International Journal of Molecular Sciences

Research

13 pages, 4223 KiB

Open AccessArticle

Fluorescence Properties of a Novel Cyanobacteriochrome GAF Domain from Spirulina that Exhibits Moderate Dark Reversion

by Xian-Jun Wu, Hong Yang, Yi Sheng, Yong-Li Zhu and Ping-Ping Li

Int. J. Mol. Sci. 2018, 19(8), 2253; https://doi.org/10.3390/ijms19082253 - 1 Aug 2018

Cited by 10 | Viewed by 4378

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 (t_1/2 = 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 (t_1/2 = 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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10 pages, 6522 KiB

Open AccessArticle

Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense

by Yuki Horiuchi, Danai Laskaratou, Michel Sliwa, Cyril Ruckebusch, Kuniyuki Hatori, Hideaki Mizuno and Jun-ichi Hotta

Int. J. Mol. Sci. 2018, 19(2), 371; https://doi.org/10.3390/ijms19020371 - 26 Jan 2018

Cited by 2 | Viewed by 5814

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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11318 KiB

Open AccessArticle

Assessing the Effectiveness of a Far-Red Fluorescent Reporter for Tracking Stem Cells In Vivo

by Jing Zhou, Jack Sharkey, Rajeev Shukla, Antonius Plagge and Patricia Murray

Int. J. Mol. Sci. 2018, 19(1), 19; https://doi.org/10.3390/ijms19010019 - 22 Dec 2017

Cited by 8 | Viewed by 6125

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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3207 KiB

Open AccessArticle

Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

by Thijs Roebroek, Sam Duwé, Wim Vandenberg and Peter Dedecker

Int. J. Mol. Sci. 2017, 18(9), 2015; https://doi.org/10.3390/ijms18092015 - 20 Sep 2017

Cited by 15 | Viewed by 7076

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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3470 KiB

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

by Christopher D. M. Hutchison, Violeta Cordon-Preciado, Rhodri M. L. Morgan, Takanori Nakane, Josie Ferreira, Gabriel Dorlhiac, Alvaro Sanchez-Gonzalez, Allan S. Johnson, Ann Fitzpatrick, Clyde Fare, Jon P. Marangos, Chun Hong Yoon, Mark S. Hunter, Daniel P. DePonte, Sébastien Boutet, Shigeki Owada, Rie Tanaka, Kensuke Tono, So Iwata and Jasper J. Van Thor

Int. J. Mol. Sci. 2017, 18(9), 1918; https://doi.org/10.3390/ijms18091918 - 7 Sep 2017

Cited by 14 | Viewed by 6137

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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6819 KiB

Open AccessArticle

Colorful Packages: Encapsulation of Fluorescent Proteins in Complex Coacervate Core Micelles

by Antsje Nolles, Adrie H. Westphal, J. Mieke Kleijn, Willem J. H. Van Berkel and Jan Willem Borst

Int. J. Mol. Sci. 2017, 18(7), 1557; https://doi.org/10.3390/ijms18071557 - 19 Jul 2017

Cited by 12 | Viewed by 7981

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 (P2MVP_n-b-PEO_m) 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 pK_a shift of the chromophore (FP variant mCherry). Full article

(This article belongs to the Special Issue Fluorescent Proteins)

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4211 KiB

Open AccessArticle

Combining Primed Photoconversion and UV-Photoactivation for Aberration-Free, Live-Cell Compliant Multi-Color Single-Molecule Localization Microscopy Imaging

by David Virant, Bartosz Turkowyd, Alexander Balinovic and Ulrike Endesfelder

Int. J. Mol. Sci. 2017, 18(7), 1524; https://doi.org/10.3390/ijms18071524 - 14 Jul 2017

Cited by 18 | Viewed by 9654

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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17385 KiB

Open AccessArticle

Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models

by Natalia V. Povarova, Natalia D. Petri, Anna E. Blokhina, Alexey M. Bogdanov, Nadya G. Gurskaya and Konstantin A. Lukyanov

Int. J. Mol. Sci. 2017, 18(7), 1503; https://doi.org/10.3390/ijms18071503 - 12 Jul 2017

Cited by 6 | Viewed by 6151

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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4134 KiB

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

by Elena Bollati, Daniel Plimmer, Cecilia D’Angelo and Jörg Wiedenmann

Int. J. Mol. Sci. 2017, 18(7), 1174; https://doi.org/10.3390/ijms18071174 - 4 Jul 2017

Cited by 11 | Viewed by 7888

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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4765 KiB

Open AccessArticle

Interaction of Biliverdin Chromophore with Near-Infrared Fluorescent Protein BphP1-FP Engineered from Bacterial Phytochrome

by Olesya V. Stepanenko, Olga V. Stepanenko, Irina M. Kuznetsova, Daria M. Shcherbakova, Vladislav V. Verkhusha and Konstantin K. Turoverov

Int. J. Mol. Sci. 2017, 18(5), 1009; https://doi.org/10.3390/ijms18051009 - 8 May 2017

Cited by 12 | Viewed by 7293

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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19 pages, 2271 KiB

Open AccessReview

Fluorescent Proteins for Investigating Biological Events in Acidic Environments

by Hajime Shinoda, Michael Shannon and Takeharu Nagai

Int. J. Mol. Sci. 2018, 19(6), 1548; https://doi.org/10.3390/ijms19061548 - 23 May 2018

Cited by 72 | Viewed by 9895

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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1572 KiB

Open AccessReview

Fluorogenic Labeling Strategies for Biological Imaging

by Chenge Li, Alison G. Tebo and Arnaud Gautier

Int. J. Mol. Sci. 2017, 18(7), 1473; https://doi.org/10.3390/ijms18071473 - 9 Jul 2017

Cited by 66 | Viewed by 10785

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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7200 KiB

Open AccessReview

Deciphering Structural Photophysics of Fluorescent Proteins by Kinetic Crystallography

by Dominique Bourgeois

Int. J. Mol. Sci. 2017, 18(6), 1187; https://doi.org/10.3390/ijms18061187 - 2 Jun 2017

Cited by 12 | Viewed by 5779

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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2278 KiB

Open AccessPerspective

Photoconvertible Fluorescent Proteins and the Role of Dynamics in Protein Evolution

by Rebekka M. Wachter

Int. J. Mol. Sci. 2017, 18(8), 1792; https://doi.org/10.3390/ijms18081792 - 18 Aug 2017

Cited by 17 | Viewed by 6614

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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