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Special Issue "Fluorescent Proteins"

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

Deadline for manuscript submissions: closed (30 April 2018).

Special Issue Editors

Guest Editor
Prof. Dr. Dominique Bourgeois

Institut de Biologie Structurale, Univ. Grenoble Alpes, CEA, CNRS, F-38044 Grenoble, France
Website | E-Mail
Interests: phototransformable fluorescent proteins; single-molecule imaging; kinetic x-ray crystallography; protein dynamics; super-resolution microscopy; photoswitching; photoblinking; photobleaching
Guest Editor
Prof. Dr. Hideaki Mizuno

Biochemistry, Molecular and Structural Biology Section, KU Leuven, Celestijnenlaan 200G bus2403, B-3001 Heverlee, Belgium
Website | E-Mail
Interests: photochemistry of fluorescent proteins; photoconversion; fluorescence probes; FRET imaging; live-cell fluorescence imaging; fluorescence correlation spectroscopy; single-molecule imaging; super-resolution microscopy

Special Issue Information

Dear Colleagues,

This Special Issue, “Fluorescent Proteins”, will cover a selection of recent research topics and current review articles in the field of fluorescent protein research and applications. Experimental papers, up-to-date review articles, and commentaries are all welcome. We propose to dedicate this issue to Roger Tsien.

Since the chemistry Nobel Prize was awarded in 2008 to Shimomura, Chalfie and Tsien to celebrate the discovery of GFP, fluorescent protein research has remained a central theme, guiding the development of fluorescence bioimaging. Although major advances are accomplished every year, optimal fluorescent proteins (FPs) are still lacking and the design of more effective markers is constantly claimed to be the major clue to the further development of fluorescence microscopy. Recent advances include the engineering of far-red and infrared FPs, which are necessary to investigate biological tissues in depth, and the development of high-performance phototransformable FPs, which largely dictates the potential of fluorescence nanoscopy. In the near future, smart fluorescent protein sensors will also provide new opportunities to image biological samples functionally at high resolution. Although “ideal” FPs differ for each imaging technique, the vast array of properties that need to be addressed when engineering FP variants require a profound understanding of their structure-function relationships at the molecular level. This Special Issue of IJMS, thus, aims at providing an up-to-date view of how advanced fluorescent proteins work and how they can possibly be engineered to work even better.

Prof. Dr. Dominique Bourgeois
Prof. Dr. Hideaki Mizuno
Guest Editors

Manuscript Submission Information

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Keywords

  • GFP chromophore dynamics
  • Fluorescent protein structure and dynamics
  • Photoactivation, conversion, switching, blinking, bleaching
  • Far-red and infrared fluorescent proteins
  • Fluorescent protein-based sensors and timers
  • Large Stokes Shift fluorescent proteins
  • Phototransformable fluorescent proteins
  • Fluorescent protein maturation and folding
  • Genome editing with fluorescent proteins

Published Papers (14 papers)

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Research

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Open AccessArticle
Fluorescence Properties of a Novel Cyanobacteriochrome GAF Domain from Spirulina that Exhibits Moderate Dark Reversion
Int. J. Mol. Sci. 2018, 19(8), 2253; https://doi.org/10.3390/ijms19082253
Received: 18 June 2018 / Revised: 11 July 2018 / Accepted: 24 July 2018 / Published: 1 August 2018
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Abstract
Cyanobacteriochromes (CBCRs) are biliproteins for photoreception that are present in cyanobacteria. These proteins possess one or more unique cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that can covalently bind the linear tetrapyrrole (bilin). Light absorption triggers the photoisomerization of bilin between the 15Z and 15E [...] Read more.
Cyanobacteriochromes (CBCRs) are biliproteins for photoreception that are present in cyanobacteria. These proteins possess one or more unique cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that can covalently bind the linear tetrapyrrole (bilin). Light absorption triggers the photoisomerization of bilin between the 15Z and 15E photostates. The 15E photoproduct of some CBCR GAF domains can revert to the stable 15Z state in the absence of light. In some cases, this property makes these domains function as sensors of light intensity or as red/dark optogenetic switches. However, there have been few reports regarding the applicability of these fluorescent properties. Here, we report a red/green cyanobacteriochrome GAF domain from Spirulina subsalsa, designated SPI1085g3, which exhibited photoconversion from the red-absorbing dark state (Pr, λmax = 642 nm) to the orange-absorbing photoproduct state (Po, λmax = 590 nm), and exhibited moderate dark reversion (t1/2 = 3.3 min) from the Po state to the Pr state. The SPI1085g3 Pr state exhibited intense red fluorescence (λmax = 662 nm), with a quantum yield of 0.14. The fluorescence was switched off by red light irradiation and increased in the dark. Replacement of Cys448 of SPI1085g3 with Ser resulted in a slightly improved fluorescence quantum yield and nearly 13-fold faster dark reversion (t1/2 = 15.2 s) than that of the wild type. This novel red/dark-switchable fluorescent biliprotein expands the present repertoire and diversity of photoswitchable fluorescent protein candidates. Full article
(This article belongs to the Special Issue Fluorescent Proteins)
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Open AccessArticle
Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense
Int. J. Mol. Sci. 2018, 19(2), 371; https://doi.org/10.3390/ijms19020371
Received: 20 December 2017 / Revised: 15 January 2018 / Accepted: 24 January 2018 / Published: 26 January 2018
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Abstract
Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To [...] Read more.
Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT)14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense. We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein “ember” from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 105 M−1·cm−1. The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers. Full article
(This article belongs to the Special Issue Fluorescent Proteins)
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Open AccessArticle
Assessing the Effectiveness of a Far-Red Fluorescent Reporter for Tracking Stem Cells In Vivo
Int. J. Mol. Sci. 2018, 19(1), 19; https://doi.org/10.3390/ijms19010019
Received: 14 November 2017 / Revised: 15 December 2017 / Accepted: 20 December 2017 / Published: 22 December 2017
Cited by 3 | PDF Full-text (11318 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Far-red fluorescent reporter genes can be used for tracking cells non-invasively in vivo using fluorescence imaging. Here, we investigate the effectiveness of the far-red fluorescent protein, E2-Crimson (E2C), for tracking mouse embryonic cells (mESCs) in vivo following subcutaneous administration into mice. Using a [...] Read more.
Far-red fluorescent reporter genes can be used for tracking cells non-invasively in vivo using fluorescence imaging. Here, we investigate the effectiveness of the far-red fluorescent protein, E2-Crimson (E2C), for tracking mouse embryonic cells (mESCs) in vivo following subcutaneous administration into mice. Using a knock-in strategy, we introduced E2C into the Rosa26 locus of an E14-Bra-GFP mESC line, and after confirming that the E2C had no obvious effect on the phenotype of the mESCs, we injected them into mice and imaged them over nine days. The results showed that fluorescence intensity was weak, and cells could only be detected when injected at high densities. Furthermore, intensity peaked on day 4 and then started to decrease, despite the fact that tumour volume continued to increase beyond day 4. Histopathological analysis showed that although E2C fluorescence could barely be detected in vivo at day 9, analysis of frozen sections indicated that all mESCs within the tumours continued to express E2C. We hypothesise that the decrease in fluorescence intensity in vivo was probably due to the fact that the mESC tumours became more vascular with time, thus leading to increased absorbance of E2C fluorescence by haemoglobin. We conclude that the E2C reporter has limited use for tracking cells in vivo, at least when introduced as a single copy into the Rosa26 locus. Full article
(This article belongs to the Special Issue Fluorescent Proteins)
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Open AccessArticle
Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization
Int. J. Mol. Sci. 2017, 18(9), 2015; https://doi.org/10.3390/ijms18092015
Received: 16 August 2017 / Revised: 8 September 2017 / Accepted: 11 September 2017 / Published: 20 September 2017
Cited by 2 | PDF Full-text (3207 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Reversibly switchable fluorescent proteins (RSFPs) enable advanced fluorescence imaging, though the performance of this imaging crucially depends on the properties of the labels. We report on the use of an existing small binding peptide, named Enhancer, to modulate the spectroscopic properties of the [...] Read more.
Reversibly switchable fluorescent proteins (RSFPs) enable advanced fluorescence imaging, though the performance of this imaging crucially depends on the properties of the labels. We report on the use of an existing small binding peptide, named Enhancer, to modulate the spectroscopic properties of the recently developed rsGreen series of RSFPs. Fusion constructs of Enhancer with rsGreen1 and rsGreenF revealed an increased molecular brightness and pH stability, although expression in living E. coli or HeLa cells resulted in a decrease of the overall emission. Surprisingly, Enhancer binding also increased off-switching speed and resistance to switching fatigue. Further investigation suggested that the RSFPs can interconvert between fast- and slow-switching emissive states, with the overall protein population gradually converting to the slow-switching state through irradiation. The Enhancer modulates the spectroscopic properties of both states, but also preferentially stabilizes the fast-switching state, supporting the increased fatigue resistance. This work demonstrates how the photo-physical properties of RSFPs can be influenced by their binding to other small proteins, which opens up new horizons for applications that may require such modulation. Furthermore, we provide new insights into the photoswitching kinetics that should be of general consideration when developing new RSFPs with improved or different photochromic properties. Full article
(This article belongs to the Special Issue Fluorescent Proteins)
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Open AccessArticle
X-ray Free Electron Laser Determination of Crystal Structures of Dark and Light States of a Reversibly Photoswitching Fluorescent Protein at Room Temperature
Int. J. Mol. Sci. 2017, 18(9), 1918; https://doi.org/10.3390/ijms18091918
Received: 14 August 2017 / Revised: 1 September 2017 / Accepted: 2 September 2017 / Published: 7 September 2017
Cited by 5 | PDF Full-text (3470 KB) | HTML Full-text | XML Full-text
Abstract
The photochromic fluorescent protein Skylan-NS (Nonlinear Structured illumination variant mEos3.1H62L) is a reversibly photoswitchable fluorescent protein which has an unilluminated/ground state with an anionic and cis chromophore conformation and high fluorescence quantum yield. Photo-conversion with illumination at 515 nm generates a meta-stable intermediate [...] Read more.
The photochromic fluorescent protein Skylan-NS (Nonlinear Structured illumination variant mEos3.1H62L) is a reversibly photoswitchable fluorescent protein which has an unilluminated/ground state with an anionic and cis chromophore conformation and high fluorescence quantum yield. Photo-conversion with illumination at 515 nm generates a meta-stable intermediate with neutral trans-chromophore structure that has a 4 h lifetime. We present X-ray crystal structures of the cis (on) state at 1.9 Angstrom resolution and the trans (off) state at a limiting resolution of 1.55 Angstrom from serial femtosecond crystallography experiments conducted at SPring-8 Angstrom Compact Free Electron Laser (SACLA) at 7.0 keV and 10.5 keV, and at Linac Coherent Light Source (LCLS) at 9.5 keV. We present a comparison of the data reduction and structure determination statistics for the two facilities which differ in flux, beam characteristics and detector technologies. Furthermore, a comparison of droplet on demand, grease injection and Gas Dynamic Virtual Nozzle (GDVN) injection shows no significant differences in limiting resolution. The photoconversion of the on- to the off-state includes both internal and surface exposed protein structural changes, occurring in regions that lack crystal contacts in the orthorhombic crystal form. Full article
(This article belongs to the Special Issue Fluorescent Proteins)
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Open AccessArticle
Colorful Packages: Encapsulation of Fluorescent Proteins in Complex Coacervate Core Micelles
Int. J. Mol. Sci. 2017, 18(7), 1557; https://doi.org/10.3390/ijms18071557
Received: 23 May 2017 / Revised: 30 June 2017 / Accepted: 13 July 2017 / Published: 19 July 2017
Cited by 4 | PDF Full-text (6819 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Encapsulation of proteins can be beneficial for food and biomedical applications. To study their biophysical properties in complex coacervate core micelles (C3Ms), we previously encapsulated enhanced green fluorescent protein (EGFP) and its monomeric variant, mEGFP, with the cationic-neutral diblock copolymer poly(2-methyl-vinyl-pyridinium)n- [...] Read more.
Encapsulation of proteins can be beneficial for food and biomedical applications. To study their biophysical properties in complex coacervate core micelles (C3Ms), we previously encapsulated enhanced green fluorescent protein (EGFP) and its monomeric variant, mEGFP, with the cationic-neutral diblock copolymer poly(2-methyl-vinyl-pyridinium)n-b-poly(ethylene-oxide)m (P2MVPn-b-PEOm) as enveloping material. C3Ms with high packaging densities of fluorescent proteins (FPs) were obtained, resulting in a restricted orientational freedom of the protein molecules, influencing their structural and spectral properties. To address the generality of this behavior, we encapsulated seven FPs with P2MVP41-b-PEO205 and P2MVP128-b-PEO477. Dynamic light scattering and fluorescence correlation spectroscopy showed lower encapsulation efficiencies for members of the Anthozoa class (anFPs) than for Hydrozoa FPs derived from Aequorea victoria (avFPs). Far-UV CD spectra of the free FPs showed remarkable differences between avFPs and anFPs, caused by rounder barrel structures for avFPs and more elliptic ones for anFPs. These structural differences, along with the differences in charge distribution, might explain the variations in encapsulation efficiency between avFPs and anFPs. Furthermore, the avFPs remain monomeric in C3Ms with minor spectral and structural changes. In contrast, the encapsulation of anFPs gives rise to decreased quantum yields (monomeric Kusabira Orange 2 (mKO2) and Tag red fluorescent protein (TagRFP)) or to a pKa shift of the chromophore (FP variant mCherry). Full article
(This article belongs to the Special Issue Fluorescent Proteins)
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Open AccessArticle
Combining Primed Photoconversion and UV-Photoactivation for Aberration-Free, Live-Cell Compliant Multi-Color Single-Molecule Localization Microscopy Imaging
Int. J. Mol. Sci. 2017, 18(7), 1524; https://doi.org/10.3390/ijms18071524
Received: 11 June 2017 / Revised: 4 July 2017 / Accepted: 5 July 2017 / Published: 14 July 2017
Cited by 5 | PDF Full-text (4211 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Super-resolution fluorescence microscopy plays a major role in revealing the organization and dynamics of living cells. Nevertheless, single-molecule localization microscopy imaging of multiple targets is still limited by the availability of suitable fluorophore combinations. Here, we introduce a novel imaging strategy which combines [...] Read more.
Super-resolution fluorescence microscopy plays a major role in revealing the organization and dynamics of living cells. Nevertheless, single-molecule localization microscopy imaging of multiple targets is still limited by the availability of suitable fluorophore combinations. Here, we introduce a novel imaging strategy which combines primed photoconversion (PC) and UV-photoactivation for imaging different molecular species tagged by suitable fluorescent protein combinations. In this approach, the fluorescent proteins can be specifically photoactivated/-converted by different light wavelengths using PC and UV-activation modes but emit fluorescence in the same spectral emission channel. We demonstrate that this aberration-free, live-cell compatible imaging method can be applied to various targets in bacteria, yeast and mammalian cells and can be advantageously combined with correlative imaging schemes. Full article
(This article belongs to the Special Issue Fluorescent Proteins)
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Open AccessArticle
Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models
Int. J. Mol. Sci. 2017, 18(7), 1503; https://doi.org/10.3390/ijms18071503
Received: 31 May 2017 / Revised: 2 July 2017 / Accepted: 10 July 2017 / Published: 12 July 2017
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Abstract
Despite great advances in practical applications of fluorescent proteins (FPs), their natural function is poorly understood. FPs display complex spatio-temporal expression patterns in living Anthozoa coral polyps. Here we applied confocal microscopy, specifically, the fluorescence recovery after photobleaching (FRAP) technique to analyze intracellular [...] Read more.
Despite great advances in practical applications of fluorescent proteins (FPs), their natural function is poorly understood. FPs display complex spatio-temporal expression patterns in living Anthozoa coral polyps. Here we applied confocal microscopy, specifically, the fluorescence recovery after photobleaching (FRAP) technique to analyze intracellular localization and mobility of endogenous FPs in live tissues. We observed three distinct types of protein distributions in living tissues. One type of distribution, characteristic for Anemonia, Discosoma and Zoanthus, is free, highly mobile cytoplasmic localization. Another pattern is seen in FPs localized to numerous intracellular vesicles, observed in Clavularia. The third most intriguing type of intracellular localization is with respect to the spindle-shaped aggregates and lozenge crystals several micrometers in size observed in Zoanthus samples. No protein mobility within those structures was detected by FRAP. This finding encouraged us to develop artificial aggregating FPs. We constructed “trio-FPs” consisting of three tandem copies of tetrameric FPs and demonstrated that they form multiple bright foci upon expression in mammalian cells. High brightness of the aggregates is advantageous for early detection of weak promoter activities. Simultaneously, larger aggregates can induce significant cytostatic and cytotoxic effects and thus such tags are not suitable for long-term and high-level expression. Full article
(This article belongs to the Special Issue Fluorescent Proteins)
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Open AccessArticle
FRET-Mediated Long-Range Wavelength Transformation by Photoconvertible Fluorescent Proteins as an Efficient Mechanism to Generate Orange-Red Light in Symbiotic Deep Water Corals
Int. J. Mol. Sci. 2017, 18(7), 1174; https://doi.org/10.3390/ijms18071174
Received: 7 April 2017 / Revised: 15 May 2017 / Accepted: 17 May 2017 / Published: 4 July 2017
Cited by 2 | PDF Full-text (4134 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Photoconvertible fluorescent proteins (pcRFPs) are a group of fluorophores that undergo an irreversible green-to-red shift in emission colour upon irradiation with near-ultraviolet (near-UV) light. Despite their wide application in biotechnology, the high-level expression of pcRFPs in mesophotic and depth-generalist coral species currently lacks [...] Read more.
Photoconvertible fluorescent proteins (pcRFPs) are a group of fluorophores that undergo an irreversible green-to-red shift in emission colour upon irradiation with near-ultraviolet (near-UV) light. Despite their wide application in biotechnology, the high-level expression of pcRFPs in mesophotic and depth-generalist coral species currently lacks a biological explanation. Additionally, reduced penetration of near-UV wavelengths in water poses the question whether light-driven photoconversion is relevant in the mesophotic zone, or whether a different mechanism is involved in the post-translational pigment modification in vivo. Here, we show in a long-term mesocosm experiment that photoconversion in vivo is entirely dependent on near-UV wavelengths. However, a near-UV intensity equivalent to the mesophotic underwater light field at 80 m depth is sufficient to drive the process in vitro, suggesting that photoconversion can occur near the lower distribution limits of these corals. Furthermore, live coral colonies showed evidence of efficient Förster Resonance Energy Transfer (FRET). Our simulated mesophotic light field maintained the pcRFP pool in a partially photoconverted state in vivo, maximising intra-tetrameric FRET and creating a long-range wavelength conversion system with higher quantum yield than other native RFPs. We hypothesise that efficient conversion of blue wavelengths, abundant at depth, into orange-red light could constitute an adaptation of corals to life in light-limited environments. Full article
(This article belongs to the Special Issue Fluorescent Proteins)
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Open AccessArticle
Interaction of Biliverdin Chromophore with Near-Infrared Fluorescent Protein BphP1-FP Engineered from Bacterial Phytochrome
Int. J. Mol. Sci. 2017, 18(5), 1009; https://doi.org/10.3390/ijms18051009
Received: 12 April 2017 / Revised: 30 April 2017 / Accepted: 4 May 2017 / Published: 8 May 2017
Cited by 3 | PDF Full-text (4765 KB) | HTML Full-text | XML Full-text
Abstract
Near-infrared (NIR) fluorescent proteins (FPs) designed from PAS (Per-ARNT-Sim repeats) and GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA transcriptional activator) domains of bacterial phytochromes covalently bind biliverdin (BV) chromophore via one or two Cys residues. We studied BV interaction with a series of NIR FP variants [...] Read more.
Near-infrared (NIR) fluorescent proteins (FPs) designed from PAS (Per-ARNT-Sim repeats) and GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA transcriptional activator) domains of bacterial phytochromes covalently bind biliverdin (BV) chromophore via one or two Cys residues. We studied BV interaction with a series of NIR FP variants derived from the recently reported BphP1-FP protein. The latter was engineered from a bacterial phytochrome RpBphP1, and has two reactive Cys residues (Cys15 in the PAS domain and Cys256 in the GAF domain), whereas its mutants contain single Cys residues either in the PAS domain or in the GAF domain, or no Cys residues. We characterized BphP1-FP and its mutants biochemically and spectroscopically in the absence and in the presence of denaturant. We found that all BphP1-FP variants are monomers. We revealed that spectral properties of the BphP1-FP variants containing either Cys15 or Cys256, or both, are determined by the covalently bound BV chromophore only. Consequently, this suggests an involvement of the inter-monomeric allosteric effects in the BV interaction with monomers in dimeric NIR FPs, such as iRFPs. Likely, insertion of the Cys15 residue, in addition to the Cys256 residue, in dimeric NIR FPs influences BV binding by promoting the BV chromophore covalent cross-linking to both PAS and GAF domains. Full article
(This article belongs to the Special Issue Fluorescent Proteins)
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Review

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Open AccessReview
Fluorescent Proteins for Investigating Biological Events in Acidic Environments
Int. J. Mol. Sci. 2018, 19(6), 1548; https://doi.org/10.3390/ijms19061548
Received: 2 May 2018 / Revised: 18 May 2018 / Accepted: 19 May 2018 / Published: 23 May 2018
Cited by 4 | PDF Full-text (2271 KB) | HTML Full-text | XML Full-text
Abstract
The interior lumen of acidic organelles (e.g., endosomes, secretory granules, lysosomes and plant vacuoles) is an important platform for modification, transport and degradation of biomolecules as well as signal transduction, which remains challenging to investigate using conventional fluorescent proteins (FPs). Due to the [...] Read more.
The interior lumen of acidic organelles (e.g., endosomes, secretory granules, lysosomes and plant vacuoles) is an important platform for modification, transport and degradation of biomolecules as well as signal transduction, which remains challenging to investigate using conventional fluorescent proteins (FPs). Due to the highly acidic luminal environment (pH ~ 4.5–6.0), most FPs and related sensors are apt to lose their fluorescence. To address the need to image in acidic environments, several research groups have developed acid-tolerant FPs in a wide color range. Furthermore, the engineering of pH insensitive sensors, and their concomitant use with pH sensitive sensors for the purpose of pH-calibration has enabled characterization of the role of luminal ions. In this short review, we summarize the recent development of acid-tolerant FPs and related functional sensors and discuss the future prospects for this field. Full article
(This article belongs to the Special Issue Fluorescent Proteins)
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Open AccessReview
Fluorogenic Labeling Strategies for Biological Imaging
Int. J. Mol. Sci. 2017, 18(7), 1473; https://doi.org/10.3390/ijms18071473
Received: 21 June 2017 / Revised: 3 July 2017 / Accepted: 6 July 2017 / Published: 9 July 2017
Cited by 25 | PDF Full-text (1572 KB) | HTML Full-text | XML Full-text
Abstract
The spatiotemporal fluorescence imaging of biological processes requires effective tools to label intracellular biomolecules in living systems. This review presents a brief overview of recent labeling strategies that permits one to make protein and RNA strongly fluorescent using synthetic fluorogenic probes. Genetically encoded [...] Read more.
The spatiotemporal fluorescence imaging of biological processes requires effective tools to label intracellular biomolecules in living systems. This review presents a brief overview of recent labeling strategies that permits one to make protein and RNA strongly fluorescent using synthetic fluorogenic probes. Genetically encoded tags selectively binding the exogenously applied molecules ensure high labeling selectivity, while high imaging contrast is achieved using fluorogenic chromophores that are fluorescent only when bound to their cognate tag, and are otherwise dark. Beyond avoiding the need for removal of unbound synthetic dyes, these approaches allow the development of sophisticated imaging assays, and open exciting prospects for advanced imaging, particularly for multiplexed imaging and super-resolution microscopy. Full article
(This article belongs to the Special Issue Fluorescent Proteins)
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Open AccessReview
Deciphering Structural Photophysics of Fluorescent Proteins by Kinetic Crystallography
Int. J. Mol. Sci. 2017, 18(6), 1187; https://doi.org/10.3390/ijms18061187
Received: 25 April 2017 / Revised: 24 May 2017 / Accepted: 26 May 2017 / Published: 2 June 2017
Cited by 5 | PDF Full-text (7200 KB) | HTML Full-text | XML Full-text
Abstract
Because they enable labeling of biological samples in a genetically-encoded manner, Fluorescent Proteins (FPs) have revolutionized life sciences. Photo-transformable fluorescent proteins (PTFPs), in particular, recently attracted wide interest, as their fluorescence state can be actively modulated by light, a property central to the [...] Read more.
Because they enable labeling of biological samples in a genetically-encoded manner, Fluorescent Proteins (FPs) have revolutionized life sciences. Photo-transformable fluorescent proteins (PTFPs), in particular, recently attracted wide interest, as their fluorescence state can be actively modulated by light, a property central to the emergence of super-resolution microscopy. PTFPs, however, exhibit highly complex photophysical behaviours that are still poorly understood, hampering the rational engineering of variants with improved performances. We show that kinetic crystallography combined with in crystallo optical spectroscopy, modeling approaches and single-molecule measurements constitutes a powerful tool to decipher processes such as photoactivation, photoconversion, photoswitching, photoblinking and photobleaching. Besides potential applications for the design of enhanced PTFPs, these investigations provide fundamental insight into photoactivated protein dynamics. Full article
(This article belongs to the Special Issue Fluorescent Proteins)
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Other

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Open AccessPerspective
Photoconvertible Fluorescent Proteins and the Role of Dynamics in Protein Evolution
Int. J. Mol. Sci. 2017, 18(8), 1792; https://doi.org/10.3390/ijms18081792
Received: 18 July 2017 / Revised: 11 August 2017 / Accepted: 17 August 2017 / Published: 18 August 2017
PDF Full-text (2278 KB) | HTML Full-text | XML Full-text
Abstract
Photoconvertible fluorescent proteins (pcFPs) constitute a large group of fluorescent proteins related to green fluorescent protein (GFP) that, when exposed to blue light, bear the capability of irreversibly switching their emission color from green to red. Not surprisingly, this fascinating class of FPs [...] Read more.
Photoconvertible fluorescent proteins (pcFPs) constitute a large group of fluorescent proteins related to green fluorescent protein (GFP) that, when exposed to blue light, bear the capability of irreversibly switching their emission color from green to red. Not surprisingly, this fascinating class of FPs has found numerous applications, in particular for the visualization of biological processes. A detailed understanding of the photoconversion mechanism appears indispensable in the design of improved variants for applications such as super-resolution imaging. In this article, recent work is reviewed that involves using pcFPs as a model system for studying protein dynamics. Evidence has been provided that the evolution of pcFPs from a green ancestor involved the natural selection for altered dynamical features of the beta-barrel fold. It appears that photoconversion may be the outcome of a long-range positional shift of a fold-anchoring region. A relatively stiff, rigid element appears to have migrated away from the chromophore-bearing section to the opposite edge of the barrel, thereby endowing pcFPs with increased active site flexibility while keeping the fold intact. In this way, the stage was set for the coupling of light absorption with subsequent chemical transformations. The emerging mechanistic model suggests that highly specific dynamic motions are linked to key chemical steps, preparing the system for a concerted deprotonation and β-elimination reaction that enlarges the chromophore’s π-conjugation to generate red color. Full article
(This article belongs to the Special Issue Fluorescent Proteins)
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