Two substitutions on the WPD-loop (V360P and S361E, see Figure ) separate Chimeras 2 and 3, and therefore give rise to the observed increase in stability of the wide-open state for Chimera 2, as seen in our MD simulations. To probe this further, we determined the ΔRMSF and the differences in the interaction networks (using KIF) for Chimeras 2 and 3 when sampling the wide-open conformational state (Figure ), in order to identify how these substitutions contributed to this population shift. Our RMSF calculations revealed the central portion of the WPD-loop Chimera 2 structure to be substantially more rigid than in Chimera 3 (Figures and). Further, the α-helix which follows the WPD-loop (which contains the two substitutions V360P and S361E) was also observed to be significantly more stable in Chimera 2 (Figures and). Interestingly, the N-terminal portion of the WPD-loop was observed to be significantly more stable in Chimera 3, although the magnitude of these changes was comparatively lower (~3Å vs 0.5 Å ΔRMSF differences, see Figure ). of the calculated ΔRMSFs, and those residues identified as significant have a black dot placed at the bottom of the graph. To account for the usage of multiple t-tests, the Benjamini-Hochberg correction was applied, using a false discovery rate of 5%, see the Methods section for further details. (B) Projection of the calculated ΔRMSFs identified as significantly different onto the structure of YopH, with residues colored from blue (more rigid in the Chimera 3), to white (equally rigid or no significant difference) to red (more rigid in Chimera 2). Our KIF calculations (Figure ) identified the largest change in the interaction network to be the removal of an inter-loop hydrogen bond between the backbone of V360P to F357 (Figures and). This interaction is present in Chimera 3 but abolished in Chimera 2 due to the proline nitrogen no longer being able to form a hydrogen bond (a proline does not have a hydrogen on the nitrogen group). Given the nature of the substitution (to a conformationally constricted residue), we also analyzed the backbone dihedral angles sampled by V360P in both the wide-open and open states of Chimeras 2 and 3 and observed no sampling of disallowed Ramachandran values (Figure ). This would suggest that the observed population shift was not a result of any steric hindrance as a result of the addition of a proline residue. Taken together, the V360P substitution would not be expected to give rise to the observed population shift, as the only change in the interaction network would be expected to increase the stability of the wide-open state for Chimera 3.