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Advanced Research in Fluorescent Proteins

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biophysics".

Deadline for manuscript submissions: closed (31 March 2023) | Viewed by 24714

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Prof. Dr. Konstantin A. Lukyanov

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1. Skolkovo Institute of Science and Technology, 121205 Moscow, Russia
2. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia
Interests: green fluorescent protein; live cell imaging; protein labeling; photoconvertible fluorescent proteins; phototoxic fluorescent proteins; genetically encoded sensors
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Dear Colleagues,

Green fluorescent proteins (GFPs) together with a broad diversity of GFP-like proteins of various colors have become a routine tool to visualize target structures and activities in living biological models. In addition, fluorescent proteins based on domains bound to endogenous or exogenous fluorophores/fluorogens have been developed. Stimulated by practical demands, this field extensively uses methods of rational design and directed molecular evolution, protein structural studies, chemistry of dyes, steady-state and time-resolved optical spectroscopy, computer modeling of structures and spectral properties, and more. Advanced imaging and optogenetics would greatly benefit from novel fluorescent probes, e.g., with extended color and fluorescence lifetime diversity for multiparameter imaging, increased stability against harsh fixation conditions (such as for electron microscopy or expansion microscopy), and enhanced photostability and photoswitching properties for super-resolution fluorescence microscopy. This Special Issue will collect original and review papers on the development, in-depth characterization, and applications of fluorescent protein-based tools.

Prof. Dr. Konstantin A. Lukyanov
Guest Editor

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Keywords

GFP-like fluorescent proteins
flavin-, biliverdin-, or bilirubin-binding fluorescent proteins
fluorogen-activating proteins
genetically encoded sensors
phototoxic fluorescent proteins
photoconversions
super-resolution fluorescence microscopy
fluorescence lifetime imaging
multiparameter live-cell imaging

Published Papers (12 papers)

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Research

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13 pages, 4223 KiB

Open AccessArticle

NanoLuc Luciferase as a Fluorogen-Activating Protein for GFP Chromophore Based Fluorogens

by Yulia A. Bogdanova, Elvira R. Zaitseva, Alexander Yu. Smirnov, Nadezhda S. Baleeva, Alexey S. Gavrikov, Ivan N. Myasnyanko, Sergey A. Goncharuk, Erik F. Kot, Konstantin S. Mineev, Alexander S. Mishin and Mikhail S. Baranov

Int. J. Mol. Sci. 2023, 24(9), 7958; https://doi.org/10.3390/ijms24097958 - 27 Apr 2023

Cited by 1 | Viewed by 1436

Abstract

In this work, we showed that the well-known NanoLuc luciferase can act as a fluorogen activating protein for various arylidene-imidazolones structurally similar to the Kaede protein chromophore. We showed that such compounds can be used as fluorescent sensors for this protein and can [...] Read more.

(This article belongs to the Special Issue Advanced Research in Fluorescent Proteins)

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13 pages, 3114 KiB

Open AccessArticle

Combined Structural and Computational Study of the mRubyFT Fluorescent Timer Locked in Its Blue Form

by Konstantin M. Boyko, Maria G. Khrenova, Alena Y. Nikolaeva, Pavel V. Dorovatovskii, Anna V. Vlaskina, Oksana M. Subach, Vladimir O. Popov and Fedor V. Subach

Int. J. Mol. Sci. 2023, 24(9), 7906; https://doi.org/10.3390/ijms24097906 - 26 Apr 2023

Cited by 1 | Viewed by 1218

Abstract

The mRubyFT is a monomeric genetically encoded fluorescent timer based on the mRuby2 fluorescent protein, which is characterized by the complete maturation of the blue form with the subsequent conversion to the red one. It has higher brightness in mammalian cells and higher photostability compared with other fluorescent timers. A high-resolution structure is a known characteristic of the mRubyFT with the red form chromophore, but structural details of its blue form remain obscure. In order to obtain insight into this, we obtained an S148I variant of the mRubyFT (mRubyFT^S148I) with the blocked over time blue form of the chromophore. X-ray data at a 1.8 Å resolution allowed us to propose a chromophore conformation and its interactions with the neighboring residues. The imidazolidinone moiety of the chromophore is completely matured, being a conjugated π-system. The methine bridge is not oxidized in the blue form bringing flexibility to the phenolic moiety that manifests itself in poor electron density. Integration of these data with the results of molecular dynamic simulation disclosed that the OH group of the phenolic moiety forms a hydrogen bond with the side chain of the T163 residue. A detailed comparison of mRubyFT^S148I with other available structures of the blue form of fluorescent proteins, Blue102 and mTagBFP, revealed a number of characteristic differences. Molecular dynamic simulations with the combined quantum mechanic/molecular mechanic potentials demonstrated that the blue form exists in two protonation states, anion and zwitterion, both sharing enolate tautomeric forms of the C=C–O⁻ fragment. These two forms have similar excitation energies, as evaluated by calculations. Finally, excited state molecular dynamic simulations showed that excitation of the chromophore in both protonation states leads to the same anionic fluorescent state. The data obtained shed light on the structural features and spectral properties of the blue form of the mRubyFT timer. Full article

(This article belongs to the Special Issue Advanced Research in Fluorescent Proteins)

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21 pages, 9208 KiB

Open AccessArticle

Structural Insights into the Binding of Red Fluorescent Protein mCherry-Specific Nanobodies

by Hui Liang, Zhiqiang Ma, Ziying Wang, Peiyu Zhong, Ran Li, He Jiang, Xin Zong, Chao Zhong, Xihuan Liu, Peng Liu, Jiayuan Liu, Haoran Zhu, Rui Liu and Yu Ding

Int. J. Mol. Sci. 2023, 24(8), 6952; https://doi.org/10.3390/ijms24086952 - 09 Apr 2023

Viewed by 2170

Abstract

Red fluorescent proteins (RFPs) have broad applications in life science research, and the manipulation of RFPs using nanobodies can expand their potential uses. However, the structural information available for nanobodies that bind with RFPs is still insufficient. In this study, we cloned, expressed, purified, and crystallized complexes formed by mCherry with LaM1, LaM3, and LaM8. Then, we analyzed the biochemical properties of the complexes using mass spectrometry (MS), fluorescence-detected size exclusion chromatography (FSEC), isothermal titration calorimetry (ITC), and bio-layer interferometry (BLI) technology. We determined the crystal structure of mCherry-LaM1, mCherry-LaM3, and mCherry-LaM8, with resolutions of 2.05 Å, 3.29 Å, and 1.31 Å, respectively. In this study, we systematically compared various parameters of several LaM series nanobodies, including LaM1, LaM3, and LaM8, with previously reported data on LaM2, LaM4, and LaM6, specifically examining their structural information. After designing multivalent tandem LaM1-LaM8 and LaM8-LaM4 nanobodies based on structural information, we characterized their properties, revealing their higher affinity and specificity to mCherry. Our research provides novel structural insights that could aid in understanding nanobodies targeting a specific target protein. This could provide a starting point for developing enhanced mCherry manipulation tools. Full article

(This article belongs to the Special Issue Advanced Research in Fluorescent Proteins)

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28 pages, 8574 KiB

Open AccessArticle

Blue-to-Red TagFT, mTagFT, mTsFT, and Green-to-FarRed mNeptusFT2 Proteins, Genetically Encoded True and Tandem Fluorescent Timers

by Oksana M. Subach, Anna V. Vlaskina, Yulia K. Agapova, Alena Y. Nikolaeva, Konstantin V. Anokhin, Kiryl D. Piatkevich, Maxim V. Patrushev, Konstantin M. Boyko and Fedor V. Subach

Int. J. Mol. Sci. 2023, 24(4), 3279; https://doi.org/10.3390/ijms24043279 - 07 Feb 2023

Cited by 1 | Viewed by 2089

Abstract

True genetically encoded monomeric fluorescent timers (tFTs) change their fluorescent color as a result of the complete transition of the blue form into the red form over time. Tandem FTs (tdFTs) change their color as a consequence of the fast and slow independent maturation of two forms with different colors. However, tFTs are limited to derivatives of the mCherry and mRuby red fluorescent proteins and have low brightness and photostability. The number of tdFTs is also limited, and there are no blue-to-red or green-to-far-red tdFTs. tFTs and tdFTs have not previously been directly compared. Here, we engineered novel blue-to-red tFTs, called TagFT and mTagFT, which were derived from the TagRFP protein. The main spectral and timing characteristics of the TagFT and mTagFT timers were determined in vitro. The brightnesses and photoconversions of the TagFT and mTagFT tFTs were characterized in live mammalian cells. The engineered split version of the TagFT timer matured in mammalian cells at 37 °C and allowed the detection of interactions between two proteins. The TagFT timer under the control of the minimal arc promoter, successfully visualized immediate-early gene induction in neuronal cultures. We also developed and optimized green-to-far-red and blue-to-red tdFTs, named mNeptusFT and mTsFT, which were based on mNeptune-sfGFP and mTagBFP2-mScarlet fusion proteins, respectively. We developed the FucciFT2 system based on the TagFT-hCdt1-100/mNeptusFT2-hGeminin combination, which could visualize the transitions between the G1 and S/G2/M phases of the cell cycle with better resolution than the conventional Fucci system because of the fluorescent color changes of the timers over time in different phases of the cell cycle. Finally, we determined the X-ray crystal structure of the mTagFT timer and analyzed it using directed mutagenesis. Full article

(This article belongs to the Special Issue Advanced Research in Fluorescent Proteins)

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11 pages, 3171 KiB

Open AccessArticle

Domain Truncation in Hispidin Synthase Orthologs from Non-Bioluminescent Fungi Does Not Lead to Hispidin Biosynthesis

by Kseniia A. Palkina, Anastasia V. Balakireva, Olga A. Belozerova, Tatiana V. Chepurnykh, Nadezhda M. Markina, Sergey I. Kovalchuk, Aleksandra S. Tsarkova, Alexander S. Mishin, Ilia V. Yampolsky and Karen S. Sarkisyan

Int. J. Mol. Sci. 2023, 24(2), 1317; https://doi.org/10.3390/ijms24021317 - 10 Jan 2023

Cited by 6 | Viewed by 1933

Abstract

Hispidin is a polyketide found in plants and fungi. In bioluminescent fungi, hispidin serves as a precursor of luciferin and is produced by hispidin synthases. Previous studies revealed that hispidin synthases differ in orthologous polyketide synthases from non-bioluminescent fungi by the absence of two domains with predicted ketoreductase and dehydratase activities. Here, we investigated the hypothesis that the loss of these domains in evolution led to the production of hispidin and the emergence of bioluminescence. We cloned three orthologous polyketide synthases from non-bioluminescent fungi, as well as their truncated variants, and assessed their ability to produce hispidin in a bioluminescence assay in yeast. Interestingly, expression of the full-length enzyme hsPKS resulted in dim luminescence, indicating that small amounts of hispidin are likely being produced as side products of the main reaction. Deletion of the ketoreductase and dehydratase domains resulted in no luminescence. Thus, domain truncation by itself does not appear to be a sufficient step for the emergence of efficient hispidin synthases from orthologous polyketide synthases. At the same time, the production of small amounts of hispidin or related compounds by full-length enzymes suggests that ancestral fungal species were well-positioned for the evolution of bioluminescence. Full article

(This article belongs to the Special Issue Advanced Research in Fluorescent Proteins)

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10 pages, 2565 KiB

Open AccessArticle

Systematic Comparison of Plant Promoters in Nicotiana spp. Expression Systems

by Ekaterina S. Shakhova, Nadezhda M. Markina, Tatiana Mitiouchkina, Evgenia N. Bugaeva, Tatiana A. Karataeva, Kseniia A. Palkina, Liliia I. Fakhranurova, Ilia V. Yampolsky, Karen S. Sarkisyan and Alexander S. Mishin

Int. J. Mol. Sci. 2022, 23(23), 15441; https://doi.org/10.3390/ijms232315441 - 06 Dec 2022

Cited by 3 | Viewed by 1870

Abstract

We report a systematic comparison of 19 plant promoters and 20 promoter-terminator combinations in two expression systems: agroinfiltration in Nicotiana benthamiana leaves, and Nicotiana tabacum BY-2 plant cell packs. The set of promoters tested comprised those not present in previously published work, including several computationally predicted synthetic promoters validated here for the first time. The expression of EGFP driven by different promoters varied by more than two orders of magnitude and was largely consistent between two tested Nicotiana systems. We confirmed previous reports of significant modulation of expression by terminators, as well as synergistic effects of promoters and terminators. Additionally, we observed non-linear effects of gene dosage on expression level. The dataset presented here can inform the design of genetic constructs for plant engineering and transient expression assays. Full article

(This article belongs to the Special Issue Advanced Research in Fluorescent Proteins)

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12 pages, 1551 KiB

Open AccessArticle

Two-Photon Excitation Spectra of Various Fluorescent Proteins within a Broad Excitation Range

by Ruth Leben, Randall L. Lindquist, Anja E. Hauser, Raluca Niesner and Asylkhan Rakhymzhan

Int. J. Mol. Sci. 2022, 23(21), 13407; https://doi.org/10.3390/ijms232113407 - 02 Nov 2022

Cited by 5 | Viewed by 2597

Abstract

Two-photon excitation fluorescence laser-scanning microscopy is the preferred method for studying dynamic processes in living organ models or even in living organisms. Thanks to near-infrared and infrared excitation, it is possible to penetrate deep into the tissue, reaching areas of interest relevant to life sciences and biomedicine. In those imaging experiments, two-photon excitation spectra are needed to select the optimal laser wavelength to excite as many fluorophores as possible simultaneously in the sample under consideration. The more fluorophores that can be excited, and the more cell populations that can be studied, the better access to their arrangement and interaction can be reached in complex systems such as immunological organs. However, for many fluorophores, the two-photon excitation properties are poorly predicted from the single-photon spectra and are not yet available, in the literature or databases. Here, we present the broad excitation range (760 nm to 1300 nm) of photon-flux-normalized two-photon spectra of several fluorescent proteins in their cellular environment. This includes the following fluorescent proteins spanning from the cyan to the infrared part of the spectrum: mCerulean3, mTurquoise2, mT-Sapphire, Clover, mKusabiraOrange2, mOrange2, LSS-mOrange, mRuby2, mBeRFP, mCardinal, iRFP670, NirFP, and iRFP720. Full article

(This article belongs to the Special Issue Advanced Research in Fluorescent Proteins)

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18 pages, 5613 KiB

Open AccessArticle

LSSmScarlet2 and LSSmScarlet3, Chemically Stable Genetically Encoded Red Fluorescent Proteins with a Large Stokes’ Shift

by Oksana M. Subach, Anna V. Vlaskina, Yulia K. Agapova, Kiryl D. Piatkevich, Maxim V. Patrushev, Valeriya R. Samygina and Fedor V. Subach

Int. J. Mol. Sci. 2022, 23(19), 11051; https://doi.org/10.3390/ijms231911051 - 21 Sep 2022

Cited by 2 | Viewed by 2546

Abstract

Red fluorescent proteins with a large Stokes’ shift (LSSRFPs) are genetically encoded and efficiently excited by 488 nm light, allowing simultaneous dual-color one- and two-photon fluorescence imaging and fluorescence correlation spectroscopy in combination with green fluorescent proteins FPs. Recently, based on the conventional bright mScarlet RFP, we developed the LSSRFP LSSmScarlet. LSSmScarlet is characterized by two pKa values at pH values of 1.9 and 5.8. In this study, we developed improved versions of LSSmScarlet, named LSSmScarlet2 and LSSmScarlet3, which are characterized by a Stokes’ shift of 128 nm and extreme pH stability with a single pKa value of 2.2. LSSmScarlet2 and LSSmScarlet3 had 1.8-fold faster and 3-fold slower maturation than LSSmScarlet, respectively. In addition, both LSSRFPs were 1.5- to 1.6-fold more photostable and more chemically resistant to denaturation by guanidinium chloride and guanidinium thiocyanate. We also compared the susceptibility of the LSSmScarlet2, LSSmScarlet3, and other LSSRFPs to the reagents used for whole-mount imaging, expansion microscopy, and immunostaining techniques. Due to higher pH stability and faster maturation, the LSSmScarlet3-LAMP3 fusion was 2.2-fold brighter than LSSmScarlet-LAMP3 in lysosomes of mammalian cells. The LSSmScarlet3-hLAMP2A fusion was similar in brightness to LSSmScarlet-hLAMP2A in lysosomes. We successfully applied the monomeric LSSmScarlet2 and LSSmScarlet3 proteins for confocal imaging of structural proteins in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet2 protein at a resolution of 1.41 Å. Site-directed mutagenesis of the LSSmScarlet2 protein demonstrated the key role of the T74 residue in improving the pH and chemical stability of the LSSmScarlet2 protein. Full article

(This article belongs to the Special Issue Advanced Research in Fluorescent Proteins)

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10 pages, 1686 KiB

Open AccessArticle

Genetically Encoded Fluorescent Sensors for SARS-CoV-2 Papain-like Protease PLpro

by Elena L. Sokolinskaya, Lidia V. Putlyaeva, Vasilisa S. Polinovskaya and Konstantin A. Lukyanov

Int. J. Mol. Sci. 2022, 23(14), 7826; https://doi.org/10.3390/ijms23147826 - 15 Jul 2022

Cited by 4 | Viewed by 1858

Abstract

In the SARS-CoV-2 lifecycle, papain-like protease PLpro cuts off the non-structural proteins nsp1, nsp2, and nsp3 from a large polyprotein. This is the earliest viral enzymatic activity, which is crucial for all downstream steps. Here, we designed two genetically encoded fluorescent sensors for the real-time detection of PLpro activity in live cells. The first sensor was based on the Förster resonance energy transfer (FRET) between the red fluorescent protein mScarlet as a donor and the biliverdin-binding near-infrared fluorescent protein miRFP670 as an acceptor. A linker with the PLpro recognition site LKGG in between made this FRET pair sensitive to PLpro cleavage. Upon the co-expression of mScarlet-LKGG-miRFP670 and PLpro in HeLa cells, we observed a gradual increase in the donor fluorescence intensity of about 1.5-fold. In the second sensor, both PLpro and its target—green mNeonGreen and red mScarletI fluorescent proteins separated by an LKGG-containing linker—were attached to the endoplasmic reticulum (ER) membrane. Upon cleavage by PLpro, mScarletI diffused from the ER throughout the cell. About a two-fold increase in the nucleus/cytoplasm ratio was observed as a result of the PLpro action. We believe that the new PLpro sensors can potentially be used to detect the earliest stages of SARS-CoV-2 propagation in live cells as well as for the screening of PLpro inhibitors. Full article

(This article belongs to the Special Issue Advanced Research in Fluorescent Proteins)

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13 pages, 2197 KiB

Open AccessArticle

Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties

by Olga V. Stepanenko, Irina M. Kuznetsova, Konstantin K. Turoverov and Olesya V. Stepanenko

Int. J. Mol. Sci. 2022, 23(13), 7347; https://doi.org/10.3390/ijms23137347 - 01 Jul 2022

Cited by 2 | Viewed by 1273

Abstract

Understanding the photophysical properties and stability of near-infrared fluorescent proteins (NIR FPs) based on bacterial phytochromes is of great importance for the design of efficient fluorescent probes for use in cells and in vivo. Previously, the natural ligand of NIR FPs biliverdin (BV) has been revealed to be capable of covalent binding to the inherent cysteine residue in the PAS domain (Cys15), and to the cysteine residue introduced into the GAF domain (Cys256), as well as simultaneously with these two residues. Here, based on the spectroscopic analysis of several NIR FPs with both cysteine residues in PAS and GAF domains, we show that the covalent binding of BV simultaneously with two domains is the reason for the higher quantum yield of BV fluorescence in these proteins as a result of rigid fixation of the chromophore in their chromophore-binding pocket. We demonstrate that since the attachment sites are located in different regions of the polypeptide chain forming a figure-of-eight knot, their binding to BV leads to shielding of many sites of proteolytic degradation due to additional stabilization of the entire protein structure. This makes NIR FPs with both cysteine residues in PAS and GAF domains less susceptible to cleavage by intracellular proteases. Full article

(This article belongs to the Special Issue Advanced Research in Fluorescent Proteins)

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19 pages, 2701 KiB

Open AccessArticle

The Functional Characterization of GCaMP3.0 Variants Specifically Targeted to Subcellular Domains

by Annika Kempmann, Thomas Gensch, Andreas Offenhäusser, Irina Tihaa, Vanessa Maybeck, Sabine Balfanz and Arnd Baumann

Int. J. Mol. Sci. 2022, 23(12), 6593; https://doi.org/10.3390/ijms23126593 - 13 Jun 2022

Cited by 2 | Viewed by 1674

Abstract

Calcium (Ca²⁺) ions play a pivotal role in physiology and cellular signaling. The intracellular Ca²⁺ concentration ([Ca²⁺]_i) is about three orders of magnitude lower than the extracellular concentration, resulting in a steep transmembrane concentration gradient. Thus, the spatial and the temporal dynamics of [Ca²⁺]_i are ideally suited to modulate Ca²⁺-mediated cellular responses to external signals. A variety of highly sophisticated methods have been developed to gain insight into cellular Ca²⁺ dynamics. In addition to electrophysiological measurements and the application of synthetic dyes that change their fluorescent properties upon interaction with Ca²⁺, the introduction and the ongoing development of genetically encoded Ca²⁺ indicators (GECI) opened a new era to study Ca²⁺-driven processes in living cells and organisms. Here, we have focused on one well-established GECI, i.e., GCaMP3.0. We have systematically modified the protein with sequence motifs, allowing localization of the sensor in the nucleus, in the mitochondrial matrix, at the mitochondrial outer membrane, and at the plasma membrane. The individual variants and a cytosolic version of GCaMP3.0 were overexpressed and purified from E. coli cells to study their biophysical properties in solution. All versions were examined to monitor Ca²⁺ signaling in stably transfected cell lines and in primary cortical neurons transduced with recombinant Adeno-associated viruses (rAAV). In this comparative study, we provide evidence for a robust approach to reliably trace Ca²⁺ signals at the (sub)-cellular level with pronounced temporal resolution. Full article

(This article belongs to the Special Issue Advanced Research in Fluorescent Proteins)

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Review

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26 pages, 5026 KiB

Open AccessReview

Photoswitchable Fluorescent Proteins: Mechanisms on Ultrafast Timescales

by Longteng Tang and Chong Fang

Int. J. Mol. Sci. 2022, 23(12), 6459; https://doi.org/10.3390/ijms23126459 - 09 Jun 2022

Cited by 11 | Viewed by 2985

Abstract

The advancement of super-resolution imaging (SRI) relies on fluorescent proteins with novel photochromic properties. Using light, the reversibly switchable fluorescent proteins (RSFPs) can be converted between bright and dark states for many photocycles and their emergence has inspired the invention of advanced SRI techniques. The general photoswitching mechanism involves the chromophore cis-trans isomerization and proton transfer for negative and positive RSFPs and hydration–dehydration for decoupled RSFPs. However, a detailed understanding of these processes on ultrafast timescales (femtosecond to millisecond) is lacking, which fundamentally hinders the further development of RSFPs. In this review, we summarize the current progress of utilizing various ultrafast electronic and vibrational spectroscopies, and time-resolved crystallography in investigating the on/off photoswitching pathways of RSFPs. We show that significant insights have been gained for some well-studied proteins, but the real-time “action” details regarding the bidirectional cis-trans isomerization, proton transfer, and intermediate states remain unclear for most systems, and many other relevant proteins have not been studied yet. We expect this review to lay the foundation and inspire more ultrafast studies on existing and future engineered RSFPs. The gained mechanistic insights will accelerate the rational development of RSFPs with enhanced two-way switching rate and efficiency, better photostability, higher brightness, and redder emission colors. Full article

(This article belongs to the Special Issue Advanced Research in Fluorescent Proteins)

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