Yousuke Takaoka

Protein organic chemistry and applications for labeling and engineering in live-cell systems.

The modification of proteins with synthetic probes is a powerful means of elucidating and engineering the functions of proteins both in vitro and in live cells or in vivo. Herein we review recent progress in chemistry-based protein modification methods and their application in protein engineering, with particular emphasis on the following four strategies: 1) the bioconjugation reactions of amino acids on the surfaces of natural proteins, mainly applied in test-tube settings; 2) the bioorthogonal reactions of proteins with non-natural functional groups; 3) the coupling of recognition and reactive sites using an enzyme or short peptide tag-probe pair for labeling natural amino acids; and 4) ligand-directed labeling chemistries for the selective labeling of endogenous proteins in living systems. Overall, these techniques represent a useful set of tools for application in chemical biology, with the methods 2-4 in particular being applicable to crude (living) habitats. Although still in its infancy, the use of organic chemistry for the manipulation of endogenous proteins, with subsequent applications in living systems, represents a worthy challenge for many chemists.

Specific Cell Surface Protein Imaging by Extended Self-Assembling Fluorescent Turn-on Nanoprobes

Visualization of tumor-specific protein biomarkers on cell membranes has the potential to contribute greatly to basic biological research and therapeutic applications. We recently reported a unique supramolecular strategy for specific protein detection using self-assembling fluorescent nanoprobes consisting of a hydrophilic protein ligand and a hydrophobic BODIPY fluorophore in test tube settings. This method is based on recognition-driven disassembly of the nanoprobes, which induces a clear turn-on fluorescent signal. In the present study, we have successfully extended the range of applicable fluorophores to the more hydrophilic ones such as fluorescein or rhodamine by introducing a hydrophobic module near the fluorophore. Increasing the range of available fluorophores allowed selective imaging of membrane-bound proteins under live cell conditions. That is, overexpressed folate receptor (FR) or hypoxia-inducible membrane-bound carbonic anhydrases (CA) on live cell surfaces as cancer-specific biomarkers were fluorescently visualized using the designed supramolecular nanoprobes in the turn-on manner. Moreover, a cell-based inhibitor-assay platform for CA on a live cell surface was constructed, highlighting the potential applicability of the self-assembling turn-on probes.

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.

Intracellular Protein-Responsive Supramolecules: Protein Sensing and In-Cell Construction of Inhibitor Assay System

Supramolecular nanomaterials responsive to specific intracellular proteins should be greatly promising for protein sensing and imaging, controlled drug release or dynamic regulation of cellular processes. However, valid design strategies to create useful probes are poorly developed, particularly for proteins inside living cells as targets. We recently reported a unique supramolecular strategy for specific protein detection using self-assembling fluorescent probes consisting of a protein ligand and a fluorophore on the live cell surface, as well as in test tube settings. Herein, we discovered that our self-assembled supramolecular probes having a rhodamine derivative (tetramethylrhodamine or rhodamine-green) can incorporate and stay as less-fluorescent aggregates inside the living cells, so as to sense the protein activity in a reversible manner. Using the overexpressed model protein (dihydrofolate reductase), we demonstrated that this turn-on/off mode is controlled by selective ligand-protein recognition inside the live cells. Not only such a model protein, but also endogenous human carbonic anhydrase and heat shock protein 90 were specifically visualized in living mammalian cells, by use of the similar ligand-tethered supramolecular probes. Furthermore, such reversibility allowed us to intracellularly construct a unique system to evaluate the inhibitors affinity toward specific endogenous proteins in live cells, highlighting the potential of dynamic supramolecules as novel intelligent biomaterials.

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.

Semisynthetic Lectin–4-Dimethylaminopyridine Conjugates for Labeling and Profiling Glycoproteins on Live Cell Surfaces

Glycoproteins on cell surfaces play important roles in biological processes, including cell-cell interaction/signaling, immune response, and cell differentiation. Given the diversity of the structure of glycans, labeling and imaging of selected glycoproteins are challenging, although several promising strategies have been developed recently. Here, we design and construct semisynthetic reactive lectins (sugar-binding proteins) that are able to selectively label glycoproteins. Congerin II, an animal galectin, and wheat germ agglutinin are conjugated with 4-dimethylaminopyridine (DMAP), a well-known acyl transfer catalyst by our affinity-guided DMAP method and Cu(I)-assisted click chemistry. Selective labeling of glycoproteins is facilitated by the DMAP-tethered lectin catalysts both in vitro and on living cells. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis enabled us to isolate labeled glycoproteins that are uniquely exposed on distinct cell lines. Furthermore, the combination of immunoprecipitation with mass spectrometry (MS)-fingerprinting techniques allowed us to characterize 48 glycoproteins endogenously expressed on HeLa cells, and some low-abundant glycoproteins, such as epidermal growth factor receptor (EGFR) and neuropilin-1, were successfully identified. Our results demonstrate that semisynthetic DMAP-tethered lectins provide a new tool for labeling and profiling glycoproteins on living cells.

Analysis of Cell-Surface Receptor Dynamics through Covalent Labeling by Catalyst-Tethered Antibody

A general technique for introducing biophysical probes into selected receptors in their native environment is valuable for the study of their structure, dynamics, function, and molecular interactions. A number of such techniques rely on genetic engineering, which is not applicable for the study of endogenous proteins, and such approaches often suffer from artifacts due to the overexpression and bulky size of the probes/protein tags used. Here we designed novel catalyst-antibody conjugates capable of introducing small chemical probes into receptor proteins such as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in a selective manner on the surface of living cells. Because of the selectivity and efficiency of this labeling technique, we were able to monitor the cellular dynamics and lifetime of HER2 endogenously expressed on cancer cells. More significantly, the current labeling technique comprises a stable covalent bond, which combined with a peptide mass fingerprinting analysis allowed epitope mapping of antibodies on living cells and identification of potential binding sites of anti-EGFR affibody. Although as yet unreported in the literature, the binding sites predicted by our labeling method were consistently supported by the subsequent mutation and binding assay experiments. In addition, this covalent labeling method provided experimental evidence that HER2 exhibits a more dynamic structure than expected on the basis of crystallographic analysis alone. Our novel catalyst-antibody conjugates are expected to provide a general tool for investigating the protein trafficking, fluctuation, and molecular interactions of an important class of cell-surface receptors on live cell surfaces.

Affinity-Labeling-Based Introduction of a Reactive Handle for Natural Protein Modification

A new chemical method to site-specifically modify natural proteins without the need for genetic manipulation is described. Our strategy involves the affinity-labeling-based attachment of a unique reactive handle at the surface of the target protein, and the subsequent selective transformation of the reactive handle by a bioorthogonal reaction to introduce a variety of functional probes into the protein. To demonstrate this approach, we synthesized labeling reagents that contain: 1) a benzenesulfonamide ligand that directs specifically to bovine carbonic anhydrase II (bCA), 2) an electrophilic epoxide group for protein labeling, 3) an exchangeable hydrazone bond linking the ligand and the epoxide group, and 4) an iodophenyl or acetylene handle. By incubating the labeling reagent with bCA, the reactive handle was covalently attached at the surface of bCA through epoxide ring opening. Either after or before removing the ligand by a hydrazone/oxime-exchange reaction, which restores the enzymatic activity, the reactive handle incorporated could be derivatized by Suzuki coupling or Huisgen cycloaddition reactions. This method is also applicable to the target-specific multiple modification in a protein mixture. The availability of various (photo)affinity-labeling reagents and bioorthogonal reactions should extend the flexibility of this strategy for the site-selective incorporation of many functional molecules into proteins.

One-step construction of caged carbonic anhydrase I using a ligand-directed acyl imidazole-based protein labeling method

Caged enzymes whose activities can be controlled by light represent a powerful tool for various biological analyses. However, limited methods are available for the construction of caged proteins and enzymes. We recently developed a novel protein labeling method termed ligand-directed acyl imidazole (LDAI) chemistry, which allows us to selectively modify natural dihydrofolate reductase and folate receptor in a test tube and in live cell contexts. In this work, we have examined in detail the reaction characteristics of the LDAI chemistry using carbonic anhydrase I (CAI) as a model enzyme. In addition to modifying Lys residues with a carbamate bond, the LDAI method modified Ser and Tyr residues with a carbonate bond. Owing to the relatively labile carbonate bond formed, the LDAI chemistry was demonstrated to be applicable for a rational one-step construction of caged enzymes. This method is simple and based on the transient tethering of an inhibitor with moderate activity that is directed to the active site on an enzyme surface. We successfully showed that the activity of the caged CAI was almost completely suppressed by LDAI-based labeling and fully recovered by photoirradiation in the crude conditions (such as cell lysates) as well as in test tube settings.

Heme Binding Mechanism of Structurally Similar Iron-Regulated Surface Determinant Near Transporter Domains of Staphylococcus aureus Exhibiting Different Affinities for Heme

Near transporter (NEAT) domains of the iron-regulated surface determinant (Isd) proteins are essential for the import of nutritional heme from host animals to Gram-positive pathogens such as Staphylococcus aureus. The order of transfer of heme between NEAT domains occurs from IsdH to IsdA to IsdC, without any energy input despite the similarity of their three-dimensional structures. We measured the free energy of binding of heme and various metalloporphyrins to each NEAT domain and found that the affinity of heme and non-iron porphyrins for NEAT domains increased gradually in the same order as that for heme transfer. To gain insight into the atomistic mechanism for the differential affinities, we performed in silico molecular dynamics simulation and in vitro site-directed mutagenesis. The simulations revealed that the negatively charged residues that are abundant in the loop between strand β1b and the 310 helix of IsdH-NEAT3 destabilize the interaction with the propionate group of heme. The higher affinity of IsdC was in part attributed to the formation of a salt bridge between its unique residue, Glu88, and the conserved Arg100 upon binding to heme. In addition, we found that Phe130 of IsdC makes the β7-β8 hairpin less flexible in the ligand-free form, which serves to reduce the magnitude of the entropy loss on binding to heme. We confirmed that substitution of these key residues of IsdC decreased its affinity for heme. Furthermore, IsdC mutants, whose affinities for heme were lower than those of IsdA, transferred heme back to IsdA. Thus, NEAT domains have evolved the characteristic residues on the common structural scaffold such that they exhibit different affinities for heme, thus promoting the efficient transfer of heme.