Flow cytometric analysis of genetic FRET detectors containing variable substrate sequences

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Post-translational modifications (PTMs) are crucial for protein function.
• Genetic Fluorescence Resonance Energy Transfer (FRET) detectors sense conformational changes.
• Quantifying PTM-induced FRET changes using flow cytometry remains challenging.

Purpose of the Study:

• To evaluate flow cytometry for quantifying PTM-dependent FRET changes.
• To demonstrate the utility of genetic FRET detectors for PTM analysis.
• To explore the application in characterizing PTM enzyme substrate specificity.

Main Methods:

• Purification and immobilization of a genetic FRET detector on microbeads.
• Flow cytometry measurement of FRET efficiency before and after Erk-2 phosphorylation.
• Coexpression of genetic detectors with PTM enzymes (Erk-2, OGT) in bacteria and cells.

Main Results:

• Flow cytometry successfully detected PTM-dependent FRET changes, indicated by an increased fluorescence ratio (R) after phosphorylation.
• The measured R value varied with substrate sequence, showing near single-residue resolution.
• Cells coexpressing O-GlcNAc transferase (OGT) and a specific genetic detector showed PTM-induced FRET efficiency changes.

Conclusions:

• Flow cytometry is a viable method for detecting and quantifying PTM-induced FRET responses.
• Genetic FRET detectors combined with flow cytometry can characterize PTM enzyme substrate specificity.
• This approach may identify in vivo PTM target sequences.