However, the relative populations of these states depends strongly on enzyme variant: already, the D10G substitution by itself appears to be sufficient to cause a conformational shift towards a closed conformation, and the SeHisA(dup13-15/D10G/Q102A/Q24L/V15[b]M) variant, which shows the highest TrpF activity of all variants studied in ref. (Table ), also shows the most significant population shift towards sampling a closed conformation of loop 1. This is consistent with structural analysis, which indicated that the Q24L substitution is important because it introduces a new stabilizing interaction with V15b, as well as an even better interaction with V15[b]M, such that the Q24L interaction is just as important as the VVR duplication for the adaptive benefit of the V15[b]M substitution to be realized. Finally, the wide-open conformation is also only rarely sampled in the SeHisA(dup13-15) variant, which does not carry the D10G substitution. We hypothesize that in the case of these variants, this is due to the presence of a Gly-Gly dyad in the hinge of loop 1, which provides the loop with enough flexibility to explore these wide-open conformations. Clearly, dup (13-15), as well as the inclusion of additional substitutions, is significantly impacting the conformational space sampled by this loop, shifting towards a loopclosed conformation of loop 1 that is favorable for TrpF activity.