Fragile protein folds: sequence and environmental factors affecting the equilibrium of two interconverting, stably folded protein conformations

Recent research on fold-switching metamorphic proteins has revealed some notable exceptions to Anfinsen's hypothesis of protein folding. We have previously described how a single point mutation can enable a well-folded protein domain, one of the two PAS (Per-ARNT-Sim) domains of the human ARNT (aryl hydrocarbon receptor nuclear translocator) protein, to interconvert between two conformers related by a slip of an internal [Formula: see text] strand. Using this protein as a test case, we advance the concept of a “fragile fold”, a protein fold that can reversibly rearrange into another fold that differs by a substantial number of hydrogen bonds, entailing reorganization of single secondary structure elements to more drastic changes seen in metamorphic proteins. Here we use a battery of biophysical tests to examine several factors affecting the equilibrium between the two conformations of the switching ARNT PAS-B Y456T protein. Of note is that we find that factors which impact the HI loop preceding the shifted I [Formula: see text] strand affect both the equilibrium levels of the two conformers and the denatured state which links them in the interconversion process. Finally, we describe small molecules that selectively bind to and stabilize the wild-type conformation of ARNT PAS-B. These studies form a toolkit for studying fragile protein folds and could enable ways to modulate the biological functions of such fragile folds, both in natural and engineered proteins.