Biosynthetic Lanthanide-Luminescent Mini-Proteins Using Genetic Code Expansion

Area of Science:

• Biochemistry
• Biophysics
• Protein Engineering

Background:

• Noncanonical amino acids (ncAA) show promise as light-harvesting antennae for lanthanide luminescence.
• Understanding antenna-lanthanide interactions is key to designing brighter luminescent proteins.

Purpose of the Study:

• To investigate antenna-lanthanide interactions for designing bright luminescent proteins.
• To develop a computationally designed mini-protein with high lanthanide-binding affinity and luminescence.

Main Methods:

• Computational protein design to create the RF2 scaffold.
• Incorporation of 6-azatryptophan (6AW) as a noncanonical amino acid antenna.
• Characterization of lanthanide luminescence enhancement (Tb3+, Eu3+, Dy3+, Sm3+) in the designed protein.

Main Results:

• A novel protein, RF2, was designed with nanomolar lanthanide-binding affinity.
• RF2 exhibited over a 2-fold increase in terbium(III) luminescence compared to lanthanide-binding tags (LBT).
• The RF2 scaffold with 6-azatryptophan (6AW) achieved a 10-fold enhancement of europium(III) luminescence *in vivo* and sensitized other lanthanides.

Conclusions:

• *De novo* protein design can yield highly luminescent lanthanide-binding mini-proteins.
• Genetically encoded noncanonical amino acids serve as effective antennas for lanthanide luminescence.
• The RF2 6AW system offers a powerful platform for developing novel luminescent biological tools.