Biosynthetic Lanthanide-Luminescent Mini-Proteins Using Genetic Code Expansion

Noncanonical amino acids (ncAA) are promising as light-harvesting antennae for lanthanide luminescence in lanthanide-binding peptides and proteins. Here, we present empirical insights into antenna–lanthanide interactions, which reveal design principles of bright luminescent proteins. Peptides designed to act as lanthanide-binding tags (LBT) show a trade-off between sensitization and lanthanide-binding affinity. We generated a new protein, termed RF2, through computational design with nanomolar binding affinity and more than a 2-fold increase in terbium(III) luminescence relative to LBT. In this scaffold, 6-azatryptophan (6AW) achieved a 10-fold enhancement of the europium(III) luminescence in vivo . The RF2 6AW mutant also sensitizes the luminescence of dysprosium(III) and samarium(III). These results demonstrate the capability of de novo protein design to produce highly luminescent lanthanide-binding mini-proteins with a genetically encoded ncAA antenna.