A Universal Protein Ladder for Standardization of Diverse FRET Assays

Area of Science:

• Biophysics
• Molecular Biology
• Spectroscopy

Background:

• Fluorescence resonance energy transfer (FRET) is a powerful spectroscopic ruler for measuring molecular distances (3-10 nm).
• Single molecule FRET (smFRET) can resolve distinct structural states, but lacks universal benchmarks for comparing in vitro and intracellular measurements.

Purpose of the Study:

• To present a modular protein ladder for harmonizing FRET data across diverse experimental platforms.
• To enable accurate translation of molecular distance measurements between in vitro and intracellular environments.

Main Methods:

• Engineered repeating tetratricopeptide repeat (TPR) motifs.
• Self-labeling enzymes and click chemistry with non-canonical amino acids for fluorophore attachment.
• Demonstrated consistent FRET efficiencies across mammalian and bacterial expression systems.

Main Results:

• The protein ladder provides a predictable calibration curve for FRET efficiencies.
• Consistent FRET efficiencies were observed across different expression systems and labeling strategies.
• Enabled interpolation between various FRET modalities, including confocal smFRET, flow cytometry FRET, and FLIM-FRET.

Conclusions:

• The developed protein ladder harmonizes FRET data, bridging the gap between in vitro and cellular environments.
• This standardization is crucial infrastructure for accurate molecular distance determination in complex biological systems.
• Facilitates reliable comparison of FRET measurements across diverse experimental setups and biological contexts.