, enzymes, and other biomolecules. Thus, we sought to assess the impact of our method on sampling of a sidechain rotamer in a ligand binding site. ries of nonpolar ligands) was chosen because it has studied extensively both crystallographically and as a computational model. A cursory search of the RCSB PDB for T4 lysozyme L99A returns over 120 structures containing nearly more than 90 distinct ligands and recently, absolute binding free energy calculations were calculated between T4 lysozyme and 141 distinct ligands, using results from a more careful search. Previously it has been observed that the rotamer states of a valine sidechain in the binding pocket of T4 lysozyme remodels itself in the presence of p-xylene, as compared with other ligands like toluene. With benzene and toluene bound, valine 111 (Val111) adopts a trans conformation (χ 1 =180 • ); however in the presence of modestly larger ligands like p-xylene and o-xylene, the Val111 rotamer flips to the gauche(-) conformation (χ 1 =-60 • ). This particular rotamer rearrangement has been used previously for testing methods for sampling sidechain rearrangement on ligand binding, and has proven challenging to adequately sample with standard MD simulations, driving applications of umbrella sampling 6 and Hamiltonian replica exchange. 8,43