Investigating Different Rational Design Approaches to Increase Brightness in Red Fluorescent Proteins

Abstract

Red fluorescent proteins (RFPs) are used extensively in biological research because their longer emission wavelengths are less phototoxic and allow deeper imaging of animal tissue. However, far-red RFPs generally display low brightness, emphasizing the need to develop brighter variants. Here, we investigate three approaches to rigidify the RFP chromophore to increase the quantum yield, and thereby brightness. We first used computational protein design on a maturation-efficient mRojo-VHSV variant previously engineered in our lab to introduce a Superdecker motif, a parallel pi-stack comprising aromatic residue side chains and the phenolate moiety of the chromophore, which we hypothesized would enhance chromophore packing and reduce non-radiative decay. The best mutants identified showed up to 1.7-fold higher quantum yield at pH 9, relative to their parent protein. We next postulated that brightness could be further increased by rigidifying the chromophore via branched aliphatic residues. Computational protein design was performed on a dim mCherry variant, mRojoA, followed by directed evolution on the brightest mutant. The combination of these methodologies yielded mSandy2, the brightest Discosoma-derived monomeric RFP with an emission maximum above 600 nm. Finally, we aimed to increase brightness by focusing on positions where residue rigidity correlated to quantum yield in mCherry-related RFPs according to NMR data that had been previously acquired in our lab. Combinatorial site-saturation mutagenesis was performed on two different surface patches of mCherry at positions 144/145/198 and 194/196/220. Our results demonstrated that surface residues may not be adequate targets for this approach. Altogether, the work herein presents unique rational design methodologies that can be used to increase brightness in RFPs.