Directed evolution of a bright variant of mCherry: Suppression of non-radiative decay by fluorescence lifetime selections

The approximately linear scaling of fluorescence quantum yield (QY) with fluorescence lifetime (τ) in fluorescent proteins (FPs) has inspired engineering of brighter fluorophores based on screening for increased lifetimes. Several recently developed FPs such as mTurquoise2, mScarlet and FusionRed-MQV which have become useful for live cell imaging are products of lifetime selection strategies. However, the underlying photophysical basis of the improved brightness has not been scrutinized. In this study, we focused on understanding the outcome of lifetime-based directed evolution of mCherry, which is a popular red-FP (RFP). We identified four positions (W143, I161, Q163, and I197) near the FP chromophore that can be mutated to create mCherry-XL (eXtended Lifetime: QY = 0.70; τ =3.9 ns). The threefold higher quantum yield of mCherry-XL is on par with that of the brightest RFP to date, mScarlet. We examined selected variants within the evolution trajectory and found a near-linear scaling of lifetime with quantum yield and consistent blue-shifts of the absorption and emission spectra. We find that the improvement in brightness is primarily due to a decrease in the non-radiative decay of the excited state. In addition, our analysis revealed the decrease in non-radiative rate is not limited to the blue-shift of the energy gap and changes in the excited state reorganization energy. Our findings suggest that non-radiative mechanisms beyond the scope of energy-gap models such the Englman-Jortner are suppressed in this lifetime evolution trajectory.