Generation of bright monomeric red fluorescent proteins <i>via</i> computational design of enhanced chromophore packing

Legault, Sandrine; Fraser-Halberg, Derek Paco; McAnelly, Ralph; Eason, Matthew G.; Thompson, Michael C.; Chica, Roberto A.

doi:10.1039/d1sc05088e

Cited by 12 publications

(21 citation statements)

References 59 publications

(99 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Spectroscopic, computational, and crystallographic evidence suggests that chromophore planarity and protein rigidity are the primary characteristics of bright FPs. − Other investigators have considered more subtle physical factors that influence brightness. − Using physical models such as the Marcus–Hush theory, these studies focused on quantifying and understanding the role of the driving force in charge transfer between the two resonance forms of the GFP chromophore in the ground and excited electronic states . This driving force is vulnerable to electrostatic control from the environment, which regulates access to nonradiative pathways of excited state depopulation, and therefore directly influences brightness.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

View full text Add to dashboard Cite

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.

show abstract

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

View full text Add to dashboard Cite

show abstract

“…6 Consequently, strategies beyond rigidifying the chromophore are necessary for the design of brighter FPs. 24 These considerations suggest that large variations in absorption and emission wavelengths are possible for anionic RFP chromophores with similar values of lifetime and quantum yield, because the non-radiative rate does not depend exclusively on the transition energy gap. The RFP mScarlet is red-shifted by 10 nm (or 315 cm -1 ) in maximum absorption and by 6 nm (or 272 cm -1 ) in maximum emission compared to mCherry-XL, yet it matches its quantum yield and fluorescence lifetime values.…”

Section: Discussionmentioning

confidence: 96%

“…knr non E-J (µs yielded the bright red-shifted RFP, mSandy2 (λem = 606 nm; ϕ =0.35). 24 The crystal structure of mCherry reveals an interaction of the indole ring on W143 with the amine group on the Q163 residue (3.4 Å).…”

Section: Discussionmentioning

confidence: 99%

“…Spectroscopic, computational, and crystallographic evidence suggest that chromophore planarity and protein rigidity are the primary characteristics of bright FPs. [22][23][24] Other investigators have considered more subtle physical factors that influence brightness. [15][16][17] Using physical models such as the Marcus-Hush theory, these studies focused on quantifying and understanding the role of the driving force in charge transfer between the two resonance forms of the GFP chromophore in the ground and excited electronic states.…”

Section: Introductionmentioning

confidence: 99%

“…Previous attempts to develop brighter and red-shifted versions of mCherry have been unable to restore its fluorescence quantum yield to that of DsRed. 22,24,28,29 In this study, we present a monomeric variant denoted mCherry-XL (eXtended Lifetime: W143S, I161V, Q163Y and I197R) which is 3-fold brighter than mCherry, and matches the molecular brightness of DsRed. We provide insight into the evolution from mCherry ( = 0.22; τ =1.6 ns) to mCherry-XL ( = 0.70; τ =3.9 ns).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Mukherjee

Manna

Hung

et al. 2022

Preprint

View full text Add to dashboard Cite

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.

show abstract

Homopolymeric Protein Phosphors: Overpassing the Stability Frontier of Deep‐Red Bio‐Hybrid Light‐Emitting Diodes

Ferrara

Fernandéz‐Blázquez

Werner

et al. 2023

Adv Funct Materials

View full text Add to dashboard Cite

Although protein-polymer phosphors are an emerging photon-management filter concept for hybrid light-emitting diodes, deep-red-emitting devices based on archetypal fluorescent proteins (FPs; mCherry) are still poorly performing with lifetimes <50 h under high photon-flux excitation and ambient conditions. Here, the challenge is two-fold: i) understanding the deactivation mechanism of red-emitting FP-polymer coatings and, in turn, ii) identifying the best polymer design for highly stable devices. This study first provides comprehensive photophysical/thermal/structural studies and device degradation (ambient/ inert) analysis, revealing the presence of photo-induced cis-trans isomerization and the effect of oxygen and water on the deactivation of mCherry in reference polymer coatings. Based on these findings, a new bio-phosphor configuration using polyvinyl alcohol derivatives, in which crystallinity and amount of trapped water (stiffness and oxygen/moisture barriers) are easily controlled by the hydroxylation degree, is successfully achieved. Compared to the prior art, these devices significantly outperform the reference stability (>50-fold enhancement), showing a brightness loss of <5% over the first 2000 h and a final device lifetime of 2600 h. Hence, this study describes a unique rationale toward designing polymers to stabilize FPs for lighting, overpassing stability frontiers in deep-red hybrid light-emitting diodes (HLEDs) going from hours to months.

show abstract

Generation of bright monomeric red fluorescent proteins via computational design of enhanced chromophore packing

Abstract: Red fluorescent proteins (RFPs) have found widespread application in chemical and biological research due to their longer emission wavelengths. Here, we use computational protein design to increase the quantum yield...

Cited by 12 publications

References 59 publications

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

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

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

Homopolymeric Protein Phosphors: Overpassing the Stability Frontier of Deep‐Red Bio‐Hybrid Light‐Emitting Diodes

Contact Info

Product

Resources

About