Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Fluorescent biosensors are crucial experimental tools for live-cell imaging and the quantification of different biological analytes. Fluorescent protein (FP)-based biosensors are widely used for imaging applications in living systems. However, the use of FP-based biosensors is hindered by their large size, poor photostability, and laborious genetic manipulations required to improve their properties. Recently, semisynthetic fluorescent biosensors have been developed to address the limitations of FP-based biosensors using chemically modified fluorescent probes and self-labeling protein tag/peptide tags or DNA/RNA-based hybrid systems. Semisynthetic biosensors have unique advantages, as they can be easily modified using different probes. Moreover, the self-labeling protein tag, which labels synthetically developed ligands via covalent bonds, has immense potential for biosensor development. This review discusses the recent progress in different types of fluorescent biosensors for metabolites, protein aggregation and degradation, DNA methylation, endocytosis and exocytosis, membrane tension, and cellular viscosity. Here, we explain in detail the design strategy and working principle of these biosensors. The information presented will help the reader to create new biosensors using self-labeling protein tags for various applications.

DOI： 10.1016/j.bios.2023.115862

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

Photoswitchable fluorescent molecules (PSFMs) are widely applicable in the life sciences for super-resolution imaging. Owing to the large and hydrophobic molecular structures of PSFMs that may aggregate in a biological medium, the development of synthetic PSFMs with persistent reversible photoswitching is challenging. Here, we established a protein-surface-assisted photoswitching strategy that allows for persistent reversible fluorescence photoswitching of a PSFM in an aqueous solution. As a first step, we applied the photochromic chromophore furylfulgimide (FF) as a photoswitchable fluorescence quencher and developed a Förster resonance energy transfer-based PSFM, named FF-TMR. Most importantly, the protein-surface modification strategy allows FF-TMR to exhibit persistent reversible photoswitching performance in an aqueous solution. In fixed cells, the fluorescence intensity of FF-TMR bound to antitubulin antibody was repetitively modulated. The protein-surface-assisted photoswitching strategy will be a useful platform to broaden the utility of functionalized synthetic chromophores enabling persistent fluorescence switching that inherits their high resistance to light irradiation.

DOI： 10.1021/acs.analchem.3c00163

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

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.

DOI： 10.1039/d1sc06274c

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Language：Others   Publishing type：Research paper (scientific journal)  

Near-infrared fluorescent probes: a next-generation tool for protein-labeling applications
<p>This minireview describes the development of NIR chemical probes for various protein-tag systems.</p>

DOI： 10.1039/d0sc04792a

Language：Others   Publishing type：Research paper (scientific journal)  

Development of photoswitchable fluorescent molecules using arylazopyrazole

Language：English   Publishing type：Research paper (scientific journal)  

Development of fluorogenic probes for rapid, high-contrast imaging of transient nuclear localization of sirtuin 3

DOI： 10.1002/cbic.201900568

Language：English   Publishing type：Research paper (scientific journal)  

Engineered Protein-tag for Rapid Live-cell Fluorogenic Visualization of Proteins by Anionic Probes

Language：English   Publishing type：Research paper (scientific journal)  

Rapid no-wash labeling of PYP-tag proteins with reactive fluorogenic ligands affords stable fluorescent protein conjugates for long-term cell imaging studies

Language：English  

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.

DOI： 10.1021/acs.bioconjchem.9b00696

Language：English   Publishing type：Research paper (scientific journal)  

Chemical Tools with Fluorescence Switches for Verifying Epigenetic Modifications

Language：English   Publishing type：Research paper (scientific journal)  

Photoactive yellow protein and its chemical probes: an approach to protein labelling in living cells.

DOI： 10.1093/jb/mvz051

Language：English   Publishing type：Research paper (scientific journal)  

Development of an effective protein-labeling system based on smart fluorogenic probes.

DOI： 10.1007/s00775-019-01669-y

Language：English   Publishing type：Research paper (scientific journal)  

SCOTfluors: Small, Conjugatable, Orthogonal, and Tunable Fluorophores for In Vivo Imaging of Cell Metabolism.

DOI： 10.1002/anie.201900465

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1021/jacs.7b09713

Language：English   Publishing type：Research paper (scientific journal)  

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'.

DOI： 10.1098/rsta.2017.0018

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/c7sc90017a

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1039/c6sc04435b

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1038/NCHEMBIO.2156

Language：English   Publishing type：Research paper (scientific journal)  

Fabrication of "clickable" Polyfluorene Nanowires with High Aspect Ratio as Biological Sensing Platforms
© 2016 American Chemical Society. "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.

DOI： 10.1021/acssensors.6b00070

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1002/anie.201506935

Language：English   Publishing type：Research paper (scientific journal)  

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 3.7min. 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.

DOI： 10.1002/chem.201406093

Language：Japanese   Publishing type：Research paper (scientific journal)  

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-tonoise 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.

Language：English   Publishing type：Research paper (scientific journal)  

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 commercially 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 beta-lactamase, which forms specific covalent bonds with beta-lactam antibiotics such as ampicillin or cephalosporin. Based on fluorescence (or Forster) 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 beta-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.

DOI： 10.1021/ar400135f

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.cbpa.2013.05.015

Language：English   Publishing type：Research paper (scientific journal)  

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. © 2013 American Chemical Society.

DOI： 10.1021/ja405745v

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1021/ja306045j

Language：English   Publishing type：Research paper (scientific journal)  

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 beta-lactamase tag (BL-tag) technology. In the present study, intracellular protein labeling with novel cell membrane permeable probes based on beta-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.

DOI： 10.1002/chem.201100973

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/cbic.201100137

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/cbic.201100021

Language：English   Publishing type：Research paper (scientific journal)  

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 he 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 beta-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 tartlet 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 he 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.

DOI： 10.1021/bc100333k

Language：English   Publishing type：Research paper (scientific journal)  

A novel photocontrolled compound release system using liposomes and a caged antimicrobial peptide was developed. The caged antimicrobial peptide was activated by UV irradiation, resulting in the formation of pores on the liposome surface to release the contained fluorophores. The compound release could be observed using fluorescence measurements and time-lapse fluorescence microscopy. UV irradiation resulted in a quick release of the inclusion compounds (within 1 min in most cases) under simulated physiological conditions. The proposed system is expected to be applicable in a wide range of fields from cell biology to clinical sciences.

DOI： 10.1021/ja102167m

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/cbic.201000007

Language：English   Publishing type：Research paper (scientific journal)  

Protein labeling provides significant information about protein function. In this research, we developed a novel protein labeling technique by utilizing photoactive yellow protein (PYP). PYP is a small protein (14 kDa) derived from purple bacteria and binds to 7-hydroxycoumarin-3-carboxylic acid as well as to a natural ligand, 4-hydroxycinnamic acid, through a thioester bond with Cys69. Based on the structure and fluorescence property of this coumarin derivative, we designed two fluorescent I probes that bind to PYP. One has an azido moiety, which allows stepwise labeling by click chemistry, and the other is a fluorogenic probe. The live-cell imaging and specific labeling of PYP were achieved by using both probes. The flexibility of the probe design and the small size of the tag protein are great advantages of this system against the existing methods. This novel labeling technique can be used in a wide variety of applications for biological research.

DOI： 10.1021/ja904800k

Language：English   Publishing type：Research paper (scientific journal)  

Techniques for labeling proteins with small molecules have attracted the attention of many life scientists. We have developed a novel protein labeling system that combines a genetically modified, noncatalytic B-lactamase variant and specific mechanism-based fluorescent probes. Rational design of the tag protein and the labeling probes enables highly specific incorporation of the fluorogen. The feasibility of our approach was confirmed by gel electrophoresis, mass spectrometry, fluorescence spectroscopy, and fluorescence microscopic imaging. Labeling techniques that satisfy the dual criteria of specificity and fluorogenicity have rarely been reported. As a consequence, this method could be a broadly useful research tool in the field of life science.

DOI： 10.1021/ja8082285

Language：English   Publishing type：Research paper (scientific journal)  

A novel design principle for F-19 MRI probes detecting protease activity was developed. This principle is based on F-19 MRI signal quenching by the intramolecular paramagnetic effect from Gd3+. The intramolecular Gd3+ dramatically attenuated the F-19 probe signal, and the paramagnetic effect was cancelled by the probe hydrolyzation by caspase-3. Using this probe, it was shown that the probe could detect caspase-3 activity spatially from a phantom image using F-19 MRI.

DOI： 10.1021/ja077058z

Language：English   Publishing type：Research paper (scientific journal)  

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-HI, 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-H I 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-HI 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.

DOI： 10.1021/bi035742u

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/ja026577t

Language：English   Publishing type：Research paper (scientific journal)  

In this review, we summarize design strategies for generating proteins with desired sequences such as long contiguous base pairs and diverse sequence specificities based on the nature of Cys(2)-His(2) zinc finger proteins. Recent progress towards artificial DNA binding proteins has been achieved by structure-based design processes and by selection strategies. Indeed, a multi-zinc finger protein with an 18 (or 27)-base pair address, and new zinc finger proteins for diverse DNA target sites (TATA-box and p53 binding site) have been created successfully. Such novel zinc finger proteins will probably be useful tools in molecular biology and potentially in human medicine. (C) 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0928-0987(00)00212-8

Language：English   Publishing type：Research paper (scientific journal)  

Cys(2)-His(2)-type zinc finger proteins have a tandemly repeated array structure consisting of independent finger modules. They are expected to elevate the DNA binding affinity and specificity by increasing the number of finger modules. To investigate the relation between the number and the DNA binding affinity of the zinc finger, we have designed the two- to four-finger peptides by connecting the central zinc finger (finger 2) of Sp1 with the canonical linker sequence, Thr-Gly-Glu-Lys-Pro. Gel mobility shift assays reveal that the cognate three- and four-finger peptides, Sp1(zf222) and Sp1(zf2222), strongly bind to the predicted target sequences, but the two-finger peptide, Sp1(zf22), does not. Of special interest is the fact that the dissociation constant for Spl(zf2222) binding to the target DNA is comparable to that for Spl(zf222). The methylation interference, DNase I and hydroxyl radical footprintings, and circular permutation analyses demonstrate that Spl(zf2222) binds to its target site with three successive zinc fingers and the binding of the fourth zinc finger is inhibited by DNA bending induced by the binding of the three-finger domain. The present results strongly indicate that the zinc finger protein binds to DNA by the three-finger domain as one binding unit. In addition, this information provides the basis for the design of a novel multifinger protein with high affinity and specificity for long DNA sequences, such as chromosomal DNAs.

DOI： 10.1021/bi001762+

Language：English   Publishing type：Research paper (scientific journal)  

Zinc finger constitutes one of the most common DNA binding motifs. Although zinc finger proteins consisting of Cys(2)His(2), Cys(3)His, Cys(4), and Cys(6) domains are known in nature, a novel His(4) zinc finger protein has never been observed. Herein, we have created the first artificial His(4)-type zinc finger protein (H(4)Sp1) engineered by Cys --> His mutations of the Cys(2)His(2)type zinc finger transcription factor Sp1. The CD features of the single finger H(4)Sp1f2 and three-finger H(4)Sp1 clearly demonstrate the folding of the mutant His(4) peptides by complexation with Zn(II). The NMR study of Zn(II)-H(4)Sp1f2 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)-H(4)Sp1 to the GC-box site of duplex DNA. The methylation interference pattern of Zn(II)-H(4)Sp1 binding significantly resembles that of the corresponding C(2)H(2)Sp1 binding. The present artificial peptide H(4)Sp1 is the first example of a zinc finger containing the His4 domain. Of special interest is the fact that the zinc finger domains of H(4)Sp1 are folded (although not identical to the native structure) and bind DNA similar to wild-type C(2)H(2)Sp1.

DOI： 10.1021/ja994009g

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1021/bi992989b

Event date： 2022.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：兵庫県姫路市   Country：Japan  

Event date： 2022.10

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Language：Japanese  

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The engineering, structure, and DNA binding properties of a novel His4-type zinc finger peptide.
We have created a novel His4-type zinc finger protein (H4Sp1) engineered by Cys-- > His mutations of the Cys2His2-type zinc finger in transcription factor Sp1. The CD and NMR studies reveal that the His4 domain has Zn(II)-dependent folding properties and similar secondary structures to wild-type Cys2His2 domain. The DNA binding experiments demonstrate that H4Sp1 can bind DNA in a specific way. The present artificial peptide H4Sp1 will provide valuable information about the interaction between a metallopeptide and DNA.

DOI： 10.1093/nass/44.1.295

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc.