mOrange

mFruit are second-generation monomeric red fluorescent proteins (mRFPs) that have improved brightness and photostability compared to the first-generation mRFP1. Their emission and excitation wavelengths are distributed over a range of about 550−650 and 540−590 nm, respectively. However, the variations in their spectra can be traced back to a few key amino acids. Spectroscopic and atomic resolution crystallographic analyses of three representatives, mOrange, mStrawberry, and mCherry, reveal that different mechanisms operate to establish the excitation and emission maxima. Undergoing a second oxidation step, each mFruit produces an acylimine linkage in the polypeptide backbone. In comparison to the progenitor DsRed, direct covalent modification to this linkage (mOrange) and indirect modification of the chromophore environment (mStrawberry and mCherry) produces strong blue- and red-shifted variants. The blue shift of mOrange is induced by a covalent modification of its protein backbone.