Influence of fluorescence lifetime selections and conformational flexibility on brightness of FusionRed variants

Fluorescent Proteins (FPs) for bioimaging are typically developed by screening mutant libraries for clones with improved photophysical properties. This approach has resulted in FPs with high brightness, but the mechanistic origins of the improvements are often unclear. We focused on improving the molecular brightness in the FusionRed family of FPs with fluorescence lifetime selections on targeted libraries, with the aim of reducing non-radiative decay rates. Our new variants show fluorescence quantum yields up to 75% and lifetimes >3.5 ns. We present a comprehensive analysis of these new FPs, including trends in spectral shifts, photophysical data, photostability, and cellular brightness resulting from codon optimization. We also performed all-atom molecular dynamics simulations to investigate the impact of sidechain mutations. The trajectories reveal that individual mutations reduce the flexibility of the chromophore and sidechains, leading to an overall reduction in non-radiative rates.