Significant differences in both the flexibility and hydrogen bonding network were observed for Chimeras 1, 3 and 4, and these changes were not just limited to the WPD-loop, but also some surrounding regions, most notably the E-loop and Loop 11 compared to WT PTP1B (Figures and). The similar stability observed for the WPD-loop across the WT and Chimeras is likely an artifact of the measurement process, in which only frames with a WPD-loop RMSD ≤ 1.5 Å to the open crystal structure conformation was taken forward for the RMSF calculations (Figure ). Analysis of the differences in the H-bonding networks of these Chimeras to WT PTP1B (Figure ) suggest the mutation G183T (present in Chimeras 1, 3 and 4) is primarily responsible for altering the stability of these regions through a new hydrogen bond between the side chain alcohol of G183T to D265. This appears to "pull" the WPD-loop, E-loop and Loop 11 apart, substantially weakening many key interactions (relative to WT PTP1B) between these loops (Figure ), thereby increasing their observed flexibility, and likely giving rise to the overall reduced stability of this conformation as seen in our Markov state models (Figure and Table ).