Engineering the native ensemble to tune protein function: Diverse mutational strategies and interlinked molecular mechanisms

Natural proteins are fragile entities, intrinsically sensitive to perturbations both at the level of sequence and their immediate environment. Here, we highlight the diverse strategies available for engineering function through mutations influencing backbone conformational entropy, charge–charge interactions, and in the loops and hinge regions, many of which are located far from the active site. It thus appears that there are potentially numerous ways to microscopically vary the identity of residues and the constituent interactions to tune function. Functional modulation could occur via changes in native-state stability, altered thermodynamic coupling extents within the folded structure, redistributed dynamics, or through modulation of the population of conformational substates. As these mechanisms are intrinsically linked and given the pervasive long-range effects of mutations, it is crucial to consider the interaction network as a whole and fully map the native conformational landscape to place mutational effects in the context of allostery and protein evolution.