Increasing the brightness of red fluorescent proteins by saturation mutagenesis

Red fluorescent proteins (RFPs) are genetically-encoded fluorophores that are widely used for in vivo imaging. For all applications of RFPs, brighter variants are desired. Previously, we improved the brightness of mRojoA, a red-shifted mutant of the widely-used RFP mCherry, by designing a triple-decker motif of aromatic rings around its chromophore. This yielded the brighter variant mRojo-VHSV, which contains a triple-decker motif consisting of His and Tyr side chains that pack against the chromophore. This improved chromophore packing resulted in an approximately 3-fold brightness increase at physiological pH. However, the His side chain in the triple-decker motif of mRojo-VHSV adopted a perpendicular arrangement to the other two, which may result in a suboptimal packing arrangement. To further improve chromophore packing in mRojo-VHSV, we performed saturation mutagenesis of residues surrounding its triple-decker motif (positions 62, 97, 165, and 199). Using a microplate fluorescence screening assay, a total of 376 colonies were screened for improved brightness. The brightest mutant found, L199M, was expressed and purified, and its spectral properties were characterized in detail. We found that the quantum yield of this variant was improved by two-fold, resulting in a two-fold brightness increase compared to mRojo-VHSV as well as a 5.3-fold increase in brightness compared to mRojoA. The L199M improved variant is the basis for continued engineering with the goal of further improving the spectral properties of this family of RFPs.