Dark proteins disturb multichromophore coupling in tetrameric fluorescent proteins

DsRed is representative of the tetrameric reef coral fluorescent proteins that constitute particularly interesting coupled multichromophoric systems. Either a green emitting or a red emitting chromophore can form within each of the monomers of the protein tetramer. Within the tetramers the chromophores are thought to be efficiently fluorescence resonance energy transfer (FRET) coupled. We have used spectrally resolved room temperature single molecule spectroscopy to address the issue of FRET and the role of dark proteins within single protein tetramers of DsRed and its variants DsRed2, DsRed_N42H and AG4. Our results show that for the majority of the tetramers the different chromophores are indeed effectively coupled. However, in a fraction of the tetramers that is characteristic for each DsRed variant analyzed, we observe a lack of effective FRET coupling. For these tetramers we invoke the existence of dark proteins lacking a functional chromophore that interrupt the energy transfer chain within the multichromophoric system. We show that these species lead to donor dequenching that strongly influences the bulk emission spectra.