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 nonradiative decay rates. Our new variants show fluorescence quantum yields of 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 side chain mutations. The trajectories reveal that individual mutations reduce the flexibility of the chromophore and side chains, leading to an overall reduction in nonradiative rates.
BT - The Journal of Physical Chemistry Letters DA - 2024-02 DO - 10.1021/acs.jpclett.3c02765 N2 -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 nonradiative decay rates. Our new variants show fluorescence quantum yields of 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 side chain mutations. The trajectories reveal that individual mutations reduce the flexibility of the chromophore and side chains, leading to an overall reduction in nonradiative rates.
PB - American Chemical Society (ACS) PY - 2024 SP - 1644 EP - 1651 T2 - The Journal of Physical Chemistry Letters TI - Influence of Fluorescence Lifetime Selections and Conformational Flexibility on Brightness of FusionRed Variants VL - 15 SN - 1948-7185, 1948-7185 ER -