Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy

Pennacchietti, Francesca; Serebrovskaya, Ekaterina O.; Faro, Aline Regis; Shemyakina, Irina I.; Bozhanova, Nina G.; Kotlobay, Alexey A.; Gurskaya, Nadya G.; Bodén, Andreas; Dreier, Jes; Chudakov, Dmitriy M.; Lukyanov, Konstantin A.; Verkhusha, Vladislav V.; Mishin, Alexander S.; Testa, Ilaria

doi:10.1038/s41592-018-0052-9

Cited by 79 publications

(81 citation statements)

References 27 publications

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“…An intriguing finding from our data is the presence of the large green and red 'blocks' in the correlation table (Table 2, green: spectroscopic parameter 1-11; red: spectroscopic parameter [13][14][15][16][17]. This suggests that the spectroscopic parameters divide into two groups that show opposite responses to mutations, though some of the correlations are comparatively weak.…”

Section: Correlation Analysis Reveals Different Responses To the Polamentioning

confidence: 65%

“…Their complex photochemistry is a direct consequence of their intricate structure-function relationship, in which the probe properties are determined not just by the structure of the chromophore, but also by the chromophore's interactions with the surrounding amino acids. Mutating just a single residue in the vicinity of the chromophore can easily lead to profound changes in the spectroscopic properties [11][12][13].…”

Section: Introductionmentioning

confidence: 99%

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Integrated structure-function dataset reveals key mechanisms underlying photochromic fluorescent proteins

Zitter

Hugelier

Duwé

et al. 2020

Preprint

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Photochromic fluorescent proteins have become versatile tools in the life sciences, though our understanding of their structure-function relation is limited. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, yet differ only in one or two mutations. We also determined 43 different crystal structures of these mutants. Correlation and principal component analysis of the spectroscopic and structural properties confirmed the complex relationship between structure and spectroscopy, suggesting that the observed variability does not arise from a limited number of mechanisms, but also allowed us to identify consistent trends and to relate these to the spatial organization around the chromophore. We find that particular changes in spectroscopic properties can come about through multiple different underlying mechanisms, of which the polarity of the chromophore environment and hydrogen bonding of the chromophore are key modulators. Furthermore, some spectroscopic parameters, such as the photochromism, appear to be largely determined by a single or a few structural properties, while other parameters, such as the absorption maximum, do not allow a clear identification of a single cause. We also highlight the role of water molecules close to the chromophore in influencing photochromism. We anticipate that our dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.

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Section: Correlation Analysis Reveals Different Responses To the Polamentioning

confidence: 65%

Section: Introductionmentioning

confidence: 99%

Integrated structure-function dataset reveals key mechanisms underlying photochromic fluorescent proteins

Zitter

Hugelier

Duwé

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…The microscope is controlled through two separate software; image acquisition and most hardware control is done through the Imspector software (Max-Planck Innovation, Göttingen, Germany) while the rest of the hardware control (SLM, focus lock and 775 nm laser output power) is done through Python-based custom-written microscope control software Tempesta (https://github.com/jonatanalvelid/Tempesta-RedSTED, adapted from https://github.com/TestaLab/Tempesta) [7,16]. Line-by-line image acquisition for the two channels is achieved by sequential illumination of each line with two excitation lasers, alternating the detector read-outs and matching the STED power for each fluorophore with the AOM.…”

Section: Focus Lock Tiling and Microscope Controlmentioning

confidence: 99%

Tiled STED Imaging of Extended Sample Regions

Alvelid

Testa

2019

Preprint

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Stimulated Emission Depletion (STED) nanoscopy has become one of the most used nanoscopy techniques over the last decade. However, most recordings are done in specimen regions no larger than 10-30 x 10-30 µm 2 due to aberrations, instability and manual mechanical stages. Here, we demonstrate automated STED nanoscopy of extended sample regions up to 0.5 x 0.5 mm 2 by using a back-aperture-stationary beam scanning system. The setup allows up to 80-100 x 80-100 µm 2 field of view (FOV) with uniform spatial resolution, a mechanical stage allowing sequential tiling to record larger sample areas, and a feedback system keeping the sample in focus at all times.Taken together, this allows automated recording of theoretically unlimited-sized sample areas and volumes, without compromising the achievable spatial resolution and image quality.

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“…Current efforts focus on developing FPs with excitation and emission in the far-red/near-infrared wavelengths and with photophysical properties such as photoswitching and fluorescence intermittency optimized for super-resolution imaging modalities. 7 891011 - 121314 15 Nevertheless, all imaging applications benefit from increased cellular brightness, which is strongly dependent on molecular brightness (defined as the product of the molar extinction coefficient () and the fluorescence quantum yield ()). 1617 Lifetime-based selection methods have exploited a correlation between fluorescence lifetime () and  to develop FPs with higher  such as NowGFP, 18 mTurquoise2, 19 and mScarlet, which is the brightest red FP.…”

Section: Introductionmentioning

confidence: 99%

Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

Mukherjee

Hung

Douglas

et al. 2020

Preprint

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The development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and appropriate localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, FusionRed-MQV, which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (~140,000 M -1 cm -1 ), increased radiative rate constant and reduced non-radiative rate constant with respect to its precursor. The properties of FusionRed-MQV derive from three mutations -M42Q, C159V and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the phenol and the acylimine ends of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the phenol end of the chromophore were mutated. The M42Q mutation is located near the acylimine end of the chromophore and was discovered using sitedirected mutagenesis guided by x-ray crystal structures. FusionRed-MQV exhibits 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells (based on FACS) compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells.

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Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy

Cited by 79 publications

References 27 publications

Integrated structure-function dataset reveals key mechanisms underlying photochromic fluorescent proteins

Integrated structure-function dataset reveals key mechanisms underlying photochromic fluorescent proteins

Tiled STED Imaging of Extended Sample Regions

Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

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