The K146E mutation lies on the tip of the loop outside of the active site, and the absence of clear electrostatic or structural impact on the scaffold has made it challenging to pin-point a reason for enhanced activity as a result of this mutation. By projecting the per feature scores onto the structures of the R1 and R4 variants, we were able to identify a network of hydrogen bonds with a strong relationship to the W50 χ2 dihedral angle, as shown in Figure . This figure depicts how the loop K146E is located on interacts with a neighboring helix (residues 103-109) through several hydrogen bonds. By analyzing the linear correlation of these interactions to the W50 χ2 dihedral angle (i.e., the target variable), we observed that the interactions between the loop and the helix and those within the helix that are shown in Figure are all inversely correlated to the χ2 dihedral angle in both the R1 and R4 variants (Table ). This means that the strengthening of these interactions would be expected to drive the conformation of W50 towards the "A" conformational state. We calculated the root mean squared fluctuations (RMSF) of both the R1 and R4 variants, to determine the difference in RMSF as a result of the 4 mutations (ΔRMSF, Figure ). Our results show that not only is the R4 variant generally more flexible, but that both the loop on which K146E is located, and the neighboring helix, are destabilized in this variant (Figure ). Taken together, our results suggest the K146E mutation destabilizes its local environment, reducing the strength of these interactions, and ultimately their ability to drive W50 towards the "A" conformational state.