Unique interactions between the chromophore and glutamate 16 lead to far‐red emission in a red fluorescent protein

mPlum is a far‐red fluorescent protein with emission maximum at ～650 nm and was derived by directed evolution from DsRed. Two residues near the chromophore, Glu16 and Ile65, were previously revealed to be indispensable for the far‐red emission. Ultrafast time‐resolved fluorescence emission studies revealed a time dependent shift in the emission maximum, initially about 625 nm, to about 650 nm over a period of 500 ps. This observation was attributed to rapid reorganization of the residues solvating the chromophore within mPlum. Here, the crystal structure of mPlum is described and compared with those of two blue shifted mutants mPlum‐E16Q and ‐I65L. The results suggest that both the identity and precise orientation of residue 16, which forms a unique hydrogen bond with the chromophore, are required for far‐red emission. Both the far‐red emission and the time dependent shift in emission maximum are proposed to result from the interaction between the chromophore and Glu16. Our findings suggest that significant red shifts might be achieved in other fluorescent proteins using the strategy that led to the discovery of mPlum.     

A new colorimetric and far‐red fluorescent probe for hydrazine with a large red‐shifted absorption spectrum

Recently, growing attention has been paid to the detection of hydrazine (NH₂NH₂) because of its important roles in industrial chemical and high toxicity to human beings. Herein, we have constructed a new colorimetric and far‐red fluorescent probe containing a receptor of 4‐bromobutanoate to selectively detect hydrazine. The probe could detect hydrazine quantitatively in the range of 40–500 μM with the detection limit of 2.9 μM. In addition, the probe could monitor hydrazine by the ratiometric method with a large (185 nm) red‐shifted absorption spectrum, and the color changes from yellow to blue make it as a ‘naked‐eye’ indicator for hydrazine. Consequently, our proposed probe would be of great benefit for monitoring hydrazine in aqueous solution.     

Rhodol‐based far‐red fluorescent probe for the detection of cysteine and homocysteine over glutathione

The simultaneous discrimination of cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) is of great importance due to their roles in biology and close link to many diseases, especially via the development of a far‐red fluorescent probe that could be used for rapid, selective, and sensitive detection of all three. Herein, we report the characterization of a far‐red fluorescent probe with turn‐on fluorescence properties and visible color changes that could be used for the detection of cysteine and homocysteine over glutathione. In this study we found that the sensor could discriminate cysteine and homocysteine over glutathione within 20 min. Function of this probe was based on the conjugate addition–cyclization reaction and showed a low detection limit to cysteine and homocysteine. Upon the addition of cysteine and homocysteine, the absorption band at 592 nm rose gradually and fluorescence was detected at 645 nm. The color changed from colorless to blue and fluorescence changed from absent to strong red fluorescence, which could be differentiated by the naked eye. All these unique features make this probe particularly potentially favorable for use in cysteine/homocysteine sensing and bioimaging applications. Copyright © 2016 John Wiley & Sons, Ltd.     

Structure‐based analysis and evolution of a monomerized red‐colored chromoprotein from the Olindias formosa jellyfish

GFP‐like chromoproteins (CPs) with non‐fluorescence ability have been used as bioimaging probes. Existing CPs have voids in the optical absorption window which limits their extensibility. The development of new CP color is therefore ongoing. Here, we cloned CPs from the jellyfish, Olindias formosa, and developed a completely non‐fluorescent monomeric red CP, R‐Velour, with an absorption peak at 528 nm. To analyze the photophysical properties from a structural aspect, we determined the crystal structure of R‐Velour at a 2.1 Å resolution. R‐Velour has a trans‐chromophore similar to the green fluorescence protein, Gamillus, derived from the same jellyfish. However, in contrast to the two coplanar chromophoric rings in Gamillus, R‐Velour has a large torsion inducing non‐fluorescence property. Through site‐directed mutagenesis, we surveyed residues surrounding the chromophore and found a key residue, Ser155, which contributes to the generation of four‐color variants with the bathochromic and hypsochromic shift of the absorption peak, ranging from 506 to 554 nm. The recently proposed spectrum shift theory, based on the Marcus–Hush model, supports the spectrum shift of these mutants. These findings may support further development of R‐Velour variants with useful absorption characteristics for bioimaging, including fluorescence lifetime imaging and photoacoustic imaging.     

The whither of bacteriophytochrome‐based near‐infrared fluorescent proteins: Insights from two‐photon absorption spectroscopy

We present one‐ and two‐photon‐absorption fluorescence spectroscopic analysis of biliverdin (BV) chromophore–based single‐domain near‐infrared fluorescent proteins (iRFPs). The results of these studies are used to estimate the internal electric fields acting on BV inside iRFPs and quantify the electric dipole properties of this chromophore, defining the red shift of excitation and emission spectra of BV‐based iRFPs. The iRFP studied in this work is shown to fit well the global diagram of the red‐shift tunability of currently available BV‐based iRFPs as dictated by the quadratic Stark effect, suggesting the existence of the lower bound for the strongest red shifts attainable within this family of fluorescent proteins. The absolute value of the two‐photon absorption (TPA) cross section of a fluorescent calcium sensor based on the studied iRFP is found to be significantly larger than the TPA cross sections of other widely used genetically encodable fluorescent calcium sensors.      

Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs

Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His-Tyr-Gly chromophore-forming triad. Here, we describe synthesis of Kaede red chromophore (2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His-Tyr-Gly, Trp-Tyr-Gly, Phe-Trp-Gly, Tyr-Trp-Gly, Asn-Tyr-Gly, Phe-Tyr-Gly, and Tyr-Tyr-Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595 nm was found for the chromophore Trp-Tyr-Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore Asn-Tyr-Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.     

Rational design of a monomeric and photostable far‐red fluorescent protein for fluorescence imaging in vivo

Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue‐shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP‐labeled nucleus and tdTomato‐labeled plasma membrane, time‐lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two‐color protein labeling in living cells and in two‐color tumor labeling in mice.     

The utility of far‐infrared illumination in oxygenation dynamics as measured with near‐infrared spectroscopy

Near‐infrared spectroscopy (NIRS) is a noninvasive method for measuring the oxygenation in muscle and other tissues in vivo. For quantitative NIRS measurement of oxygenation dynamics, the vessel‐occlusion test was usually applied as physiological intervention. There are several drawbacks of the vessel‐occlusion method that include skin contact, uncomfortable and microcirculation block of patients. Thus, we propose the far‐infrared (FIR) illumination as a new physiological intervention method in this paper. Our preliminary result shows a linear correlation of oxygenation dynamic signals between FIR illumination and arterial‐occlusion test (AOT) that implies the FIR illumination could be applied for hemodynamic response measurement in clinical diagnosis. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Structure‐guided design of a reversible fluorogenic reporter of protein‐protein interactions

A reversible green fluorogenic protein‐fragment complementation assay was developed based on the crystal structure of UnaG, a recently discovered fluorescent protein. In living mammalian cells, the nonfluorescent fragments complemented and rapidly became fluorescent upon rapamycin‐induced FKBP and Frb protein interaction, and lost fluorescence when the protein interaction was inhibited. This reversible fluorogenic reporter, named uPPI [UnaG‐based protein‐protein interaction (PPI) reporter], uses bilirubin (BR) as the chromophore and requires no exogenous cofactor. BR is an endogenous molecule in mammalian cells and is not fluorescent by itself. uPPI may have many potential applications in visualizing spatiotemporal dynamics of PPIs.     

A fluorescent reporter for mapping cellular protein‐protein interactions in time and space

We introduce a fluorescent reporter for monitoring protein–protein interactions in living cells. The method is based on the Split‐Ubiquitin method and uses the ratio of two auto‐fluorescent reporter proteins as signal for interaction (SPLIFF). The mating of two haploid yeast cells initiates the analysis and the interactions are followed online by two‐channel time‐lapse microscopy of the diploid cells during their first cell cycle. Using this approach we could with high spatio‐temporal resolution visualize the differences between the interactions of the microtubule binding protein Stu2p with two of its binding partners, monitor the transient association of a Ran‐GTPase with its receptors at the nuclear pore, and distinguish between protein interactions at the polar cortical domain at different phases of polar growth. These examples further demonstrate that protein–protein interactions identified from large‐scale screens can be effectively followed up by high‐resolution single‐cell analysis.     

Spectral diversity among members of the green fluorescent protein family in hydroid jellyfish (Cnidaria,Hydrozoa)

The cDNAs encoding the genes of new proteins, homologous to the well-known Green Fluorescent Protein (GFP) from the hydroid jellyfish Aequorea victoria, were cloned. Two green fluorescent proteins from one unidentified anthomedusa, a yellow fluorescent protein from Phialidium sp., and a nonfluorescent chromoprotein from another unidentified anthomedusa were characterized. Thus, a broad diversity of GFP-like proteins among the organisms of the class Hydrozoa in both spectral properties and primary structure was shown.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 49–53.Original Russian Text Copyright © 2005 by Yanushevich, Shagin, Fradkov, Shakhbazov, Barsova, Gurskaya, Labas, Matz, K. Lukyanov, S. Lukyanov.     

The mito::mKate2 mouse: A far‐red fluorescent reporter mouse line for tracking mitochondrial dynamics in vivo

Mitochondria are incredibly dynamic organelles that undergo continuous fission and fusion events to control morphology, which profoundly impacts cell physiology including cell cycle progression. This is highlighted by the fact that most major human neurodegenerative diseases are due to specific disruptions in mitochondrial fission or fusion machinery and null alleles of these genes result in embryonic lethality. To gain a better understanding of the pathophysiology of such disorders, tools for the in vivo assessment of mitochondrial dynamics are required. It would be particularly advantageous to simultaneously image mitochondrial fission‐fusion coincident with cell cycle progression. To that end, we have generated a new transgenic reporter mouse, called mito::mKate2 that ubiquitously expresses a mitochondria localized far‐red mKate2 fluorescent protein. Here we show that mito::mKate2 mice are viable and fertile and that mKate2 fluorescence can be spectrally separated from the previously developed Fucci cell cycle reporters. By crossing mito::mKate2 mice to the ROSA26R‐mTmG dual fluorescent Cre reporter line, we also demonstrate the potential utility of mito::mKate2 for genetic mosaic analysis of mitochondrial phenotypes.     

The 2.1A crystal structure of the far-red fluorescent protein HcRed: inherent conformational flexibility of the chromophore

We have determined the crystal structure of HcRed, a far-red fluorescent protein isolated from Heteractis crispa, to 2.1A resolution. HcRed was observed to form a dimer, in contrast to the monomeric form of green fluorescent protein (GFP) or the tetrameric forms of the GFP-like proteins (eqFP611, Rtms5 and DsRed). Unlike the well-defined chromophore conformation observed in GFP and the GFP-like proteins, the HcRed chromophore was observed to be considerably mobile. Within the HcRed structure, the cyclic tripeptide chromophore, Glu(64)-Tyr(65)-Gly(66), was observed to adopt both a cis coplanar and a trans non-coplanar conformation. As a result of these two conformations, the hydroxyphenyl moiety of the chromophore makes distinct interactions within the interior of the beta-can. These data together with a quantum chemical model of the chromophore, suggest the cis coplanar conformation to be consistent with the fluorescent properties of HcRed, and the trans non-coplanar conformation to be consistent with non-fluorescent properties of hcCP, the chromoprotein parent of HcRed. Moreover, within the GFP-like family, it appears that where conformational freedom is permissible then flexibility in the chromophore conformation is possible.     

A new red fluorescent protein that allows efficient marking of murine hematopoietic stem cells

Background

Genetic marking of hematopoietic stem cells (HSCs) with multiple fluorescent proteins (FPs) would allow analysis of their features, including interaction with adjacent cells. However, there are few red FPs that are comparable to green FPs in terms of low toxicity and high fluorescent intensity. This study has evaluated the usefulness of Kusabira Orange (KO) originated from the coral stone Fungia concinna as a red FP for marking of HSCs

Methods

A vector used was the MSCV‐type retroviral vector, DΔNsap that has the PCC4 cell‐passaged myeloproliferative sarcoma virus derived long terminal repeat devoid of a binding site for YY1 and the primer‐binding site derived from the dl587rev, respectively. The vector was cloned with the codon‐optimized KO cDNA for higher expression in mammalian cells (huKO) and converted to the corresponding retroviruses pseudotyped with the vesicular stomatitis virus G envelope protein, then transduced into c‐KIT⁺Sca‐1⁺Lineage^? cells obtained from C57BL/6 (Ly5.1) mice followed by transplantation into lethally irradiated Ly5.2 mice.

Results

Approximately 70% of donor‐derived cells highly expressed huKO at 16 weeks post‐transplantation. Furthermore, the high expression of huKO was also detected in serially transplanted mice, suggesting that expression of huKO per se had little deleterious effect on murine hematopoiesis. In double marking experiments, huKO‐expressing hematopoietic cells were easily distinguished from those expressing EGFP by flow cytometery and fluorescent microscope analysis.

Conclusions

Overall, the results obtained from the present study suggest that huKO can be used as a valuable and versatile red fluorescent marker for HSCs. Copyright © 2008 John Wiley & Sons, Ltd.

     

Temperature-pressure stability of green fluorescent protein: a Fourier transform infrared spectroscopy study

Green fluorescent protein (GFP) is widely used as a marker in molecular and cell biology. For its use in high-pressure microbiology experiments, its fluorescence under pressure was recently investigated. Changes in fluorescence with pressure were found. To find out whether these are related to structural changes, we investigated the pressure stability of wild-type GFP (wtGFP) and three of its red shift mutants (AFP, GFP(mut1), and GFP(mut2)) using Fourier transform infrared spectroscopy. For the wt GFP, GFP(mut1), and GFP(mut2) we found that up to 13-14 kbar the secondary structure remains intact, whereas AFP starts unfolding around 10 kbar. The 3-D structure is held responsible for this high-pressure stability. Previously observed changes in fluorescence at low pressure are rationalized in terms of the pressure-induced elastic effect. Above 6 kbar, loss of fluorescence is due to aggregation. Revisiting the temperature stability of GFP, we found that an intermediate state is populated along the unfolding pathway of wtGFP. At higher temperatures, the unfolding resulted in the formation of aggregates of wtGFP and its mutants.     

Crystal structure and Raman studies of dsFP483, a cyan fluorescent protein from Discosoma striata

To better understand the diverse mechanisms of spectral tuning operational in fluorescent proteins (FPs), we determined the 2.1-Å X-ray structure of dsFP483 from the reef-building coral Discosoma. This protein is a member of the cyan class of Anthozoa FPs and exhibits broad, double-humped excitation and absorbance bands, with a maximum at 437-440 nm and a shoulder at 453 nm. Although these features support a heterogeneous ground state for the protein-intrinsic chromophore, peak fluorescence occurs at 483 nm for all excitation wavelengths, suggesting a common emissive state. Optical properties are insensitive to changes in pH over the entire range of protein stability. The refined crystal structure of the biological tetramer (space group C2) demonstrates that all protomers bear a cis-coplanar chromophore chemically identical with that in green fluorescent protein (GFP). To test the roles of specific residues in color modulation, we investigated the optical properties of the H163Q and K70M variants. Although absorbance bands remain broad, peak excitation maxima are red shifted to 455 and 460 nm, emitting cyan light and green light, respectively. To probe chromophore ground-state features, we collected Raman spectra using 752-nm excitation. Surprisingly, the positions of key Raman bands of wild-type dsFP483 are most similar to those of the neutral GFP chromophore, whereas the K70M spectra are more closely aligned with the anionic form. The Raman data provide further evidence of a mixed ground state with chromophore populations that are modulated by mutation. Possible internal protonation equilibria, structural heterogeneity in the binding sites, and excited-state proton transfer mechanisms are discussed. Structural alignments of dsFP483 with the homologs DsRed, amFP486, and zFP538-K66M suggest that natural selection for cyan is an exquisitely fine-tuned and highly cooperative process involving a network of electrostatic interactions that may vary substantially in composition and arrangement.     

Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals

Spectral variants of green fluorescent protein are widely used in live samples for a broad range of applications: from visualization of protein interactions, through following gene expression, to marking particular cells in complex tissues. Higher wavelength emissions (such as red) are preferred due to the lower background-autofluorescence in tissues (Miyawaka et al., Nat Cell Biol Suppl S1-7, 2003). Until now, however, red fluorescent proteins (RFP) have displayed toxicity in murine embryos, which has hampered its application in this model. Here we report strong expression of a recently developed RFP variant, DsRed.T3, in mouse ES cells, embryos, and adult mice. Our results show that the red fluorescent wavelength has a superior tissue penetrance compared with spectral variants of lower wavelength. Furthermore, we have generated an ES cell line and a corresponding transgenic mouse line in which red fluorescence is activated upon Cre excision. Finally, we introduced cell type-specifically expressed Cre transgenes into this Cre recombinase reporter cell line, and by using the tetraploid embryo complementation assay, we could directly verify the Cre recombinase specificity on ES cell-derived embryos/animals.     

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.     

pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves

We have constructed a matched set of binary vectors designated pGD, pGDG and pGDR for the expression and co-localization of native proteins and GFP or DsRed fusions in large numbers of plant cells. The utility of these vectors following agroinfiltration into leaves has been demonstrated with four genes from Sonchus yellow net virus, a plant nucleorhabdovirus, and with a nucleolar marker protein. Of the three SYNV proteins tested, sc4 gave identical localization patterns at the cell wall and nucleus when fused to GFP or DsRed. However, some differences in expression patterns were observed depending on whether DsRed or GFP was the fusion partner. In this regard, the DsRed:P fusion showed a similar pattern of localization to GFP:P, but localized foci appeared in the nucleus and near the periphery of the nucleus. Nevertheless, the viral nucleocapsid protein, expressed as a GFP:N fusion, co-localized with DsRed:P in a subnuclear locale in agreement with our previous observations (Goodin et al., 2001). This locale appears to be distinct from the nucleolus as indicated by co-expression of the N protein, DsRed:P and a nucleolar marker AtFib1 fused to GFP. The SYNV M protein, which is believed to be particularly prone to oligomerization, was detectable only as a GFP fusion. Our results indicate that agroinfiltration with bacteria containing the pGD vectors is extremely useful for transient expression of several proteins in a high proportion of the cells of Nicotiana benthamiana leaves. The GFP and DsRed elements incorporated into the pGD system should greatly increase the ease of visualizing co-localization and interactions of proteins in a variety of experimental dicotyledonous hosts.     

mCherry红色荧光标记乳酸菌的融合表达系统构建及应用

为开发新型荧光蛋白标记乳酸菌以填补国内研究空白,利用pSIP载体,构建了以红色荧光蛋白mCherry为标记,并以乳酸菌胆盐水解酶基因bsh为报告基因的乳酸菌融合表达系统。在4种不同启动子(P_(sppA)、P_(ldhL)、P_(32)和P_(slpA))调节下,相继实现了融合基因的诱导型和组成型表达,表达的融合重组蛋白mCherry-BSH同时检测出红色荧光活性和胆盐水解酶BSH活性。mCherry红色荧光标记的乳酸菌融合表达系统的成功构建不仅为研究乳酸菌在生物体内的分布、定植及存活情况从而揭示其益生功能的作用机理提供有利条件,也为更多活性蛋白在乳酸菌宿主中的表达、细胞定位、功能鉴定的研究奠定基础。