Dual Illumination Enhances Transformation of an Engineered Green‐to‐Red Photoconvertible Fluorescent Protein

Krueger, Taylor D.; Tang, Longteng; Zhu, Lingbo; Breen, Isabella L.; Wachter, Rebekka M.; Fang, Chong

doi:10.1002/anie.201911379

Cited by 22 publications

(83 citation statements)

References 61 publications

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“…The I‐ring, however, has limited options and is rather challenging to modify because the two contributing amino acids are anchoring points to the β‐barrel enclosing the protein pocket. Nevertheless, most photoconvertible red FPs entail either a strong electron‐withdrawing group (e. g., asFP595, −COR) or a large conjugated group (e. g., Kaede‐like FPs, 4‐vinylimidazole), or both (e. g., far‐red PSmOrange) in the converted red form, which substantiates that the “one acceptor” at C‐2 position red‐shifts the FP emission by promoting a further delocalized and stabilized ICT state of the protein chromophore. Encouraged by these existing RFPs with an engineerable residue sidechain at the C‐2 site, we expect that the combination of double donor and one acceptor could further red‐shift FPs while retaining the photoconvertibility to benefit bioimaging applications.…”

Section: Discussionmentioning

confidence: 99%

Devising Efficient Red‐Shifting Strategies for Bioimaging: A Generalizable Donor‐Acceptor Fluorophore Prototype

Chen

Fang

2020

Chemistry An Asian Journal

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Long emission wavelengths, high fluorescence quantum yields (FQYs), and large Stokes shifts are highly desirable features for fluorescent probes in biological imaging. However, the current development of many fluorescent probes remains largely trial-and-error and lacks efficiency. Moreover, to achieve far-red/near-infrared emission, a significant extension in the p-conjugation is usually adopted but accompanied by other drawbacks such as fluorescence loss. In this review, we discuss an effective red-shifting strategy built upon the green fluorescent protein chromophore, which enables a synergistic tuning of both the electronic ground and excited states. This approach could shorten the path toward redder emission in comparison to the conventional intramolecular charge transfer (ICT) strategy. We envision that this spectroscopy and computation-aided strategy may advance the noncanonical fluorescent protein design and be generalized to various fluorophore scaffolds for redder emission while preserving other superior properties such as high FQYs.[a] C.

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Section: Discussionmentioning

confidence: 99%

Devising Efficient Red‐Shifting Strategies for Bioimaging: A Generalizable Donor‐Acceptor Fluorophore Prototype

Chen

Fang

2020

Chemistry An Asian Journal

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“…Blinking in the green state of mEos variants could affect photoconversion to the red state (39,40). The interplay between blinking and photoconversion has also been described in other photoconvertible fluorescent proteins, such as SAASoti (43) and LEA (44). Thus, combining our approach with singlemolecule measurements will offer a more complete and quantitative understanding of the photophysics of PAFPs or PCFPs.…”

Section: Intermediate State Of Meos32 That Converts To the Red Fluorescent State Upon 561-nm Illuminationmentioning

confidence: 65%

Sample Preparation and Imaging Conditions Affect mEos3.2 Photophysics in Fission Yeast Cells

Sun

Bewersdorf

et al. 2021

Biophysical Journal

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Photoconvertible fluorescent proteins (PCFPs) are widely used in super-resolution microscopy and studies of cellular dynamics. However, our understanding of their photophysics is still limited, hampering their quantitative application. For example, we do not know the optimal sample preparation methods or imaging conditions to count protein molecules fused to PCFPs by single-molecule localization microscopy in live and fixed cells. We also do not know how the behavior of PCFPs in live cells compares with fixed cells. Therefore, we investigated how formaldehyde fixation influences the photophysical properties of the popular green-to-red PCFP mEos3.2 in fission yeast cells under a wide range of imaging conditions. We estimated photophysical parameters by fitting a 3-state model of photoconversion and photobleaching to the time course of fluorescence signal per yeast cell expressing mEos3.2. We discovered that formaldehyde fixation makes the fluorescence signal, photoconversion rate and photobleaching rate of mEos3.2 sensitive to the buffer conditions by permeabilizing the yeast cell membrane.Under some imaging conditions, the time-integrated mEos3.2 signal per yeast cell is similar in live cells and fixed cells imaged in buffer at pH 8.5 with 1 mM DTT, indicating that light chemical fixation does not destroy mEos3.2 molecules. We also discovered that some red-state mEos3.2 molecules entered an intermediate dark state that is converted back to the red fluorescent state by 561-nm illumination. Our findings provide a guide to compare quantitatively conditions for imaging and counting of mEos3.2-tagged molecules in yeast cells. Our imaging assay and mathematical model are easy to implement and provide a simple quantitative approach to measure the time-integrated signal and the photoconversion and photobleaching rates of fluorescent proteins in cells. STATEMENT OF SIGNIFICANCEMaking quantitative measurements with single-molecule localization microscopy (SMLM) has been impeded by limited understanding of the photophysics of the fluorophores, which is very sensitive to the sample preparation and imaging conditions. We characterized the photophysics of the green-to-red photoconvertible fluorescent protein mEos3.2, which is widely used in SMLM. We combined quantitative fluorescence microscopy and mathematical modeling to measure the fluorescence signal and rate constants for photoconversion and photobleaching of mEos3.2 in live and fixed cells under a wide range of illumination intensities. Our findings

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“…A protonated and twisted chromophore was also generated by irradiation of the green anionic cis chromophore in the FP LEA. 127 In this work, it was suggested that this state may be able to photoconvert upon 405 nm irradiation, but that photoswitching back to the anionic cis state is much more likely. For green mEos4b, a long-lived dark trans state was found upon 488 nm irradiation and characterized in detail.…”

Section: Photoactivation Mechanismsmentioning

confidence: 91%

Fluorescent proteins of the EosFP clade: intriguing marker tools with multiple photoactivation modes for advanced microscopy

Nienhaus

2021

RSC Chem. Biol.

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Dual Illumination Enhances Transformation of an Engineered Green‐to‐Red Photoconvertible Fluorescent Protein

Cited by 22 publications

References 61 publications

Devising Efficient Red‐Shifting Strategies for Bioimaging: A Generalizable Donor‐Acceptor Fluorophore Prototype

Devising Efficient Red‐Shifting Strategies for Bioimaging: A Generalizable Donor‐Acceptor Fluorophore Prototype

Sample Preparation and Imaging Conditions Affect mEos3.2 Photophysics in Fission Yeast Cells

Fluorescent proteins of the EosFP clade: intriguing marker tools with multiple photoactivation modes for advanced microscopy

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