Our MD simulations (Figure ) identified two major population shifts as a result of the WPDloop substitutions: one in the closed state as a result of the mutations that separate wild-type YopH and Chimera 3 (as Chimeras 3 and 2 behave similarly in this state); and one in the wide-open state as a result of the two mutations that separate Chimeras 3 and 2 (as wild-type YopH and Chimera 3 behave similarly in this state). Having observed that there is a significant population shift, the next important question is how the WPD-loop substitutions which separate these enzymes gave rise to these observed population shifts. Focusing first on the differences between wild-type YopH and Chimera 3, we determined the average difference in the Cα-root mean squared fluctuations (ΔRMSFs) for every residue in each enzyme from our simulations of the closed WPD-loop conformation (Figure ). To evaluate if each residue's calculated ΔRMSF was significant, we performed a two-sided t-test using the 15 replicas performed for each enzyme as input.