Directed Evolution of a Bright Variant of mCherry: Suppression of Nonradiative Decay by Fluorescence Lifetime Selections

Mukherjee, Srijit; Manna, Premashis; Hung, Sheng-Ting; Vietmeyer, Felix; Friis, Pia; Palmer, Amy E.; Jimenez, Ralph

doi:10.1021/acs.jpcb.2c01956

Cited by 31 publications

(54 citation statements)

References 46 publications

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“…As observed previously, 42 the quantum yield scales with lifetime (Figure 1). The increase in the quantum yield correlates with progressive increases in the time constant corresponding to the major amplitude component of the fit and the fraction of this component in the overall fit (Supplemental Figure S3.3).…”

Section: Results and Discussion: A Photophysical Measurements And Ami...supporting

confidence: 83%

“…In the photophysical analysis of mCherry-XL, we found the bulk of improvement in the fluorescence quantum yield came from the suppression of non-radiative rate, which was beyond the scope of an energy gap law. 42 The chemical nature of the point mutations acquired in the development of mCherry-XL agreed with the model of electron flow by Lin et.al. 23 In this study, we provide a structural framework, from the dynamic point of view, for these substitutions acquired in mCherry-XL and several of the intermediate mutants.…”

Section: Introductionsupporting

confidence: 82%

“…The mCherry variants investigated here were developed using lifetime-based microfluidic cell sorting of libraries constructed using site-directed and error-prone mutagenesis. 42 The photophysical properties were found to be sensitive to substitutions at positions 143, 161, 163 and 197 on the mCherry scaffold. These residues are located near the phenol end of the chromophore (Table 1).…”

Section: Results and Discussion: A Photophysical Measurements And Ami...mentioning

confidence: 97%

“…In short, mCherry variants generated using fluorescence lifetime selections in yeast (Saccharomyces cerevisiae BY4741; transformed with the pYestDest52 vector) from our previous study were cloned into the pBad-His+ vector for bacterial expression. 12,42,43 Protein was extracted from bacteria using Ni-NTA-based column chromatography. Absorption data was collected using a Cary 5000 UV−vis near-IR spectrophotometer in the double beam mode.…”

Section: Methodsmentioning

confidence: 99%

“…We recently-developed bright FPs, such as FusionRed-MQV and mCherry-XL using high-throughput microfluidic fluorescence lifetime selections, both of which are ~3x higher molecular brightness than their parents FusionRed and mCherry, respectively. 12,42,43 In this study, we focus on mCherry-XL, where we investigate the role and the chemical nature of the substitutions that were acquired in the course of its development using all atom MD simulations. In the photophysical analysis of mCherry-XL, we found the bulk of improvement in the fluorescence quantum yield came from the suppression of non-radiative rate, which was beyond the scope of an energy gap law.…”

Section: Introductionmentioning

confidence: 99%

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Conformational dynamics of mCherry variants: a link between sidechain motions and fluorescence brightness

Mukherjee

Manna

Douglas

et al. 2022

Preprint

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We recently developed the red fluorescent protein (RFP) mCherry-XL, which is threefold brighter than its predecessor, mCherry, with a directed evolution approach using fluorescence-lifetime flow cytometry selections. The enhanced brightness is due to a significant decrease in the non-radiative decay rate underlying its increase in fluorescence quantum yield. To examine the dynamic role of the four mutations that distinguish the two RFPs and closely-related variants, we employed microsecond-timescale, all-atom molecular dynamics simulations to sample their ground state conformational landscapes. The simulations revealed the significance of the I197R mutation, which leads to the formation of multiple hydrogen-bonded contacts. The triad of interactions observed between residues K70, E148 and 197R is also seen in mScarlet, another RFP of unrelated origin, but of comparably high brightness. These substitutions in mCherry-XL increased the rigidity of the β-barrel, for example as shown by increased hydrogen-bonding in the chromophore region and decreased root-mean-square backbone deviations. Furthermore, mCherry-XL showed significantly less ns-timescale breathing of the gap between β-7 and β-10 strands. This gap was previously shown to be the most flexible region of mCherry, permitting entry of O2 into the barrel. We also characterize the role played by the sidechain of residue 161 using a combination of MD simulations and in-vitro experimental characterization. We find this position is critical to the steric interactions that perturb the chromophore electronic structure. MD simulations also help us to recognize a network of hydrogen-bonded interactions between the chromophore, the residue 143, 163 and 59, which can potentially impact the electron distribution of the chromophore. Finally, we shed light on the conformational dynamics of the conserved residues R95 and S146, which are hydrogen bonded to the chromophore, and provide physical insights into the observed photophysics. To the best of our knowledge, this is the first study that evaluates the conformational space for a set of closely related FPs generated by directed evolution.

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Section: Results and Discussion: A Photophysical Measurements And Ami...supporting

confidence: 83%

Section: Introductionsupporting

confidence: 82%

Section: Results and Discussion: A Photophysical Measurements And Ami...mentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Conformational dynamics of mCherry variants: a link between sidechain motions and fluorescence brightness

Mukherjee

Manna

Douglas

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

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Multiplexed In Vivo Imaging with Fluorescence Lifetime‐Modulating Tags

El Hajji,

Lam,

Avtodeeva

et al. 2024

Advanced Science

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Fluorescence lifetime imaging microscopy (FLIM) opens new dimensions for highly multiplexed imaging in live cells and organisms using differences in fluorescence lifetime to distinguish spectrally identical fluorescent probes. Here, a set of fluorescence‐activating and absorption‐shifting tags (FASTs) capable of modulating the fluorescence lifetime of embedded fluorogenic 4‐hydroxybenzylidene rhodanine (HBR) derivatives is described. It is shown that changes in the FAST protein sequence can vary the local environment of the chromophore and lead to significant changes in fluorescence lifetime. These fluorescence lifetime‐modulating tags enable multiplexed imaging of up to three targets in one spectral channel using a single HBR derivative in live cells and live zebrafish larvae. The combination of fluorescence lifetime multiplexing with spectral multiplexing allows to successfully image six targets in live cells, opening great prospects for multicolor fluorescence lifetime multiplexing.

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Targeting Ultrafast Spectroscopic Insights into Red Fluorescent Proteins

Krueger,

Chen,

Fang

2023

Chemistry An Asian Journal

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Red fluorescent proteins (RFPs) represent an increasingly popular class of genetically encodable bioprobes and biomarkers that can advance next‐generation breakthroughs across the imaging and life sciences. Since the rational design of RFPs with improved functions or enhanced versatility requires a mechanistic understanding of their working mechanisms, while fluorescence is intrinsically an ultrafast event, a suitable toolset involving steady‐state and time‐resolved spectroscopic techniques has become powerful in delineating key structural features and dynamic steps which govern irreversible photoconverting or reversible photoswitching RFPs, and large Stokes shift (LSS)RFPs. The pertinent cis‐trans isomerization and protonation state change of RFP chromophores in their local environments, involving key residues in protein matrices, lead to rich and complicated spectral features across multiple timescales. In particular, ultrafast excited‐state proton transfer in various LSSRFPs showcases the resolving power of wavelength‐tunable femtosecond stimulated Raman spectroscopy (FSRS) in mapping a photocycle with crucial knowledge about the red‐emitting species. Moreover, recent progress in noncanonical RFPs with a site‐specifically modified chromophore provides an appealing route for efficient engineering of redder and brighter RFPs, highly desirable for bioimaging. Such an effective feedback loop involving physical chemists, protein engineers, and biomedical microscopists will enable future successes to expand fundamental knowledge and improve human health.

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Directed Evolution of a Bright Variant of mCherry: Suppression of Nonradiative Decay by Fluorescence Lifetime Selections

Cited by 31 publications

References 46 publications

Conformational dynamics of mCherry variants: a link between sidechain motions and fluorescence brightness

Conformational dynamics of mCherry variants: a link between sidechain motions and fluorescence brightness

Multiplexed In Vivo Imaging with Fluorescence Lifetime‐Modulating Tags

Targeting Ultrafast Spectroscopic Insights into Red Fluorescent Proteins

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