Molecular dynamics (MD) simulations (50 µs of accumulated sampling per system) were used to generate Markov State Models (MSMs) describing how the conformational sampling of the WPD-loop is altered for each chimera. The MD simulations were performed at the phosphocysteine intermediate (the starting point for the rate-limiting hydrolysis step of the reaction, Figure ), We note that WPD-loop closure is essential for correct positioning of the catalytic aspartic acid (Figure ) in the active site, therefore conformational fluctuations of the WPD-loop at this intermediate have the potential to adversely affect turnover. MSMs for each system were constructed using the Cα-atom distances of the WPD-loop and neighboring E-loop residues. The E-loop residues were included due to the importance of the "correct" sampling of several E-loop residues to provide transition state stabilization to WT-PTP1B. MSMs for each system are shown in Figure , with the free energy landscapes (FELs) of each system described by the first two time-lagged independent components (TICs), which represent the slowest motions of the systems (see the Materials and Methods). We note that the TICs used to describe each system are identical (i.e., TIC1 in WT-PTP1B is identical to that of TIC1 in all the chimeras). Analysis of the FELs of WT-PTP1B and Chimera 7 (which differ only by the F182Q substitution, see Focusing first on changes in the closed WPD-loop conformation for each chimera as compared to the WT (Figure ); the ΔRMSF values between WT PTP1B and Chimera 7 which differ from one another by a single point mutation (F182Q) are highly similar with the exception of the Eloop. The H-bonding analysis identifies a newly formed hydrogen bond (H-bond) between the side chains of F182Q and D181 in Chimera 7 (Figure ) and a reduction in the occupancy of the Hbonds between D181 to R221 (on the P-loop) and K120 (on the E-loop), which may have therefore altered the stability of the E-loop. For Chimeras 1, 3 and 4, however, there are differences in the rigidity of several regions around the WPD-loop as well as the WPD-loop itself, including the Eloop, Loop 11 and the N-terminal section of the α7-helix (Figure ). Both Loop 11 and α7-helix are known parts of the PTP1B allosteric network that regulates the WPD-loop conformation, suggesting the mutations present in these chimeras have also impacted this allosteric network. The new intra WPD-loop H-bond between G183T and P180 is likely partially responsible for this rigidification alongside the new side chain H-bond between T178N and T177 (Figure ). RMSFs in the open WPD-loop conformation and their H-bonding interaction networks are virtually identical (Figure ). Given the single point mutation separating the two enzymes (F182Q) points out to solvent in this conformation, these results are perhaps unsurprising.