The input files for valine-alanine were prepared using tleap from AmberTools 15. A linear valine-alanine peptide was generated with an N-terminal valine and a C-terminal alanine and parameterized using ff99SBildn. The dipeptide was explicitly solvated in tleap with a 14Årectangular box of TIP3P water, extending from the surface of the peptide to the box edge, and Cl atoms were added to neutralize the charge of the system. The system was then minimized using sander from AMBER14 with steepest descents running for 20,000 steps with 2fs timesteps and heated from 100K to 300K with constant volume for 25,000 steps using 2fs timesteps. Equilibration proceeded with sander for 500,000 steps and 2fs timesteps under constant pressure with positional restraints initially applied on all non-water atoms with a force constant of 50 kcal/mol/Å 2 . and progressively lifted in increments of 5 kcal/mol/Å 2 . over ten 50,000 step segments. The resulting topology and coordinate files of valine-alanine were used as inputs for umbrella sampling, MD, and BLUES runs.