Yuichiro Hori

Covalent Protein Labeling Based on Noncatalytic β-Lactamase and a Designed FRET Substrate

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 beta-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.

Photoactive Yellow Protein-Based Protein Labeling System with Turn-On Fluorescence Intensity

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 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.

Development of fluorogenic probes for quick no-wash live-cell imaging of intracellular proteins.

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.

Small-Molecule-Based Protein-Labeling Technology in Live Cell Studies: Probe-Design Concepts and Applications

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.

Development of a fluorogenic probe with a transesterification switch for detection of histone deacetylase activity.

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.

Multicolor Protein Labeling in Living Cells Using Mutant β-Lactamase-Tag Technology

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.

Development of Protein‐Labeling Probes with a Redesigned Fluorogenic Switch Based on Intramolecular Association for No‐Wash Live‐Cell Imaging

Fluorescence labeling of proteins is a powerful technique for studying precise protein localization and movement in living cells. Currently, fluorescent proteins (FPs) are the primary tools in this field owing to their technical feasibility and convenience. Although various FPs have been reported, their properties such as protein size and brightness are not completely satisfactory for some biological applications. As an alternative to FPs, chemical methods utilizing synthetic fluorescence probes and fusion proteins have recently emerged. In this imaging technique, a ligand-binding domain is fused to the protein of interest (POI) as a protein (peptide) tag and is reacted with a fluorophore-conjugated ligand. Thus, the POI is labeled by the fluorophore through the linkage of the tag and the ligand. Representative examples of previously commercialized protein (peptide) tags are the HaloTag, the SNAP tag, and the tetracysteine tag. The key characteristics of these techniques are that POIs are conditionally labeled by the temporal addition of probes, and various fluorophores can easily be incorporated into probes by replacing just the fluorophore moiety. However, since the fluorescence of free probes or probes bound to nontarget biomolecules interfere with the identification of the labeled target protein, thorough washing of cells is necessary to remove free probes. This is a time-consuming process, and incomplete washing causes a decrease in the signal-to-noise ratio. To solve this problem, we have developed proteinlabeling probes that do not require any washing procedure for live-cell imaging. As possible approaches to the problem, we and other groups have reported turn-on fluorescence labeling systems, in which the fluorescence of a probe is quenched in a free state and is recovered in a protein-tag-bound state. These fluorogenic probes minimize background fluorescence and overcome the limitation of conventional protein-labeling systems. Although many protein-labeling methods are known, fluorogenic methods that do not require any washing are still restricted to a few protein-tagging systems. Therefore, the further development of novel fluorogenic systems is required. Based on the quenching mechanism of intramolecular association, we have recently created a fluorogenic probe, FCTP, for labeling the photoactive yellow protein (PYP) tag (Scheme 1a). The PYP tag is derived from purple bacteria and binds to the thioester derivatives of 4-hydroxycinnamic acid, a natural cofactor, or 7-hydroxycoumarin through transthioesterification with residue Cys69. 13] The small size of the PYP tag (14 kDa; half the size of the green fluorescent protein, GFP) makes this protein particularly interesting, as

Photocontrolled Compound Release System Using Caged Antimicrobial Peptide

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.

Protein labeling with fluorogenic probes for no-wash live-cell imaging of 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.

Intracellular Protein Labeling with Prodrug‐Like Probes Using a Mutant β‐Lactamase Tag

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.