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Directed Evolution of a Bright Variant of mCherry: Suppression of Nonradiative Decay by Fluorescence Lifetime
Srijit Mukherjee1,2, Premashis Manna3, Sheng-Ting Hung4
1JILA, University of Colorado, Boulder and National Institute of Standards and Technology, 440 UCB, Boulder, Colorado 80309, United States.
Abstract:
The approximately linear scaling of fluorescence quantum yield (ϕ) 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: ϕ = 0.70; τ = 3.9 ns). The 3-fold 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 nonradiative decay of the excited state. In addition, our analysis revealed the decrease in nonradiative rate is not limited to the blue-shift of the energy gap and changes in the excited state reorganization energy. Our findings suggest that nonradiative mechanisms beyond the scope of energy-gap models such the Englman-Jortner model are suppressed in this lifetime evolution trajectory.
Insights
Researchers engineered a brighter red fluorescent protein (RFP) called mCherry-XL by increasing its fluorescence lifetime. This enhanced protein exhibits a 3-fold higher quantum yield, making it comparable to the brightest RFPs available for live cell imaging.
Area of Science:
- Biophysics
- Photochemistry
- Molecular Biology
Background:
- Fluorescent proteins (FPs) are engineered for brighter fluorophores by increasing fluorescence lifetime (τ) and quantum yield (ϕ).
- Recent FPs like mTurquoise2 and mScarlet utilize lifetime selection strategies for improved live cell imaging.
- The photophysical basis for brightness enhancement in lifetime-selected FPs remains underexplored.
Purpose of the Study:
- To investigate the photophysical mechanisms behind lifetime-based directed evolution of red fluorescent proteins (RFPs).
- To understand the outcome of lifetime-based directed evolution applied to the mCherry RFP.
Main Methods:
- Directed evolution of mCherry focusing on increasing fluorescence lifetime.
- Identification of key amino acid positions influencing protein properties.
- Spectroscopic analysis of engineered variants, including mCherry-XL.
Main Results:
- Engineered mCherry-XL with an extended lifetime (τ = 3.9 ns) and significantly increased quantum yield (ϕ = 0.70).
- Achieved a 3-fold higher quantum yield, matching the brightest known RFPs like mScarlet.
- Observed a near-linear scaling between lifetime and quantum yield, with spectral blue-shifts.
- Determined that reduced nonradiative decay is the primary driver of increased brightness.
Conclusions:
- The brightness enhancement in mCherry-XL is mainly due to decreased nonradiative decay rates.
- Nonradiative mechanisms suppressed involve factors beyond simple energy gap models.
- Findings suggest novel strategies for engineering brighter FPs by targeting nonradiative decay pathways.
