TY - JOUR AU - Srijit Mukherjee AU - Sheng-Ting Hung AU - Nancy Douglas AU - Premashis Manna AU - Connor Thomas AU - Annika Ekrem AU - Amy Palmer AU - Ralph Jimenez AB - 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-1cm-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 site-directed 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.  BT - Biochemistry DA - 2020-09 DO - 10.1021/acs.biochem.0c00484 IS - 39 M3 - doi: 10.1021/acs.biochem.0c00484 N1 - doi: 10.1021/acs.biochem.0c00484 N2 - 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-1cm-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 site-directed 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.  PB - American Chemical Society PY - 2020 SN - 0006-2960 EP - 3669–3682 T2 - Biochemistry TI - Engineering of a brighter variant of the FusionRed fluorescent protein using lifetime flow-cytometry and structure-guided mutations UR - https://doi.org/10.1021/acs.biochem.0c00484 VL - 59 ER -