IsPETase in all of the H/F and S/I PETase mutants (Figure ). In contrast, the space created by the S/I substitution enables the tryptophan to rotate into the A conformer in all PETases, as well as exploring new conformations (Figure and 7) that have not been captured in experimental structures. This is consistent with the expected plasticity of this residue in the unliganded form of the enzyme. This plasticity is lost in the H/F variant of IsPETase, consistent with the observed loss of activity of the IsPETase S187H variant. This data highlights that the S/I residues alone are sufficient to facilitate W185 and the equivalent residue in all other enzymes to rotate, without the need for other modifications to the scaffold, and that this "wobbling effect" appears to be transferable to other PETases. Given the observed conformational plasticity of the conserved tryptophan residue in loop W, we opted to fully explore its torsional space via well-tempered metadynamics simulations, in which the ꭓ1 and ꭓ2 dihedrals were used as collective variables (CVs). These simulations reached convergence within 400 ns, in terms of the diffusive behavior of the simulations (Figure ). The free energy surfaces (FES) for each enzyme as a function of the two chosen CVs are presented in Figure . The overall landscape is conserved across all enzyme variants, with several common basins (labeled from A to I), separated by high energy barriers of up to about 40 kcal/mol observed in our simulations (Table ). The D, E, and F basins correspond to the tryptophan side chain being oriented toward S/H214 and I/F218. In the F basin, the tryptophan is also proximal to the aspartic acid, in the I basin to the histidine and in the G basin to the serine of the catalytic triad, respectively. The H/F mutant of LCC and TfCut (either as H/F and S/I) feature neither the F nor the I basins. The I basin is only present in the H/F variants of IsPETase and BurPL. For LCC and TfCut, the S/I mutation lowers the barriers that connect the basins B, G, and E (Table ). This is connected to an outward torsion of the Trp side chain (i.e., in the direction of the solvent), with the G basin corresponding to a conformation where the side chain shields the catalytic histidine from the solvent. The F basin in the S/I mutant of LCC consists of the Trp side chain located in proximity to the catalytic residues. Free energy landscapes were generated as a function of the ꭓ1 and ꭓ2 dihedral angles. The minimum free energy value is set to zero in every graph. The corresponding energy barriers are tabulated in Table .