Phanta: a non-fluorescent photochromic acceptor for pcFRET

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Photo-convertible Förster Resonance Energy Transfer (pcFRET) requires specific donor and acceptor pairs.
• Existing non-fluorescent acceptors for pcFRET are limited.
• Genetically encoded proteins offer advantages for live-cell imaging.

Purpose of the Study:

• To develop and characterize a novel orange non-fluorescent photochromic protein for pcFRET.
• To demonstrate the utility of this protein as an acceptor in a FRET pair.
• To apply the developed FRET system for monitoring caspase 3 activation in live cells.

Main Methods:

• Development of a genetically encoded orange photochromic protein, named Phanta.
• Characterization of Phanta's photochromic properties and spectral overlap with donor fluorescent proteins.
• Construction and testing of a Phanta-EGFP FRET pair for live-cell imaging.
• Monitoring caspase 3 activation using two-color imaging of the FRET pair.

Main Results:

• Phanta, an orange non-fluorescent protein with a quantum yield of 0.003, was successfully developed.
• Phanta can be reversibly switched between two states using cyan and violet light.
• Phanta exhibits spectral overlap with common donor green fluorescent proteins like EGFP.
• The Phanta-EGFP FRET pair effectively monitored caspase 3 activation in live cells.

Conclusions:

• Phanta is the first reported genetically encoded non-fluorescent acceptor for pcFRET.
• The Phanta-EGFP pair provides a robust system for live-cell caspase 3 activity monitoring.
• This system is compatible with standard instrumentation and simple protocols, facilitating broader research applications.