A Monomeric Photoconvertible Fluorescent Protein for Imaging of Dynamic Protein Localization

The use of green-to-red photoconvertible fluorescent proteins (FPs) enables researchers to highlight a subcellular population of a fusion protein of interest and to image its dynamics in live cells. In an effort to enrich the arsenal of photoconvertible FPs and to overcome the limitations imposed by the oligomeric structure of natural photoconvertible FPs, we designed and optimized a new monomeric photoconvertible FP. Using monomeric versions of Clavularia sp. cyan FP as template, we employed sequence-alignment-guided design to create a chromophore environment analogous to that shared by known photoconvertible FPs. The designed gene was synthesized and, when expressed in Escherichia coli, found to produce green fluorescent colonies that gradually switched to red after exposure to white light. We subjected this first-generation FP [named mClavGR1 (monomeric Clavularia-derived green-to-red photoconvertible 1)] to a combination of random and targeted mutageneses and screened libraries for efficient photoconversion using a custom-built system for illuminating a 10-cm Petri plate with 405-nm light. Following more than 15 rounds of library creation and screening, we settled on an optimized version, known as mClavGR2, that has eight mutations relative to mClavGR1. Key improvements of mClavGR2 relative to mClavGR1 include a 1.4-fold brighter red species, 1.8-fold higher photoconversion contrast, and dramatically improved chromophore maturation in E. coli. The monomeric status of mClavGR2 has been demonstrated by gel-filtration chromatography and the functional expression of a variety of mClavGR2 chimeras in mammalian cells. Furthermore, we have exploited mClavGR2 to determine the diffusion kinetics of the membrane protein intercellular adhesion molecule 1 both when the membrane is in contact with a T-lymphocyte expressing leukocyte-function-associated antigen 1 and when it is not. These experiments clearly establish that mClavGR2 is well suited for rapid photoconversion of protein subpopulations and subsequent tracking of dynamic changes in localization in living cells.     

Semi-rational engineering of a coral fluorescent protein into an efficient highlighter

Kaede is a natural photoconvertible fluorescent protein found in the coral Trachyphyllia geoffroyi. It contains a tripeptide, His 62-Tyr 63-Gly 64, which acts as a green chromophore that is photoconvertible to red following (ultra-) violet irradiation. Here, we report the molecular cloning and crystal structure determination of a new fluorescent protein, KikG, from the coral Favia favus, and its in vitro evolution conferring green-to-red photoconvertibility. Substitution of the His 62-Tyr 63-Gly 64 sequence into the native protein provided only negligible photoconversion. On the basis of the crystal structure, semi-rational mutagenesis of the amino acids surrounding the chromophore was performed, leading to the generation of an efficient highlighter, KikGR. Within mammalian cells, KikGR is more efficiently photoconverted and is several-fold brighter in both the green and red states than Kaede. In addition, KikGR was successfully photoconverted using two-photon excitation microscopy at 760 nm, ensuring optical cell labelling with better spatial discrimination in thick and highly scattering tissues.     

Reversible photoswitching in fluorescent proteins: a mechanistic view

Phototransformable fluorescent proteins (FPs) have received considerable attention in recent years, because they enable many new exciting modalities in fluorescence microscopy and biotechnology. On illumination with proper actinic light, phototransformable FPs are amenable to long-lived transitions between various fluorescent or nonfluorescent states, resulting in processes known as photoactivation, photoconversion, or photoswitching. Here, we review the subclass of photoswitchable FPs with a mechanistic perspective. These proteins offer the widest range of practical applications, including reversible high-density data bio-storage, photochromic FRET, and super-resolution microscopy by either point-scanning, structured illumination, or single molecule-based wide-field approaches. Photoswitching can be engineered to occur with high contrast in both Hydrozoan and Anthozoan FPs and typically results from a combination of chromophore cis-trans isomerization and protonation change. However, other switching schemes based on, for example, chromophore hydration/dehydration have been discovered, and it seems clear that ever more performant variants will be developed in the future.     

A structural basis for reversible photoswitching of absorbance spectra in red fluorescent protein rsTagRFP

rsTagRFP is the first monomeric red fluorescent protein (FP) with reversibly photoswitchable absorbance spectra. The switching is realized by irradiation of rsTagRFP with blue (440 nm) and yellow (567 nm) light, turning the protein fluorescence ON and OFF, respectively. It is perhaps the most useful probe in this color class that has yet been reported. Because of the photoswitchable absorbance, rsTagRFP can be used as an acceptor in photochromic Förster resonance energy transfer. Yellow FPs, YPet and mVenus, are demonstrated to be excellent photochromic Förster resonance energy transfer donors for the rsTagRFP acceptor in its fusion constructs. Analysis of X-ray structures has shown that photoswitching of rsTagRFP is accompanied by cis–trans isomerization and protonation/deprotonation of the chromophore, with the deprotonated cis- and protonated trans-isomers corresponding to its ON and OFF states, respectively. Unlike in other photoswitchable FPs, both conformers of rsTagRFP chromophore are essentially coplanar. Two other peculiarities of the rsTagRFP chromophore are an essentially hydrophobic environment of its p-hydroxyphenyl site and the absence of direct hydrogen bonding between this moiety and the protein scaffold. The influence of the immediate environment on rsTagRFP chromophore was probed by site-directed mutagenesis. Residues Glu145 and His197 were found to participate in protonation/deprotonation of the chromophore accompanying the photoswitching of rsTagRFP fluorescence, whereas residues Met160 and Leu174 were shown to spatially restrict chromophore isomerization, favoring its radiative decay.     

Modern fluorescent proteins: from chromophore formation to novel intracellular applications

The diverse biochemical and photophysical properties of fluorescent proteins (FPs) have enabled the generation of a growing palette of colors, providing unique opportunities for their use in a variety of modern biology applications. Modulation of these FP characteristics is achieved through diversity in both the structure of the chromophore as well as the contacts between the chromophore and the surrounding protein barrel. Here we review our current knowledge of blue, green, and red chromophore formation in permanently emitting FPs, photoactivatable FPs, and fluorescent timers. Progress in understanding the interplay between FP structure and function has allowed the engineering of FPs with many desirable features, and enabled recent advances in microscopy techniques such as super-resolution imaging of single molecules, imaging of protein dynamics, photochromic FRET, deep-tissue imaging, and multicolor two-photon microscopy in live animals.     

Crystallographic evidence for water-assisted photo-induced peptide cleavage in the stony coral fluorescent protein Kaede

A coral fluorescent protein from Trachyphyllia geoffroyi, Kaede, possesses a tripeptide of His62-Tyr63-Gly64, which forms a chromophore with green fluorescence. This chromophore's fluorescence turns red following UV light irradiation. We have previously shown that such photoconversion is achieved by a formal beta-elimination reaction, which results in a cleavage of the peptide bond found between the amide nitrogen and the alpha-carbon at His62. However, the stereochemical arrangement of the chromophore and the precise structural basis for this reaction mechanism previously remained unknown. Here, we report the crystal structures of the green and red form of Kaede at 1.4 A and 1.6 A resolutions, respectively. Our structures depict the cleaved peptide bond in the red form. The chromophore conformations both in the green and red forms are similar, except a well-defined water molecule in the proximity of the His62 imidazole ring in the green form. We propose a molecular mechanism for green-to-red photoconversion, which is assisted by the water molecule.     

X-Ray Crystal Structure and Properties of Phanta,a Weakly Fluorescent Photochromic GFP-Like Protein

Phanta is a reversibly photoswitching chromoprotein (Φ_F, 0.003), useful for pcFRET, that was isolated from a mutagenesis screen of the bright green fluorescent eCGP123 (Φ_F, 0.8). We have investigated the contribution of substitutions at positions His193, Thr69 and Gln62, individually and in combination, to the optical properties of Phanta. Single amino acid substitutions at position 193 resulted in proteins with very low Φ_F, indicating the importance of this position in controlling the fluorescence efficiency of the variant proteins. The substitution Thr69Val in Phanta was important for supressing the formation of a protonated chromophore species observed in some His193 substituted variants, whereas the substitution Gln62Met did not significantly contribute to the useful optical properties of Phanta. X-ray crystal structures for Phanta (2.3 Å), eCGP123^T69V (2.0 Å) and eCGP123^H193Q (2.2 Å) in their non-photoswitched state were determined, revealing the presence of a cis-coplanar chromophore. We conclude that changes in the hydrogen-bonding network supporting the cis-chromophore, and its contacts with the surrounding protein matrix, are responsible for the low fluorescence emission of eCGP123 variants containing a His193 substitution.     

绿色荧光蛋白及其应用

随着对绿色荧光蛋白(green fluorescent protein,GFP)研究的不断深入,人们对其结构、荧光产生机理等已有较为全面的认识。近年来利用GFP及其它荧光蛋白(FPs)发展了诸如荧光互补技术(FC)、荧光共振能量转移技术(FRET)和超分辨成像(super-resolution imaging)等一系列新技术,极大地促进了生物学、医药科学的研究。主要介绍了荧光蛋白的结构,荧光产生的机理,不同类型的荧光蛋白和基于荧光蛋白产生的新技术等方面的最新研究进展。     

Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein

Green fluorescent protein from the jellyfish (Aequorea GFP) and GFP-like proteins from coral species encode light-absorbing chromophores within their protein sequences. A coral fluorescent protein, Kaede, contains a tripeptide, His(62)-Tyr(63)-Gly(64), which acts as a green chromophore that is photoconverted to red. Here, we present the structural basis for the green-to-red photoconversion. As in Aequorea GFP, a chromophore, 4-(p-hydroxybenzylidene)-5-imidazolinone, derived from the tripeptide mediates green fluorescence in Kaede. UV irradiation causes an unconventional cleavage within Kaede protein between the amide nitrogen and the alpha carbon (Calpha) at His(62) via a formal beta-elimination reaction, which requires the whole, intact protein for its catalysis. The subsequent formation of a double bond between His(62)-Calpha and -Cbeta extends the pi-conjugation to the imidazole ring of His(62), creating a new red-emitting chromophore, 2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolinone. The present study not only reveals diversity in the chemical structure of fluorescent proteins but also adds a new dimension to posttranslational modification mechanisms.     

Ultrafast Photoconversion of the Green Fluorescent Protein Studied by Accumulative Femtosecond Spectroscopy

The irreversible photoconversion of T203V green fluorescent protein (GFP) via decarboxylation is studied under femtosecond excitation using an accumulative product detection method that allows us to measure small conversion efficiencies of down to ΔOD = 10⁻⁷ absorbance change per pulse. Power studies with 800- and 400-nm pulse excitation reveal that excitation to higher states of the neutral form of the GFP chromophore induces photoconversion very efficiently. The singly excited neutral chromophore is a resonant intermediate of the two-step excitation process that leads to efficient photoconversion. We determine the dynamics of this two-step process by separating the excitation step of the neutral chromophore from the further excitation step to the reactive state in a time-resolved two-color experiment. The dynamics show that a further excitation to the very reactive higher excited state is only possible from the initially excited neutral chromophore and not from the fluorescent intermediate state. For applications of GFP in two-photon fluorescence microscopy, the found photochemical behavior implies that the high intensity conditions used in microscopy can lead to photoconversion easily and care has to be taken to avoid unwanted photoconversion.     

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state¹ and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish^2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.     

Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging

The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 A crystal structure (1 A=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.     

Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells

The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for fluorescence (or F?rster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, the potential for photoconversion artifacts and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs.     

Genetically encoded optical sensors of neuronal activity and cellular function

Fluorescent proteins (FPs) have been engineered to produce an optical report in response to cellular signals. FP fluorescence can be made directly sensitive to the chemical environment, via specific mutations of or around the chromophore. Alternatively, FPs can be made indirectly sensitive to cellular signals by their fusion to 'detector' proteins that respond to specific cellular signals with structural rearrangements that act on the FP to alter fluorescence. These optical sensors of membrane voltage, neurotransmitter release, and intracellular messengers, including powerful new sensors of Ca(2+), cyclic nucleotides and nitric oxide, are likely to provide new insights into the workings of cellular signals and of information processing in neural circuits.     

Structural characterization of the photoswitchable fluorescent protein Dronpa-C62S

The photoswitching behavior of green fluorescent proteins (GFPs) or GFP-like proteins is increasingly recognized as a new technique for optical marking. Recently, Ando and his colleagues developed a new green fluorescent protein Dronpa, which possesses the unique photochromic property of being photoswitchable in a non-destructive manner. To better understand this mechanism, we determined the crystal structures of a new GFP Dronpa and its mutant C62S, at 1.9 Angstroms and 1.8 Angstroms, respectively. Determination of the structures demonstrates that a unique hydrogen-bonding network and the sulfur atom of the chromophore are critical to the photoswitching property of Dronpa. Reversible photoswitching was lost in cells expressing the Dronpa-C62S upon repetitive irradiation compared to the native protein. Structural and mutational analyses reveal the chemical basis for the functional properties of photoswitchable fluorescent proteins and provide the basis for subsequent coherent engineering of this subfamily of Dronpa homologs.     

Color recovery after photoconversion of H2B::mEosFP allows detection of increased nuclear DNA content in developing plant cells

Many higher plants are polysomatic whereby different cells possess variable amounts of nuclear DNA. The conditional triggering of endocycles results in higher nuclear DNA content (C value) that in some cases has been correlated to increased cell size. While numerous multicolored fluorescent protein (FP) probes have revealed the general behavior of the nucleus and intranuclear components, direct visualization and estimation of changes in nuclear-DNA content in live cells during their development has not been possible. Recently, monomeric Eos fluorescent protein (mEosFP) has emerged as a useful photoconvertible protein whose color changes irreversibly from a green to a red fluorescent form upon exposure to violet-blue light. The stability and irreversibility of red fluorescent mEosFP suggests that detection of green color recovery would be possible as fresh mEosFP is produced after photoconversion. Thus a ratiometric evaluation of the red and green forms of mEosFP following photoconversion could be used to estimate production of a core histone such as H2B during its concomitant synthesis with DNA in the synthesis phase of the cell cycle. Here we present proof of concept observations on transgenic tobacco (Nicotiana tabacum) Bright Yellow 2 cells and Arabidopsis (Arabidopsis thaliana) plants stably expressing H2B::mEosFP. In Arabidopsis seedlings an increase in green fluorescence is observed specifically in cells known to undergo endoreduplication. The detection of changes in nuclear DNA content by correlating color recovery of H2B::mEosFP after photoconversion is a novel approach involving a single FP. The method has potential for facilitating detailed investigations on conditions that favor increased cell size and the development of polysomaty in plants.     

The 1.7 A crystal structure of Dronpa: a photoswitchable green fluorescent protein

The green fluorescent protein (GFP), its variants, and the closely related GFP-like proteins possess a wide variety of spectral properties that are of widespread interest as biological tools. One desirable spectral property, termed photoswitching, involves the light-induced alteration of the optical properties of certain GFP members. Although the structural basis of both reversible and irreversible photoswitching events have begun to be unraveled, the mechanisms resulting in reversible photoswitching are less clear. A novel GFP-like protein, Dronpa, was identified to have remarkable light-induced photoswitching properties, maintaining an almost perfect reversible photochromic behavior with a high fluorescence to dark state ratio. We have crystallized and subsequently determined to 1.7 A resolution the crystal structure of the fluorescent state of Dronpa. The chromophore was observed to be in its anionic form, adopting a cis co-planar conformation. Comparative structural analysis of non-photoactivatable and photoactivatable GFPs, together with site-directed mutagenesis of a position (Cys62) within the Dronpa chromophore, has provided a basis for understanding Dronpa photoactivation. Specifically, we propose a model of reversible photoactivation whereby irradiation with light leads to subtle conformational changes within and around the environment of the chromophore that promotes proton transfer along an intricate polar network.     

Design of small monomeric and highly bright near-infrared fluorescent proteins

Due to the low absorbance in the far-red (FR) and near-infrared (NIR) “optical window”, NIR fluorescent proteins (FPs) are powerful tools for deep imaging. Here, we report three new, highly bright NIR FPs termed BDFP1.8, BDFP1.8:1.8 (tandem BDFP1.8) and BDFP1.9, which evolved from a previously reported FR FP, BDFP1.6: a derivative of ApcF2 from Chroococcidiopsis thermalis sp. PCC7203. ApcF2 binds phycocyanobilin (PCB) non-covalently, while BDFPs, the derivatives of ApcF2, can bind biliverdin (BV) covalently. We identified that dimeric BDFP1.8 and monomeric BDFP1.8:1.8 have a 2.4-and 4.4-fold higher effective brightness, respectively, than iRFP720, which has the highest effective brightness among the reported NIR FPs. Monomeric DBFP1.9 (17 kDa) has one of the smallest masses among highly bright FPs in the FR and NIR regions. Enhancing the affinity between the apo-proteins and the BV chromophore is an effective method to improve the effective brightness of biliprotein FPs. Moreover, BDFP1.8 and 1.9 exhibit higher stability to temperature, pH and light than iRFP720. Finally, the highly bright NIR BDFP1.8 together with FR BDFP1.6 could effectively biolabel cells in dual colors.     

Structure‐guided evolution of Green2 toward photostability and quantum yield enhancement by F145Y substitution

Quantum yield is a determinant for fluorescent protein (FP) applications and enhancing FP brightness through gene engineering is still a challenge. Green2, our de novo FP synthesized by microfluidic picoarray and cloning, has a significantly lower quantum yield than enhanced green FP, though they have high homology and share the same chromophore. To increase its quantum yield, we introduced an F145Y substitution into Green2 based on rational structural analysis. Y145 significantly increased the quantum yield (0.22 vs. 0.18) and improved the photostability (t_1/2, 73.0 s vs. 46.0 s), but did not affect the excitation and emission spectra. Further structural analysis showed that the F145Y substitution resulted in a significant electrical field change in the immediate environment of the chromophore. The perturbation of electrostatic charge around the chromophore lead to energy barrier changes between the ground and excited states, which resulted in the enhancement of quantum yield and photostability. Our results illustrate a typical example of engineering an FP based solely on fluorescence efficiency optimization and provide novel insights into the rational evolution of FPs.