An orange fluorescent protein (FP) with a large Stokes shift (LSS), called LSSmOrange, fills up a spectral gap in a palette of proteins with LSS. It extends the possibilities of multicolor microscopy and spectroscopy and allows designing Forster resonance energy transfer (FRET) biosensor to combine with widely-used CFP-YFP based sensors for simultaneous FRET imaging. LSSmOrange was developed from conventional orange FP by engineering an excited state proton transfer (ESPT) followed by refinement of the spectral and biochemical properties with directed molecular evolution. Previous studies on LSS red FPs such as mKeima, LSSmKate1 and LSSmKate2, revealed that specific proton relays are responsible for ESPT in these proteins. It was also demonstrated that ESPT can be introduced into conventional red FPs by site-specific mutagenesis resulting in LSS FP phenotype. Mutagenesis, low-temperature and isotope effect studies confirmed that ESPT is responsible for LSS phenotype of LSSmOrange. They also revealed a proton relay for the ESPT from protonated chromophore to Ser148 and Asp165 (numbering according to EGFP). Crystallographic studies uncovered the role of an ensemble of the key amino acid substitutions resulted in the LSS phenotype. These findings form the foundation for a future design of advanced orange and red LSS FPs with improved properties and new phenotypes.

Red-shifted fluorescent proteins with a large Stokes shift: Chemical mechanism and imaging applications of LSSmOrange

248th National Meeting (2014)

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