We investigated the donor effects (mCerulean3 versus mTurquoise2.1) on the spectroscopy and dynamics of mCerulean3-linker-mCitrine constructs using integrated fluorescence spectroscopy methods. Here, mCerulean3 (a cyan fluorescent protein) and mCitrine (a yellow fluorescent protein) act as Förster resonance energy transfer (FRET) pair, separated by flexible linker region. We hypothesize that the construct with mTurquoise2.1 would have many advantages as a donor, which include a higher FRET efficiency as compared with the mCerulean3 due to the enhanced spectral overlap with mCitrine. To test this hypothesis, we used steady-state spectroscopy, time-resolved fluorescence, and fluorescence correlation spectroscopy of both mCerulean3-linker-mCitrine and mTurquoise2.1-linker-mCitrine to investigate the donor effect on the FRET efficiency and translational diffusion as a means for developing a rational design for hetero-FRET constructs for environmental sensing.
|