Figure 1: Tuning an allosteric ensemble through mutations that promote conformational entropy.

(a) The fusion proteins comprise two domains connected by two linkers (since one domain is inserted into the other). Each domain can be active or inactive (together with or separately from the other domain) resulting in four categories of representative states as shown. (b) Non-allosteric fusions lack allostery because states with an inactive enzyme domain are much less stable than states with an active enzyme domain regardless of the presence of ligand. (c) Allostery can be introduced though the introduction of conformational flexibility through mutation, where the flexibility increases the probability of the inactive states. Thus, the desired mutations remodel the energy landscape to increase the conformational entropy of the enzyme domain in the absence of ligand. The effector remodels the landscape by increasing the stability of states that bind the effector and thus their population in the ensemble, resulting in an effector-dependent increase in the observed enzyme activity. Although the schematic depicts the creation of positive allostery, negative allostery can be established in the same manner, as can be illustrated by switching the active and inactive domains in this figure (see Hilser and Thompson²⁹).