Blue-Shifted Green Fluorescent Protein Homologues Are Brighter than Enhanced Green Fluorescent Protein under Two-Photon Excitation

Molina, Rosana S.; Tran, Tam M; Campbell, Robert E.; Lambert, G.; Salih, Anya; Shaner, Nathan C.; Hughes, Thom; Drobizhev, Mikhail

doi:10.1021/acs.jpclett.7b00960

Cited by 41 publications

(66 citation statements)

References 52 publications

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“…A well-behaved FRET donor would be easily excited by the 2-photon laser, be stable under a variety of 2-photon laser powers, and display a stable, mono-exponential lifetime[32]. To our knowledge, there is only one report of mNeonGreen’s performance using two-photon illumination[33]. This study demonstrated that blue shifted fluorescent proteins tend to perform better under 2-photon excitation than more yellow shifted fluorescent proteins but did not preclude mNeonGreen’s use from two-photon based studies.…”

Section: Resultsmentioning

confidence: 99%

Comparing the Performance of mScarlet-I, mRuby3, and mCherry as FRET Acceptors for mNeonGreen

McCullock

MacLean

Kammermeier

2019

Preprint

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19 Förster Resonance Energy Transfer (FRET) has become an immensely powerful tool to 20 profile intra-and inter-molecular interactions. Through fusion of genetically encoded 21 fluorescent proteins (FPs) researchers have been able to detect protein oligomerization, 22 receptor activation, and protein translocation among other biophysical phenomena.23 Recently, two bright monomeric red fluorescent proteins, mRuby3 and mScarlet-I, have 24 been developed. These proteins offer much improved physical properties compared to 25 previous generations of monomeric red FPs that should help facilitate more general 26 adoption of Green/Red FRET. Here we assess the ability of these two proteins, along 27 with mCherry, to act as a FRET acceptor for the bright, monomeric, green-yellow FP 28 mNeonGreen using intensiometric FRET and 2-photon Fluorescent Lifetime Imaging 29 Microscopy (FLIM) FRET techniques. We first determined that mNeonGreen was a 30 stable donor for 2-photon FLIM experiments under a variety of imaging conditions. We 31 then tested the red FP's ability to act as FRET acceptors using mNeonGreen-Red FP 32 tandem construct. With these constructs we found that mScarlet-I and mCherry are able 33 to efficiently FRET with mNeonGreen in spectroscopic and FLIM FRET. In contrast, 34 mNeonGreen and mRuby3 FRET with a much lower efficiency than predicted in these 35 same assays. We explore possible explanations for this poor performance but are 36 unable to definitively determine the cause, all though protein maturation seems to play a 37 role. Overall, we find that mNeonGreen is an excellent FRET donor, and both mCherry 38 and mScarlet-I, but not mRuby3, act as practical FRET acceptors, with mScarlet-I out 39 performing mCherry due it's higher brightness. 40 41 Introduction 42 Genetically encoded Fluorescent Proteins (FPs) have advanced basic and translational 43 biology immensely. Starting with the cloning of the Aequorea victoria green FP[1], a 44 massive and continual effort to expand the number of available FPs with a different 45 physical and spectral properties began. Currently, there is an enormous variety of FPs 46 at all parts of the visible spectrum, and even some parts of the ultraviolet and infrared 47 spectrums. These new proteins were either cloned from other organisms[2-4], or48 developed through evolution of already identified FPs [4][5][6][7][8][9][10][11]. This ever expanding 49 catalog of FPs has been reviewed by others [12][13][14], and new efforts and archives have 50 been created to organize this information, such as the FPbase database [15]. 51As the catalogue of FPs has expanded, so have the potential uses. Of particular 52 note is the use of FPs as biosensors which can measure signaling events, cell 53 metabolites, pH, voltage and more [16]. Many of these biosensors employ Förster 54 Resonance Energy Transfer (FRET) as part of their reporting mechanism, producing a 55 change in acceptor emission upon donor excitation when the quantity of interest 56 changes. FRET is also orientation and distance dep...

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Section: Resultsmentioning

confidence: 99%

Comparing the Performance of mScarlet-I, mRuby3, and mCherry as FRET Acceptors for mNeonGreen

McCullock

MacLean

Kammermeier

2019

Preprint

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“…When the F 2 ratio is greater than the F 1 ratio, it is because the σ 2 of the anionic form increases upon Ca 2+ -binding while the ε changes little or not at all. The increase in σ 2 can be explained by its higher sensitivity to the local electric field compared to ε (25)(26)(27) . This sensitivity is due to a factor describing the change of the permanent dipole moment upon excitation that enters the expression for σ 2 , and the electric field affects this factor through polarizability.…”

Section: Discussionmentioning

confidence: 99%

“…Notably, the increase in σ 2 upon binding Ca 2+ is always paralleled by a shift of the one-photon absorption peak to a shorter wavelength. We previously developed a physical model ( (25)(26)(27) ) that predicts that both of these changes will happen in the anionic chromophore if the electric field directed from the center of the phenolate ring to the center of the imidazole ring increases. In red GECIs, the chromophore is oriented such that its phenolate oxygen points toward the opening of the barrel at the site of circular permutation where the Ca 2+ -sensing domain is fused to the FP (as observed for RCaMP (PDB ID: 3U0K (21) ), R-GECO1 (PDB ID: 4I2Y (21) ), and K-GECO1 (PDB ID: 5UKG (22) ).…”

Section: Discussionmentioning

confidence: 99%

“…The first two factors, ρ e and φ e , are the same for both one-photon and two-photon excitation. However, the ratios ε e,sat /ε e,free and σ 2,e,sat /σ 2,e,free might not be equal since ε and σ 2 can depend differently on the local environment of the chromophore (25)(26)(27) . Therefore, the overall change in fluorescence could depend on the mode of excitation.…”

Section: Background and Analytical Considerationsmentioning

confidence: 99%

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Understanding the Ca²⁺-dependent Fluorescence Change in Red Genetically Encoded Ca²⁺ Indicators

Molina

Qian

et al. 2018

Preprint

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Genetically encoded Ca 2+ indicators (GECIs) are widely used to illuminate dynamic Ca 2+ signaling activity in living cells and tissues. Various fluorescence colors of GECIs are available, including red. Red GECIs are promising because longer wavelengths of light scatter less in tissue, making it possible to image deeper. They are engineered from a circularly permuted red fluorescent protein fused to a Ca 2+ sensing domain, calmodulin and a calmodulin-binding peptide. A conformational change in the sensing domain upon binding Ca 2+ causes a change in the fluorescence intensity of the fluorescent protein. Three factors could contribute to this change in fluorescence: 1) a shift in the protonation state of the chromophore, 2) a change in fluorescence quantum yield, and 3) a change in the extinction coefficient for one-photon excitation or the two-photon cross section for two-photon excitation. We conducted a systematic study of the photophysical properties of a select cohort of red GECIs in their Ca 2+ -free and Ca 2+ -saturated states to determine which factors are most important for the Ca 2+ -dependent change in fluorescence. In total, we analyzed nine red GECIs, including jRGECO1a, K-GECO1, jRCaMP1a, R-GECO1, R-GECO1.2, CAR-GECO1, O-GECO1, REX-GECO1, and a new variant termed jREX-GECO1. We found that these red GECIs could be separated into three classes that each rely on a particular set of factors. Furthermore, in some cases the magnitude of the change in fluorescence was different depending on one-photon excitation or two-photon excitation by up to a factor of two.

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“…A well-behaved FRET donor would be easily excited by the 2-photon laser, be stable under a variety of 2-photon laser powers, and display a stable, mono-exponential lifetime [32]. To our knowledge, there is only one report of mNeon-Green's performance using two-photon illumination [33]. This study demonstrated that blue shifted fluorescent proteins tend to perform better under 2-photon excitation than more yellow shifted fluorescent proteins but did not preclude mNeonGreen's use from two-photon based studies.…”

Section: Mneongreen Is a Suitable Donor For Two-photon Flimmentioning

confidence: 99%

Comparing the performance of mScarlet-I, mRuby3, and mCherry as FRET acceptors for mNeonGreen

2020

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Fö rster Resonance Energy Transfer (FRET) has become an immensely powerful tool to profile intra-and inter-molecular interactions. Through fusion of genetically encoded fluorescent proteins (FPs) researchers have been able to detect protein oligomerization, receptor activation, and protein translocation among other biophysical phenomena. Recently, two bright monomeric red fluorescent proteins, mRuby3 and mScarlet-I, have been developed. These proteins offer much improved physical properties compared to previous generations of monomeric red FPs that should help facilitate more general adoption of Green/Red FRET. Here we assess the ability of these two proteins, along with mCherry, to act as a FRET acceptor for the bright, monomeric, green-yellow FP mNeonGreen using intensiometric FRET and 2-photon Fluorescent Lifetime Imaging Microscopy (FLIM) FRET techniques. We first determined that mNeonGreen was a stable donor for 2-photon FLIM experiments under a variety of imaging conditions. We then tested the red FP's ability to act as FRET acceptors using mNeonGreen-Red FP tandem construct. With these constructs we found that mScarlet-I and mCherry are able to efficiently FRET with mNeonGreen in spectroscopic and FLIM FRET. In contrast, mNeonGreen and mRuby3 FRET with a much lower efficiency than predicted in these same assays. We explore possible explanations for this poor performance and determine mRuby3's protein maturation properties are a major contributor. Overall, we find that mNeonGreen is an excellent FRET donor, and both mCherry and mScarlet-I, but not mRuby3, act as practical FRET acceptors, with the brighter mScarlet-I out performing mCherry in intensiometric studies, but mCherry out performing mScarlet-I in instances where consistent efficiency in a population is critical.

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Blue-Shifted Green Fluorescent Protein Homologues Are Brighter than Enhanced Green Fluorescent Protein under Two-Photon Excitation

Cited by 41 publications

References 52 publications

Comparing the Performance of mScarlet-I, mRuby3, and mCherry as FRET Acceptors for mNeonGreen

Comparing the Performance of mScarlet-I, mRuby3, and mCherry as FRET Acceptors for mNeonGreen

Understanding the Ca²⁺-dependent Fluorescence Change in Red Genetically Encoded Ca²⁺ Indicators

Comparing the performance of mScarlet-I, mRuby3, and mCherry as FRET acceptors for mNeonGreen

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Blue-Shifted Green Fluorescent Protein Homologues Are Brighter than Enhanced Green Fluorescent Protein under Two-Photon Excitation

Cited by 41 publications

References 52 publications

Comparing the Performance of mScarlet-I, mRuby3, and mCherry as FRET Acceptors for mNeonGreen

Comparing the Performance of mScarlet-I, mRuby3, and mCherry as FRET Acceptors for mNeonGreen

Understanding the Ca2+-dependent Fluorescence Change in Red Genetically Encoded Ca2+ Indicators

Comparing the performance of mScarlet-I, mRuby3, and mCherry as FRET acceptors for mNeonGreen

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Understanding the Ca²⁺-dependent Fluorescence Change in Red Genetically Encoded Ca²⁺ Indicators