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.