Dark proteins disturb multichromophore coupling in tetrameric fluorescent proteins

Area of Science:

• Biophysics
• Molecular Biology
• Protein Engineering

Background:

• DsRed and its variants are tetrameric coral fluorescent proteins with coupled multichromophoric systems.
• These proteins can form green or red emitting chromophores within monomers, potentially linked by fluorescence resonance energy transfer (FRET).

Purpose of the Study:

• To investigate FRET efficiency and the role of non-fluorescent proteins in DsRed tetramers using single molecule spectroscopy.
• To analyze DsRed variants (DsRed2, DsRed_N42H, AG4) for FRET coupling and the presence of dark proteins.

Main Methods:

• Spectrally resolved room temperature single molecule spectroscopy.
• Analysis of DsRed and its variants (DsRed2, DsRed_N42H, AG4) at the single tetramer level.

Main Results:

• The majority of DsRed tetramers demonstrate effective FRET coupling between chromophores.
• A characteristic fraction of tetramers for each variant showed a lack of effective FRET.
• The presence of non-fluorescent (dark) proteins was identified as the cause for interrupted energy transfer in some tetramers.
• Dark proteins lead to donor dequenching, significantly influencing bulk emission spectra.

Conclusions:

• DsRed tetramers are generally well-coupled via FRET, but the presence of dark proteins is a significant factor affecting energy transfer.
• Understanding the role of dark proteins is crucial for interpreting bulk spectral properties and for protein engineering applications.
• Single molecule spectroscopy is effective in elucidating the heterogeneity of FRET efficiency within DsRed tetramer populations.