Biosynthetic lanthanide-luminescent mini-proteins using genetic code expansion

Abstract

Non-canonical 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 nano-molar binding affinity and more than two-fold increase in terbium(III) luminescence. In this scaffold, 6-azatryptophan (6AW) achieved a ten-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.