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Fluorescent probes with a designed unnatural warhead for binding to PYP-tag enabled intracellular/cell-surface selective protein labeling. This unique imaging tool was successfully applied to reveal multiple subcellular localizations of GLUT4.

The development of near-infrared (NIR) fluorescent probes over the past few decades has changed the way that biomolecules are imaged, and thus represents one of the most rapidly progressing areas of research. Presently, NIR fluorescent probes are routinely used to visualize and understand intracellular activities. The ability to penetrate tissues deeply, reduced photodamage to living organisms, and a high signal-to-noise ratio characterize NIR fluorescent probes as efficient next-generation tools for elucidating various biological events. The coupling of self-labeling protein tags with synthetic fluorescent probes is one of the most promising research areas in chemical biology. Indeed, at present, protein-labeling techniques are not only used to monitor the dynamics and localization of proteins but also play a more diverse role in imaging applications. For instance, one of the dominant technologies employed in the visualization of protein activity and regulation is based on protein tags and their associated NIR fluorescent probes. In this mini-review, we will discuss the development of several NIR fluorescent probes used for various protein-tag systems., This minireview describes the development of NIR chemical probes for various protein-tag systems.

Photoswitchable fluorescent molecules (PSFMs) are important tools for fluorescence imaging of biomolecules. To date, PSFMs have been applied for pulse-chase experiments and super-resolution imaging...

Live-cell protein labeling using a protein tag and a fluorescent probe is a powerful approach for studying protein localization and dynamics. Herein, we engineered a protein tag, PYP-tag, to image ...

The ability to monitor proteolytic pathways that remove unwanted and damaged proteins from cells is essential for understanding the multiple processes used to maintain cellular homeostasis. In this study, we have developed a new protein-labeling probe that employs an 'OFF-ON-OFF' fluorescence switch to enable real-time imaging of the expression (fluorescence ON) and degradation (fluorescence OFF) of PYP-tagged protein constructs in living cells. Fluorescence switching is modulated by intramolecular contact quenching interactions in the unbound probe (fluorescence OFF) being disrupted upon binding to the PYP-tag protein, which turns fluorescence ON. Quenching is then restored when the PYP-tag-probe complex undergoes proteolytic degradation, which results in fluorescence being turned OFF. Optimization of probe structures and PYP-tag mutants has enabled this fast reacting 'OFF-ON-OFF' probe to be used to fluorescently image the expression and degradation of short-lived proteins.

Protein degradation plays various roles in cellular homeostasis and signal transduction. Real-time monitoring of the degradation process not only contributes to the elucidation of relevant biological phenomena but also offers a powerful tool for drug discoveries targeting protein degradation. Fluorescent protein labeling with a protein tag and a synthetic fluorescent probe is a powerful technique that enables the direct visualization of proteins of interest in living cells. Although a variety of protein tags and their labeling probes have been reported, techniques for the visualization of protein degradation in living cells remain limited. In order to overcome this limitation, we herein employed a PYP-tag labeling probe with a fluorescence turn-off switch that enables the imaging of protein degradation. Furthermore, we performed a structure-based design of a PYP-tag to stabilize a complex formed by the probe and the protein tag for long-term live-cell imaging. We successfully applied this technique to live-cell imaging of the degradation process of Regnase-1 in response to immunostimulation.

Labelling technologies developed over the past few years have changed the way of looking at biomolecules and have made a considerable contribution to our understanding of the functions and regulation of dynamic biological processes. One of the robust technologies employed to image proteins in a cellular environment is based on the use of chemical tags and their fluorescent probes, which provides flexibility in developing probes with a wide range of synthetic fluorophores. A variety of chemical tags, ranging from short amino acid sequences to small proteins, have been employed to generate protein-labelling systems. One such chemical tag is the photoactive yellow protein (PYP)-tag, which is a small bacterial protein, developed for the selective labelling and imaging of proteins. Herein, we briefly discuss the protein-labelling system developed based on PYP-tag technology, with a focus on the design strategy for PYP-tag labelling probes and their applications in protein imaging.

Proteins are an important component of living systems and play a crucial role in various physiological functions. Fluorescence imaging of proteins is a powerful tool for monitoring protein dynamics. Fluorescent protein (FP)-based labeling methods are frequently used to monitor the movement and interaction of cellular proteins. However, alternative methods have also been developed that allow the use of synthetic fluorescent probes to target a protein of interest (POI). Synthetic fluorescent probes have various advantages over FP-based labeling methods. They are smaller in size than the fluorescent proteins, offer a wide variety of colors and have improved photochemical properties. There are various chemical recognition-based labeling techniques that can be used for labeling a POI with a synthetic probe. In this review, we focus on the development of protein-labeling systems, particularly the SNAP-tag, BL-tag, and PYP-tag systems, and understanding the fluorescence behavior of the fluorescently labeled target protein in these systems. We also discuss the smart fluorogenic probes for these protein-labeling systems and their applications. The fluorogenic protein labeling will be a useful tool to investigate complex biological phenomena in future work on cell biology.

Reversible enzymatic post-translational modification of the ε-amino groups of lysine residues (e.g. N-acylation reactions) plays an important role in regulating the cellular activities of numerous proteins. This study describes how enzyme catalyzed N-deprotection of lysine residues of non-fluorescent peptide-coumarin probes can be used to generate N-deprotected peptides that undergo spontaneous O- to N-ester transfer reactions (uncatalyzed) to generate a highly fluorescent N-carbamoyl peptide. This enables detection of enzyme catalyzed N-deacetylation, N-demalonylation, N-desuccinylation and N-demethylation reactions activities towards the N-modified lysine residues of these probes using simple ‘turn on’ fluorescent assays., We developed “turn-on” fluorescent probes that detect enzymatic lysine deacylation and demethylation critical for epigenetic and other cellular phenomena, using intramolecular O- to N-ester transfer reactions.

Epigenetic DNA and histone modifications alter chromatin conformation and regulate gene expression. A major DNA modification is methylation, which is catalyzed by DNA methyltransferase (Dnmt) and results in gene suppression. Compared to DNA, histones undergo a greater variety of modification types, one of which is the acetylation of lysine. While histone acetyltransferase (HAT) catalyzes acetylation and activates gene expression, histone deacetylase (HDAC) removes the modification and causes gene suppression. As precise regulation of these epigenetic marks on DNA and histones is critical for cellular functions, their dysregulation causes various diseases including cancer, metabolic syndromes, immune diseases, and psychiatric diseases. Therefore, elucidation of the epigenetic phenomena is important not only in the field of biology but also in medical and pharmaceutical sciences. Furthermore, this field is also attracting industrial interest, because small-molecule inhibitors modulate enzymatic activity for epigenetic modification and are used for cancer treatment. Under these circumstances, various methods for detecting epigenetic modifications have been developed. However, most methods require cell lysis, which is not suitable for real-time detection of enzymatic activity. Since fluorescent probes are attractive chemical tools to solve this issue, chemists made considerable efforts to create fluorescent probes for epigenetics. To date, we have particularly focused on HDAC activity and DNA methylation and have developed fluorescent probes for their detection. The first part of this review describes our recent efforts to develop fluorescent probes for detecting HDAC activity. Since the discovery of HDAC activity in the late 1960s, no fluorescent probe has been developed that can detect enzymatic reactions in a simple, one-step procedure despite its biological and medical importance. We designed fluorescent probes to overcome this limitation by devising two different types of fluorescence switching mechanisms, which are based on aggregation-induced emission (AIE) and intramolecular transesterification. Using these probes, we detected HDAC activity simply by mixing the probes and HDAC for the first time. In the second part, a hybrid approach using a protein-labeling system was employed to detect DNA methylation in living cells. So far, live-cell detection of DNA methylation was conducted by imaging the localization of Fluorescent Proteins (FPs) fused to a methylated DNA-binding domain. However, FP lacks a fluorescence switch and emits fluorescence without binding to methylated DNA. We created a hybrid probe that comprises a fluorogen and a protein and enhances fluorescence intensity upon binding to methylated DNA. To create the hybrid probe, we applied our protein labeling system using the PYP-tag that we previously developed. This method successfully visualized methylated DNA in living cells and verified its dynamics during cell division. Both of the above-mentioned fluorescent probes have great potential for use not only in HDAC and DNA methylation but also in other epigenetics-associated modifications. For example, the mechanism of the HDAC probes can be used to detect histone demethylation. The hybrid probe can be converted to a sensor for imaging acetylated or methylated histones. In this review, we mainly describe how we designed the probes using chemical principles and solved the current obstacles with the probe design and discuss the future prospects of these probes.

Protein labeling using fluorogenic probes enables the facile visualization of proteins of interest. Herein, we report new fluorogenic probes consisting of a rationally designed coumarin ligand for the live-cell fluorogenic labeling of the photoactive yellow protein (PYP)-tag. On the basis of the photochemical mechanisms of coumarin and the probe-tag interactions, we introduced a hydroxy group into an environment-sensitive coumarin ligand to modulate its spectroscopic properties and increase the labeling reaction rate. The resulting probe had a higher labeling reaction rate constant and a greater fluorescence OFF-ON ratio than any previously developed PYP-tag labeling probe. The probe enabled the fluorogenic labeling of intracellular proteins within minutes. Furthermore, we used our probe to investigate the localization of sirtuin 3 (SIRT3), a mitochondrial deacetylase. Although the nuclear localization of SIRT3 has been controversial, this transient nuclear localization was clearly captured by the rapid, high-contrast imaging enabled by our probe.

Controlled release is one of the key technologies for medical innovation, and many stimulus-responsive nanocarriers have been developed to utilize this technology. Enzyme activity is one of the most useful stimuli, because many enzymes are specifically activated in diseased tissues. However, controlled release stimulated by enzyme activity has not been frequently reported. One of the reasons for this is the lack of versatility of carriers. Most of the reported stimulus-responsive systems involve a sophisticated design and a complicated process for the synthesis of stimulus-responsive nanocarrier components. The purpose of this study was to develop versatile controlled release systems triggered by various stimuli, including enzyme activity, without modifying the nanocarrier components. We developed two controlled release systems, both of which comprised a liposome as the nanocarrier and a membrane-damaging peptide, temporin L (TL), and its derivatives as the release-controllers. One system utilized branched peptides for proteases, and the other utilized phosphopeptides for phosphatases. In our systems, the target enzymes converted the non-membrane-damaging TL derivatives into membrane-damaging peptides and released the liposome inclusion. We demonstrated the use of our antimicrobial peptide-based controlled release systems for different enzymes and showed the promise of this technology as a novel theranostic tool.

Glucose transporter 4 (GLUT4) is an N-glycosylated protein that maintains glucose homeostasis by regulating the protein translocation. To date, it has been unclear whether the N-glycan of GLUT4 contributes to its intracellular trafficking. Here, to clarify the role of the N-glycan, we developed fluorogenic probes that label cytoplasmic and plasma-membrane proteins for multicolor imaging of GLUT4 translocation. One of the probes, which is cell impermeant, selectively detected exocytosed GLUT4. Using this probe, we verified the 'log' of the trafficking, in which N-glycan-deficient GLUT4 was transiently translocated to the cell membrane upon insulin stimulation and was rapidly internalized without retention on the cell membrane. The results strongly suggest that the N-glycan functions in the retention of GLUT4 on the cell membrane. This study showed the utility of the fluorogenic probes and indicated that this imaging tool will be applicable for research on various membrane proteins that show dynamic changes in localization.

“Clickable” nanowires with well-defined and uniform structures made of conjugated polyfluorene polymers were successfully fabricated by single particle nanofabrication technique (SPNT). Poly[(9,9-dihex-5-yn-1-ylfluorenyl-2,7-diyl)-co-(9,9′-di-n-octylfluorenyl-2,7-diyl)] (F6E8) and poly[(9,9-dihex-5-yn-1-ylfluorenyl-2,7-diyl)-co-(2,2′-bithiophene)] (F6E2T) underwent an efficient cross-linking reaction upon irradiation, resulting in formation of one-dimensional nanostructures with high and desired aspect ratio reaching up to 200. Alkyne groups on the surface of nanowires were functionalized effectively by click reaction with fluorescent 5-TAMRA-PEG3-azide, which was confirmed by confocal microscopy. Substrates functionalized with the nanowires provide dramatic expansion of “clickable” surface area immobilized directly with TAMRA, and the fluorescence resonance energy transfer (FRET) processes between TAMRA and nanowire backbones are demonstrated as biological sensing platforms.

The transport and trafficking of metabolites are critical for the correct functioning of live cells. However, in situ metabolic imaging studies are hampered by the lack of fluorescent chemical structures that allow direct monitoring of small metabolites under physiological conditions with high spatial and temporal resolution. Herein, we describe SCOTfluors as novel small-sized multi-colored fluorophores for real-time tracking of essential metabolites in live cells and in vivo and for the acquisition of metabolic profiles from human cancer cells of variable origin. © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA., The authors acknowledge funding from Medical Research Scotland (S.B.: 879-2015), MSCA Individual Fellowship (A.F.: 704912), OPTIMA (N.D.B.: EP/L016559/1), Wellcome Trust Sir Henry Dale Fellowship (Y.F.: 100104/Z/12/Z) and the Spanish Ministry of Science, Innovation and Universities (J.L.A, A.D.: CTQ2017-85378-R). M.V. acknowledges funds from ERC Consolidator Grant (771443), Biotechnology and Biological Sciences Research Council (BB/M025160/1) and the Royal Society (IEC\R3\170132). The authors thank the technical support from the Flow Cytometry and the Confocal Advanced Light Microscopy units at the University of Edinburgh.

The epigenetic modification of DNA involves the conversion of cytosine to 5-methylcytosine, also known as DNA methylation. DNA methylation is important in modulating gene expression and thus, regulating genome and cellular functions. Recent studies have shown that aberrations in DNA methylation are associated with various epigenetic disorders or diseases including cancer. This stimulates great interest in the development of methods that can detect and visualize DNA methylation. For instance, fluorescent proteins (FPs) in conjugation with methyl-CpG-binding domain (MBD) have been employed for live-cell imaging of DNA methylation. However, the FP-based approach showed fluorescence signals for both the DNA-bound and -unbound states and thus differentiation between these states is difficult. Synthetic-molecule/protein hybrid probes can provide an alternative to overcome this restriction. In this article, we discuss the synthetic-molecule/protein hybrid probe that we developed recently for live-cell imaging of DNA methylation, which exhibited fluorescence enhancement only after binding to methylated DNA.

Quick and no-wash labeling of intracellular proteins was achieved in live cells using a PYP-tag mutant and a membrane-permeable fluorogenic probe with modular structures., Conditional fluorescence imaging is a powerful technique for precise spatiotemporal analysis of proteins in live cells upon administration of a synthetic probe. To be applicable to various biological phenomena, probes must be membrane-permeable, have a high signal-to-noise ratio, and work quickly. To date, few probes meet all of these requirements. Here, we designed a fluorogenic probe (AcFCANB) that could label intracellular proteins fused to the photoactive yellow protein (PYP) tag in live cells within 30 min and used it to image heterochromatin protein 1 localization in nuclei. AcFCANB is based on a modular platform consisting of fluorophore, ligand and quencher. We accelerated the labeling reaction by strategic mutations of charged residues on the surface of PYP. A simple model based on molecular dynamics simulations quantitatively reproduced the cooperative effect of multiple mutations on labeling rate.

Protein labeling with fluorogenic probes is a powerful method for the imaging of cellular proteins. The labeling time and fluorescence contrast of the fluorogenic probes are critical factors for the precise spatiotemporal imaging of protein dynamics in living cells. To address these issues, we took mutational and chemical approaches to increase the labeling kinetics and fluorescence intensity of fluorogenic PYP-tag probes. Because of charge-reversal mutations in PYP-tag and probe redesign, the labeling reaction was accelerated by a factor of 18 in vitro, and intracellular proteins were detected with an incubation period of only 1 min. The brightness of the probe both in vitro and in living cells was enhanced by the mutant tag. Furthermore, we applied this system to the imaging analysis of bromodomains. The labeled mutant tag successfully detected the localization of bromodomains to acetylhistone and the disruption of the bromodomain-acetylhistone interaction by a bromodomain inhibitor.

Hybrid probes consisting of synthetic molecules and proteins are powerful tools for detecting biological molecules and signals in living cells. To date, most targets of the hybrid probes have been limited to pH and small analytes. Although biomacromolecules are essential to the physiological function of cells, the hybrid-probe-based approach has been scarcely employed for live-cell detection of biomacromolecules. Here, we developed a hybrid probe with a chemical switch for live-cell imaging of methylated DNA, an important macromolecule in the repression of gene expression. Using a protein labeling technique, we created a hybrid probe containing a DNA-binding fluorogen and a methylated-DNA-binding domain. The hybrid probe enhanced fluorescence intensity upon binding to methylated DNA and successfully monitored methylated DNA during mitosis. The hybrid probe offers notable advantages absent from probes based on small molecules or fluorescent proteins and is useful for live-cell analyses of epigenetic phenomena and diseases related to DNA methylation.

Two strategies have been proposed to develop enzyme-triggered compound release systems., Controlled release is one of the key technologies for medical innovation, and many stimulus-responsive nanocarriers have been developed to utilize this technology. Enzyme activity is one of the most useful stimuli, because many enzymes are specifically activated in diseased tissues. However, controlled release stimulated by enzyme activity has not been frequently reported. One of the reasons for this is the lack of versatility of carriers. Most of the reported stimulus-responsive systems involve a sophisticated design and a complicated process for the synthesis of stimulus-responsive nanocarrier components. The purpose of this study was to develop versatile controlled release systems triggered by various stimuli, including enzyme activity, without modifying the nanocarrier components. We developed two controlled release systems, both of which comprised a liposome as the nanocarrier and a membrane-damaging peptide, temporin L (TL), and its derivatives as the release-controllers. One system utilized branched peptides for proteases, and the other utilized phosphopeptides for phosphatases. In our systems, the target enzymes converted the non-membrane-damaging TL derivatives into membrane-damaging peptides and released the liposome inclusion. We demonstrated the use of our antimicrobial peptide-based controlled release systems for different enzymes and showed the promise of this technology as a novel theranostic tool.

An acylated flavonol glycoside, helichrysoside, at a dose of 10 mg/kg/day per os for 14 days, improved the glucose tolerance in mice without affecting the food intake, visceral fat weight, liver weight, and other plasma parameters. In this study, using hepatoblastoma-derived HepG2 cells, helichrysoside, trans-tiliroside, and kaempferol 3-O-&beta, D-glucopyranoside enhanced glucose consumption from the medium, but their aglycones and p-coumaric acid did not show this activity. In addition, several acylated flavonol glycosides were synthesized to clarify the structural requirements for lipid metabolism using HepG2 cells. The results showed that helichrysoside and related analogs significantly inhibited triglyceride (TG) accumulation in these cells. The inhibition by helichrysoside was more potent than that by other acylated flavonol glycosides, related flavonol glycosides, and organic acids. As for the TG metabolism-promoting activity in high glucose-pretreated HepG2 cells, helichrysoside, related analogs, and their aglycones were found to significantly reduce the TG contents in HepG2 cells. However, the desacyl flavonol glycosides and organic acids derived from the acyl groups did not exhibit an inhibitory impact on the TG contents in HepG2 cells. These results suggest that the existence of the acyl moiety at the 6'' position in the D-glucopyranosyl part is essential for glucose and lipid metabolism-promoting activities.

We developed novel fluorogenic probes for no-wash live-cell imaging of proteins fused to PYP-tag, which is a small protein tag recently reported by our group. Through the design of a new PYP-tag ligand, specific intracellular protein labeling with rapid kinetics and fluorogenic response was accomplished. The probes crossed the cell membrane, and cytosolic and nuclear localizations of PYP-tagged proteins without cell washing were visualized within a 6-min reaction time. The fluorogenic response was due to the environmental effect of fluorophore upon binding to PYP-tag. Furthermore, the PYP-tag-based method was applied to the imaging of methyl-CpG-binding domain localization. This rapid protein-labeling system combined with the small protein tag and designed fluorogenic probes offers a powerful method to study the localization, movement, and function of cellular proteins.

Protein labeling by using a protein tag and its specific fluorescent probe is increasingly becoming a useful technique for the real-time imaging of proteins in living cells. Recently, fluorogenic probes for protein labeling were developed. When using these probes, a washing step is not required for the removal of free probes from the cells, thus, allowing rapid detection of proteins in living cells with high signal-to-noise ratio. Various chemical principles have been applied in the designing of probes to include a turn-on fluorescence switch that is activated by the protein labeling reaction. In this review, we describe about the design strategy of the probes and the advances in fluorogenic protein labeling systems.

Histone deacetylases (HDACs) are key enzymatic regulators of many cellular processes such as gene expression, cell cycle, and tumorigenesis. These enzymes are attractive targets for drug development. However, very few simple methods for monitoring HDAC activity have been reported. Here, we have developed a fluorogenic probe, K4(Ac)-CCB, which consists of the histone H3 peptide containing acetyl-Lys and a coumarin fluorophore with a carbonate ester. By the simple addition of the probe to a HDAC solution, enzyme activity was clearly detected through spontaneous intramolecular transesterification, which renders the probe fluorescent. In addition, K4(Ac)-CCB can be applied to the evaluation of HDAC inhibitor activity. This is the first report to demonstrate the monitoring of HDAC activity by using a one-step procedure. Thus, our novel fluorogenic probe will provide a powerful tool for epigenetic research and the discovery of HDAC-targeted drugs.

Fluorescence imaging using synthetic probes is becoming increasingly popular as a chemistry-based technique for the analysis of biomolecules. Real-time visual information on various biological molecules can be obtained by designing probes with high flexibility. The focus of our research is to design fluorogenic probes which are non-fluorescent in their initial intact form, but exhibit enhanced fluorescence intensity upon reactions with target biomolecules. The fluorescence switch of these probes allows the detection of the function and the localization of biomolecules with a high signal-to-noise ratio. Thus far, we have succeeded in designing and synthesizing fluorogenic probes that visualize molecules involved in epigenetics. Histone deacetylases (HDACs) play an important role in the epigenetic regulation of gene expression. A significant amount of epigenetic information can be obtained by the detection of enzyme activity. However, the existing methods require complicated multistep procedures. To overcome this limitation, we developed fluorogenic probes for the detection of HDAC activity in a one-step procedure. In this review, the details of the strategy for probe design and the detection method have been described.

Ligand-induced conformation change is a general strategy for controlling protein function. In this work, we demonstrate the relationships between ligand binding and conformational stability using a previously designed protein, Ant-F, which undergoes a conformation change upon Zn(II) binding. To investigate the effect of stabilization of the apo structure on the conformation change, we also created a novel protein, Ant-F-H1, into which mutations are introduced to increase its stability over that of Ant-F. The chemical denaturation experiments clarified that apo-Ant-F-H1 is more stable than apo-Ant-F (DeltaDeltaG = -1.28 kcal/mol) and that the stability of holo-Ant-F-H1 is almost the same as that of holo-Ant-F. The Zn(II) binding assay shows that the affinity of Zn(II) for Ant-F-H1 is weaker than that for Ant-F (DeltaDeltaG = 1.40 kcal/mol). A large part of the increased value of free energy in stability corresponds to the decreased value of free energy in Zn(II) binding, indicating that the stability of the apo structure directly affects the conformation change. The denaturation experiments also reveal that Zn(II) destabilizes the conformation of both proteins. From the thermodynamic linkage, Zn(II) is thought to bind to the unfolded state with high affinity. These results suggest that the binding of Zn(II) to the unfolded state is an important factor in the conformational change as well as the stability of the apo and holo structures.

A multicolour protein labelling technique using a protein tag and fluorogenic probes is a powerful approach for spatio-temporal analyses of proteins in living cells. Since cyanine fluorophores have attractive properties for multicolour imaging of proteins, there is a huge demand to develop fluorogenic cyanine probes for specific protein labelling in living cells. Herein, we develop fluorogenic cyanine probes for labelling a protein tag by using a dinitrobenzene fluorescence quencher. The probes enhanced fluorescence intensity upon labelling reactions and emitted orange or far-red fluorescence. Intramolecular interactions between the cyanine fluorophores and the dinitrobenzene quencher led not only to fluorescence quenching of the probes in the free state but also to promotion of labelling reactions. Furthermore, the probes successfully imaged cell-surface proteins without a washing process. These findings offer valuable information on the design of fluorogenic cyanine probes and indicate that the probes are useful as novel live-cell imaging tools. This article is part of the themed issue ‘Challenges for chemistry in molecular imaging’.

Long-distance intramolecular nucleophilic reactions are promising strategies for the design of fluorogenic probes to detect enzymatic activity involved in lysine modifications. However, such reactions have been challenging and hence have not been established. In this study, we have prepared fluorogenic peptides that induce intramolecular reactions between lysine nucleophiles and electrophiles in distal positions. These peptides contain a lysine and fluorescence-quenched fluorophore with a carbonate ester, which triggers nucleophilic transesterification resulting in fluorogenic response. Transesterification occurred under mild aqueous conditions despite the presence of a long nine-amino-acid spacer between the lysine and fluorophore. In addition, one of the peptides showed the fastest reaction kinetics with a half-life time of 3.7 min. Furthermore, the incorporation of this fluorogenic switch into the probes allowed rapid fluorogenic detection of histone deacetylase (HDAC) activity. These results indicate that the transesterification reaction has great potential for use as a general fluorogenic switch to monitor the activity of lysine-targeting enzymes.

Zinc finger constitutes one of the most common DNA binding motifs. Although zinc finger proteins consisting of Cys2His2, Cys3His, Cys4, and Cys6 domains are known in nature, a novel His4 zinc finger protein has never been observed. Herein, we have created the first artificial His4-type zinc finger protein (H4Sp1) engineered by Cys → His mutations of the Cys2His2-type zinc finger transcription factor Sp1. The CD features of the single finger H4Sp1f2 and three-finger H4Sp1 clearly demonstrate the folding of the mutant His4 peptides by complexation with Zn(II). The NMR study of Zn(II)-H4Sp1f2 reveals that some distortions of the helical region occur due to Zn(II) coordination. The gel mobility shift assay and DNase I footprinting analysis strongly show the binding of Zn(II)-H4Sp1 to the GC-box site of duplex DNA. The methylation interference pattern of Zn(II)-H4Sp1 binding significantly resembles that of the corresponding C2H2Sp1 binding. The present artificial peptide H4Sp1 is the first example of a zinc fi...

DNA structural changes such as bending play an important role in various biological reactions. Not only protein binding to its specific DNA sequence but also DNA bending induced by the protein is indispensable for unique gene expression. Therefore, an artificial protein that induces a DNA conformational change is interesting as a transcriptional regulator of a specific gene. We created 6-zinc finger proteins, Sp1ZF6(Gly)n (n = 4, 7, 10), by connecting two DNA binding domains of transcription factor Sp1 with flexible polyglycine peptide linkers, and their effects on DNA structure were compared with that of native 3-zinc finger Sp1(530-623). Gel electrophoretic methods revealed that Sp1ZF6(Gly)7 and Sp1ZF6(Gly)10 bind to two distal GC boxes and result in DNA bending. Evidently, the hydroxyl radical footprinting analysis demonstrated that hypersensitive cleavage was observed at the 5'-TA step in the intervening region bound by Sp1ZF6(Gly)7 or Sp1ZF6(Gly)10. The phasing assays strongly suggested that the induced DNA bending was directed toward the major groove and that Sp1ZF6(Gly)7 caused the most drastic directional change in DNA bending. Of special interest are the facts that the newly designed 6-finger peptides Sp1ZF6(Gly)7 and Sp1ZF6(Gly)10 can induce DNA bending at the intervening region of the two distal binding sites and that the linker length between two 3-zinc finger motifs has a crucial effect on the entire DNA-bending direction. Such DNA-bending fingers may be feasible for use as a gene expression regulator based on the structural change in DNA in the future.

The use of genetic engineering techniques allows researchers to combine functional proteins with fluorescent proteins (FPs) to produce fusion proteins that can be visualized in living cells, tissues, and animals. However, several limitations of FPs, such as slow maturation kinetics or issues with photostability under laser illumination, have led researchers to examine new technologies beyond FP-based imaging. Recently, new protein-labeling technologies using protein/peptide tags and tag-specific probes have attracted increasing attention. Although several protein-labeling systems are com mercially available, researchers continue to work on addressing some of the limitations of this technology. To reduce the level of background fluorescence from unlabeled probes, researchers have pursued fluorogenic labeling, in which the labeling probes do not fluoresce until the target proteins are labeled. In this Account, we review two different fluorogenic protein-labeling systems that we have recently developed. First we give a brief history of protein labeling technologies and describe the challenges involved in protein labeling. In the second section, we discuss a fluorogenic labeling system based on a noncatalytic mutant of β-lactamase, which forms specific covalent bonds with β-lactam antibiotics such as ampicillin or cephalosporin. Based on fluorescence (or Förster) resonance energy transfer and other physicochemical principles, we have developed several types of fluorogenic labeling probes. To extend the utility of this labeling system, we took advantage of a hydrophobic β-lactam prodrug structure to achieve intracellular protein labeling. We also describe a small protein tag, photoactive yellow protein (PYP)-tag, and its probes. By utilizing a quenching mechanism based on close intramolecular contact, we incorporated a turn-on switch into the probes for fluorogenic protein labeling. One of these probes allowed us to rapidly image a protein while avoiding washout. In the future, we expect that protein-labeling systems with finely designed probes will lead to novel methodologies that allow researchers to image biomolecules and to perturb protein functions.

We propose using the least square method for approximating the electrical field distribution of the fundamental mode in photonic crystal fibers (PCFs) by using a Gaussian and a hyperbolic-secant (sech) function. It is shown that the mode field can be fitted closely to a Gaussian for large d//spl Lambda/ values (i.e., for strongly guiding structures). For small d//spl Lambda/ values (for weakly guiding structures), a sech function fits the mode field well because the field gradually extends into the cladding region, implying that the analogy of PCFs with step index fibers is not applicable in this regime.

We report a substantial increase in the heat resistance in a connector-type single-wall carbon nanotube (SWNT) saturable absorber by sealing SWNT/P3HT composite with siloxane. By applying the saturable absorber to a passively mode-locked Er fiber laser, we successfully demonstrated 280 fs, 31 mW pulse generation with a fivefold improvement in heat resistance.

A tetraphenylethylene-derivative fluorescent probe for the one-step detection of histone deacetylases (HDAC) was developed. The deacetylation of the probe triggers electrostatic interaction between the molecules and automatically leads to fluorescence enhancement based on aggregation-induced emission (AIE).

Intracellular protein labeling with small molecular probes that do not require a washing step for the removal of excess probe is greatly desired for real-time investigation of protein dynamics in living cells. Successful labeling of proteins on the cell membrane has been performed using mutant β-lactamase tag (BL-tag) technology. In the present study, intracellular protein labeling with novel cell membrane permeable probes based on β-lactam prodrugs is described. The prodrug-based probes quickly permeated the plasma membranes of living mammalian cells, and efficiently labeled intracellular proteins at low probe concentrations. Because these cell-permeable probes were activated only inside cells, simultaneous discriminative labeling of intracellular and cell surface BL-tag fusion proteins was attained by using cell-permeable and impermeable probes. Thus, this technology enables adequate discrimination of the location of proteins labeled with the same protein tag, in conjunction with different color probes, by dual-color fluorescence. Moreover, the combination of BL-tag technology and the prodrug-based probes enabled the labeling of target proteins without requiring a washing step, owing to the efficient entry of probes into cells and the fast covalent labeling achieved with BL-tag technology after bioactivation. This prodrug-based probe design strategy for BL-tags provides a simple experimental procedure with application to cellular studies with the additional advantage of reduced stress to living cells.

Protein labeling techniques using small molecule probes have become important as practical alternatives to the use of fluorescent proteins (FPs) in live cell imaging. These labeling techniques can be applied to more sophisticated fluorescence imaging studies such as pulse-chase imaging. Previously, we reported a novel protein labeling system based on the combination of a mutant β-lactamase (BL-tag) with coumarin-derivatized probes and its application to specific protein labeling on cell membranes. In this paper, we demonstrated the broad applicability of our BL-tag technology to live cell imaging by the development of a series of fluorescence labeling probes for this technology, and the examination of the functions of target proteins. These new probes have a fluorescein or rhodamine chromophore, each of which provides enhanced photophysical properties relative to coumarins for the purpose of cellular imaging. These probes were used to specifically label the BL-tag protein and could be used with other small molecule fluorescent probes. Simultaneous labeling using our new probes with another protein labeling technology was found to be effective. In addition, it was also confirmed that this technology has a low interference with respect to the functions of target proteins in comparison to GFP. Highly specific and fast covalent labeling properties of this labeling technology is expected to provide robust tools for investigating protein functions in living cells, and future applications can be improved by combining the BL-tag technology with conventional imaging techniques. The combination of probe synthesis and molecular biology techniques provides the advantages of both techniques and can enable the design of experiments that cannot currently be performed using existing tools.