Different mCherry isoforms show distinct photophysical properties in FRET tandem constructs

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Fluorescent proteins like mCherry are vital for cellular research.
• Alternative translation initiation sites (aTIS) in mCherry can lead to shorter isoforms with altered properties, potentially biasing experimental results.
• Understanding these variations is crucial for accurate interpretation of biological data.

Purpose of the Study:

• To investigate the impact of aTIS on mCherry's photophysical properties and functionality.
• To analyze mCherry isoforms in both prokaryotic and eukaryotic systems using spectroscopic techniques.
• To assess the influence of translation initiation sites on Förster resonance energy transfer (FRET) efficiency.

Main Methods:

• Design and analysis of FRET tandem constructs using mNeonGreen (donor) and mCherry (acceptor).
• Systematic evaluation using steady-state spectroscopy, time- and spectrally-resolved fluorescence measurements.
• Fluorescence lifetime imaging (FLIM) based FRET measurements to quantify energy transfer.

Main Results:

• Longer mCherry isoforms maintained photophysical properties and FRET acceptor functionality.
• Shorter isoforms generated by aTIS were non-fluorescent and non-functional.
• The choice of translation initiation site significantly affected donor fluorescence lifetime, intensity, and FRET efficiency.

Conclusions:

• Alternative translation initiation sites in mCherry can produce non-functional protein variants.
• These variations can substantially impact the reliability of mCherry as a fluorescent reporter.
• Considering translation initiation mechanisms is essential for accurate interpretation of biological measurements in both prokaryotic and eukaryotic systems.