A Luminescence-Based Screening Platform for Lanthanide-Binding Peptides and Proteins.

Area of Science:

• Biochemistry
• Biotechnology
• Materials Science

Background:

• Lanthanide ions offer unique properties like luminescence and paramagnetism for biomolecule functionalization.
• Natural lanthanide-binding proteins are scarce, and de novo design faces challenges with non-specific binding.
• Developing efficient screening methods is crucial for discovering and engineering lanthanide-binding proteins.

Purpose of the Study:

• To create an efficient workflow for screening protein scaffolds that coordinate lanthanide ions.
• To identify de novo designed protein variants with high-affinity lanthanide-binding capabilities.
• To enable the discovery of novel lanthanide-binding peptides and proteins for various applications.

Main Methods:

• A microtiter plate-based assay utilizing filter plates and dual readout based on sensitized terbium (Tb3+) luminescence.
• Benchmarking the assay with control proteins to differentiate site-specific coordination from surface binding.
• Screening a de novo scaffold library derived from a helical bundle protein.

Main Results:

• The assay successfully distinguished site-specific lanthanide coordination from non-specific surface binding.
• Identified lanthanide-binding variants with nanomolar affinity and distinct lanthanide specificity.
• Observed increased thermostability in metal-binding variants, indicating functional protein evolution.

Conclusions:

• The developed screening platform efficiently identifies lanthanide-binding peptides and proteins.
• This approach facilitates the discovery and engineering of biomolecules with tailored lanthanide-binding properties.
• The identified variants hold potential for applications in vitro and within living cells.