Yanai, Y.; Tsukada, M.; Kimura, Y.; Umeno, D.

The fusion of different proteins can result in the linkage-dependent emergence of molecular switches. In cases where allosteric regulation is designed between the input and output modules of fusion proteins, it is hard to predict whether on-switching or off-switching will occur. However, binding-induced folding, a non-allosteric molecular switch mechanism, has the potential to quickly establish a mutually regulatory relationship between the two fused proteins, in the way whether on-switching or off-switching will occur would be predictable. We inserted chloramphenicol acetyltransferase (CAT) from E. coli into a loop of a D-fucose-responsive mutant of transcription factor AraC, using linker libraries with various lengths. We found that on-switches tend to emerge when two proteins are fused with a small pitch gap at the junction, while fusion designs with a large pitch gap result in the frequent emergence of off-switches. Both types of switches rapidly evolved their switching efficiency upon mutations, establishing the D-fucose-on and -off regulation of CAT activity without disrupting the D-fucose-inducible logic of AraC function. To our surprise, both one-input/two-output split gates thus obtained could be easily inter-converted upon mutations. Through mutations, proteins not only frequently acquire properties as binding-induced folders, but also rapidly establish and evolve a mutual regulatory relationship with unrelated fusion partners, as well as transform their regulatory logic.