Designing artificial fluorescent proteins and biosensors by genetically encoding molecular rotor-based amino acids

Fluorescent proteins are indispensable tools in biological and medical research. The fluorophores are typically encoded by the primary amino acid sequence, from which a fluorescent molecular rotor structure forms upon protein folding. Here, inspired by the fluorogenic property exhibited by native fluorophores, we designed a collection of fluorogenic non-canonical amino acids that feature this molecular rotor structure-termed fluorescent molecular rotor amino acids (FMR-AAs)-akin to native fluorescent protein fluorophores. By incorporating FMR-AAs into target proteins through an expanded genetic code, we use them as encoded fluorophore analogues within a confined protein microenvironment, thus readily transforming diverse non-fluorescent proteins into artificial fluorescent proteins. We also use FMR-AAs in selected proteins as sensitive fluorescent probes for monitoring protein-protein interactions and detecting protein conformational changes in vitro and in living cells. This approach enables the generation of artificial fluorescent proteins and the development of biosensors from potentially any protein of interest with minor modifications.