Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

Abstract

The development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and appropriate localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, FusionRed-MQV, which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (~140,000 M -1 cm -1 ), increased radiative rate constant and reduced non-radiative rate constant with respect to its precursor. The properties of FusionRed-MQV derive from three mutations -M42Q, C159V and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the phenol and the acylimine ends of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the phenol end of the chromophore were mutated. The M42Q mutation is located near the acylimine end of the chromophore and was discovered using sitedirected mutagenesis guided by x-ray crystal structures. FusionRed-MQV exhibits 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells (based on FACS) compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells.

The two key substitutions in the chromophore environment of mKate2 to produce an enhanced FusionRed-like red fluorescent protein

“…In the meantime, there are definite drawbacks which take from the value of FusionRed as a multipurpose fluorescence tag and call for further improvement of this RFP. Thus, an issue of the modest molecular brightness possessed by FusionRed has been extensively addressed in the elegant studies by the Jimenez lab, where both a directed evolution [17] and semirational design [18] were utilized to engineer the brighter variants of FusionRed (specifically, the FusionRed-MQV [19] shows ~4-fold brightness advantage over the parental RFP, though its emission peak has a 20-nm hypsochromic shift). The high-resolution spatial structure of FusionRed revealed that almost a half of its molecules carries an immature chromophore and possesses a protein backbone cleavage in a vicinity of the chromophore-forming amino acid triad [20].…”