Shinya Tsukiji

Sortase-mediated ligation: a gift from Gram-positive bacteria to protein engineering.

A new enzymatic protein ligation tool, sortase, has recently emerged from Gram‐positive bacteria. This article outlines the technique, sortase‐mediated ligation, and its applications in protein engineering, which include the introduction of unnatural molecules into proteins, protein immobilization, protein–protein conjugation, protein cyclization, as a self‐cleavable tag for protein expression, protein–PNA hybrids, neoglycoconjugates, and cell‐surface protein labeling, etc.

Site‐Specific Protein Modification on Living Cells Catalyzed by Sortase

The use of enzymes is a promising approach for site‐specific protein modification on living cells owing to their substrate specificity. Herein we describe a general strategy for the site‐specific modification of cell surface proteins with synthetic molecules by using Sortase, a transpeptidase from Staphylococcus aureus. The short peptide tag LPETGG is genetically introduced to the C terminus of the target protein, expressed on the cell surface. Subsequent addition of Sortase and an N‐terminal triglycine‐containing probe results in the site‐specific labeling of the tagged protein. We were successful in the C‐terminal‐specific labeling of osteoclast differentiation factor (ODF) with a biotin‐ or fluorophore‐containing short peptide on the living cell surface. The labeling reaction occurred efficiently in serum‐containing medium, as well as serum‐free medium or PBS. The labeled products were detected after incubation for 5 min. In addition, site‐specific protein–protein conjugation was successfully demonstrated on a living cell surface by the Sortase‐catalyzed reaction. This strategy provides a powerful tool for cell biology and cell surface engineering.

An alcohol dehydrogenase ribozyme

We report an RNA molecule that exhibits activity analogous to that of alcohol dehydrogenase (ADH). Directed in vitro evolution was used to enrich nicotinamide adenine dinucleotide (NAD+)–dependent redox-active RNAs from a combinatorial pool. The most active ribozyme in the population forms a compact pseudoknotted structure and oxidizes an alcohol seven orders of magnitude faster than the estimated spontaneous rate. Moreover, this ADH RNA was coupled with a redox relay between NADH and flavin adenine dinucleotide to give a NAD+-regeneration system. Our demonstration of the redox ability of RNA adds support to an RNA-based metabolic system in ancient life.

Quenched ligand-directed tosylate reagents for one-step construction of turn-on fluorescent biosensors.

Semisynthetic fluorescent biosensors consisting of a protein framework and a synthetic fluorophore are powerful analytical tools for specific detection of biologically relevant molecules. We report herein a novel method that allows for the construction of turn-on fluorescent semisynthetic biosensors in a one-step manner. The strategy is based on the ligand-directed tosyl (LDT) chemistry, a new type of affinity-guided protein labeling scheme which can site-specifically introduce synthetic probes to the surface of proteins with concomitant release of the affinity ligands. Novel quenched ligand-directed tosylate (Q-LDT) reagents were designed by connecting an organic dye to a conjugate of a protein ligand and a fluorescence quencher through a tosyl linker. The Q-LDT-mediated labeling directly converts a natural protein to a fluorescently labeled protein that remains noncovalently complexed with the cleaved ligand-tethered quencher. The fluorescence of this labeled protein is initially quenched and only in the presence of specific analytes is the fluorescence enhanced (turned on) due to the expulsion of the ligand-quencher fragment. Using a single labeling step, this approach was successfully applied to carbonic anhydrase II (CAII) and a Src homology 2 (SH2) domain to generate turn-on fluorescent biosensors toward CAII inhibitors and phosphotyrosine peptides, respectively. Detailed investigations revealed that the obtained biosensors exhibit their natural ligand selectivity. The high target-specificity of the LDT chemistry also allowed us to prepare the SH2 domain-based biosensor not only in a purified form but also in a bacterial cell lysate. These results demonstrate the utility of the Q-LDT-based approach to expand the applications of semisynthetic biosensors.

Site-specific covalent labeling of His-tag fused proteins with a reactive Ni(II)-NTA probe.

A new method for covalent labeling of a His-tag fused protein with a small reactive probe was developed; this method is based on the complementary interaction between the His-tag and Ni(II)-NTA, which facilitates a nucleophilic reaction between a histidine residue of the tag and the electrophilic tosyl group of the Ni(II)-NTA probe by the proximity effect.

Fluorophore Labeling of Native FKBP12 by Ligand-Directed Tosyl Chemistry Allows Detection of Its Molecular Interactions in Vitro and in Living Cells

Introducing synthetic fluorophores into specific endogenous proteins and analyzing their function in living cells are a great challenge in chemical biology. Toward this end, we demonstrate the target-selective and site-specific fluorescent labeling of native FKBP12 (FK506-binding protein 12) in vitro and in living cells using ligand-directed tosyl (LDT) chemistry. The LDT-mediated labeling yielded a semisynthetic FKBP12 containing the Oregon green (OG) dye near the catalytic pocket. The OG-labeled FKBP12 (OG-FKBP12) acted as a fluorescent reporter that allows monitoring of its interaction with rapamycin and FRB (FKBP-rapamycin-binding domain) in vitro. We also successfully demonstrated the visualization of the rapamycin-mediated complexation of the OG-FKBP12 and FRB inside of living cells by the combined use with fluorescent protein-tag technology and Förster resonance energy-transfer imaging.

Mechanisms of chemical protein 19F-labeling and NMR-based biosensor construction in vitro and in cells using self-assembling ligand-directed tosylate compounds

Chemical labeling methods that convert a specific endogenous protein into a semisynthetic biosensor offer numerous new opportunities for biological research and drug discovery. We recently developed a novel protein labeling scheme, termed ligand-directed tosyl (LDT) chemistry, which can site-specifically introduce a synthetic probe to a protein with the concomitant release of the affinity ligand. In previous work, we demonstrated that LDT reagent 1 can be used to modify carbonic anhydrase I (CAI) with a 19F probe, converting it into a 19F NMR-based biosensor for CAI inhibitors either in vitro or in red blood cells (RBCs). We herein report the chemical properties of 1, and the mechanisms controlling biosensor construction. It was revealed that the LDT reagent forms self-assembled aggregates in the absence of the target protein. In the aggregated state, nonproductive hydrolysis of the reagent was significantly suppressed, which suggests the potential utility of self-assembly in the design of labeling reagents that have increased stability. In the presence of the target protein, the aggregates were disrupted to form a noncovalent protein–reagent complex, and protein 19F-labeling proceeded to generate 19F-labeled CAI. The ligand-binding pocket of the labeled CAI retained the cleaved ligand fragment in vitro, whereas the pocket was vacant in RBC. Further biochemical studies suggested that an anion transporter might play a role in eliminating the cleaved ligand from the interior to the exterior of the cells. The findings provide a fundamental basis for the rational design of reagents applicable to selective protein labeling and biosensor construction in biological contexts.

Successful Control of Aggregation and Folding Rates during Refolding of Denatured Lysozyme by Adding N-Methylimidazolium Cations with Various N'-Substituents

The present study aimed to obtain more effective refolding agents and to understand the influence of their chemical structures on their function as refolding agents. To achieve these aims, we investigated the effects of a large variety of N′‐substituted N‐methylimidazolium chlorides on the oxidative refolding of lysozyme in a high throughput manner. Among the molecules examined, N‐methylimidazolium cations with a short N′‐alkyl chain, such as an N′‐ethyl or N′‐butyl chain, significantly enhanced the refolding yield compared to conventional refolding additives such as arginine hydrochloride and Triton X‐100. Detailed kinetic analyses revealed that the effective cations selectively decreased the aggregation rate constant ( kA) without any large decreases in the folding rate constant ( kN). However, when the hydrophobicity of the N′‐substituent of the cations was increased, the desirable properties of the short N′‐alkyl chain‐type cations for protein refolding were diminished. Furthermore, increases in the N′‐alkyl chain length to an N′‐octyl or N′‐dodecyl chain drastically decreased the kA values, thereby increasing the ratio of kN to kA, despite the very small kN values and resulting in enhanced refolding yields. Thus, by tuning the chemical structure of the N′‐substituents of N‐methylimidazolium chloride, five effective refolding agents ( N′‐ethyl‐, N′‐propyl‐, N′‐butyl‐, N′‐pentyl‐ and N′‐isobutyl‐ N‐methylimidazolium chlorides) were successfully obtained, and the kinetic parameters of folding and aggregation during the refolding process could be controlled using three different modes.

Synthetic Self-Localizing Ligands That Control the Spatial Location of Proteins in Living Cells

Small-molecule ligands that control the spatial location of proteins in living cells would be valuable tools for regulating biological systems. However, the creation of such molecules remains almost unexplored because of the lack of a design methodology. Here we introduce a conceptually new type of synthetic ligands, self-localizing ligands (SLLs), which spontaneously localize to specific subcellular regions in mammalian cells. We show that SLLs bind their target proteins and relocate (tether) them rapidly from the cytoplasm to their targeting sites, thus serving as synthetic protein translocators. SLL-induced protein translocation enables us to manipulate diverse synthetic/endogenous signaling pathways. The method is also applicable to reversible protein translocation and allows control of multiple proteins at different times and locations in the same cell. These results demonstrate the usefulness of SLLs in the spatial (and temporal) control of intracellular protein distribution and biological processes, opening a new direction in the design of small-molecule tools or drugs for cell regulation.

Zn(II) dipicolylamine-based artificial receptor as a new entry for surface recognition of α-helical peptides in aqueous solution

Abstract It is clear by CD spectral titration that Zn(II)dipicolylamine-based dinuclear complexes selectively bind and stabilize the α-helix conformation of peptides having two histidine (His) residues at specific positions ( H-i and i +4 or i +7 or i +11).