Katsuyoshi Masuda

Crystal Structure of Squid Rhodopsin with Intracellularly Extended Cytoplasmic Region

G-protein-coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical G-protein-coupled receptor involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7Å resolution, which transduces signals through the Gq protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to Gt, and humanβ2-adrenergic receptor, coupling to Gs. Notably, squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and humanβ2-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic α-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr-111, Asn-87, and Asn-185 residues are located within hydrogen-bonding distances from the nitrogen atom of the Schiff base.

Pairing of oligosaccharides in the Fc region of immunoglobulin G

The Fc portion of immunoglobulin G (IgG) expresses paired oligosaccharides with microheterogeneities, which are associated with efficiencies of effector functions and with pathological states. A comparison of electrospray ionization mass spectrometry data obtained using a variety of Fc fragments derived from human and mouse IgG that do and do not retain the inter‐chain disulfide bridge(s) revealed that (1) the Fc portion can be asymmetric as well as symmetric with respect to glycosylation and (2) the ratios of the individual glycoforms are different from what is expected from the random pairing.

Characterization of the β-d-Glucopyranoside Binding Site of the Human Bitter Taste Receptor hTAS2R16

G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point mutants of hTAS2R16 to identify its binding sites. The docking simulation predicted two candidate binding structures for a salicin-hTAS2R16 complex, and at least seven amino acid residues in transmembrane 3 (TM3), TM5, and TM6 were shown to be involved in ligand recognition. We also identified the probable salicin-hTAS2R16 binding mode using a mutated receptor experiment. This study characterizes the molecular interaction between hTAS2R16 and β-d-glucopyranoside and will also facilitate rational design of bitter blockers.

Characterization of the Modes of Binding between Human Sweet Taste Receptor and Low-Molecular-Weight Sweet Compounds

One of the most distinctive features of human sweet taste perception is its broad tuning to chemically diverse compounds ranging from low-molecular-weight sweeteners to sweet-tasting proteins. Many reports suggest that the human sweet taste receptor (hT1R2–hT1R3), a heteromeric complex composed of T1R2 and T1R3 subunits belonging to the class C G protein–coupled receptor family, has multiple binding sites for these sweeteners. However, it remains unclear how the same receptor recognizes such diverse structures. Here we aim to characterize the modes of binding between hT1R2–hT1R3 and low-molecular-weight sweet compounds by functional analysis of a series of site-directed mutants and by molecular modeling–based docking simulation at the binding pocket formed on the large extracellular amino-terminal domain (ATD) of hT1R2. We successfully determined the amino acid residues responsible for binding to sweeteners in the cleft of hT1R2 ATD. Our results suggest that individual ligands have sets of specific residues for binding in correspondence with the chemical structures and other residues responsible for interacting with multiple ligands.

Comparative thermodynamic analyses of the Fv, Fab* and Fab and Fab fragments of anti-dansyl mouse monoclonal antibody

In order to investigate the role of the constant domainson the antigen‐binding property of the variable domains, we have carried out a comparative thermodynamic study of the anti‐dansyl Fv, Fab* and Fab fragments that possess the identical amino acid sequence of the variable domains. The thermodynamic analyses have shown that binding constants, enthalphy changes and entropy changes are similar for the three antigen‐binding fragments, whereas the thermal stability of Fab is much higher than that of Fv and Fab*. We have concluded that (i) the variable domains of the three antigen‐binding fragments possess identical intrinsic capability for antigen binding and (ii) the two constant domains serve to improve the stability of the variable domains.

pKa measurements from nuclear magnetic resonance of tyrosine-150 in class C beta-lactamase.

13C-NMR spectroscopy was used to estimate the p K a values for the Tyr(150) (Y150) residue in wild-type and mutant class C beta-lactamases. The tyrosine residues of the wild-type and mutant lactamases were replaced with (13)C-labelled L-tyrosine ([ phenol -4-(13)C]tyrosine) in order to observe the tyrosine residues selectively. Spectra of the wild-type and K67C mutant (Lys(67)-->Cys) enzyme were compared with the Y150C mutant lactamase spectra to identify the signal originating from Tyr(150). Titration experiments showed that the chemical shift of the Tyr(150) resonance in the wild-type enzyme is almost invariant in a range of 0.1 p.p.m. up to pH 11 and showed that the p K (a) of this residue is well above 11 in the substrate-free form. According to solvent accessibility calculations on X-ray-derived structures, the phenolic oxygen of Tyr(150), which is near the amino groups of Lys(315) and Lys(67), appears to have low solvent accessibility. These results suggest that, in the native enzyme, Tyr(150) in class C beta-lactamase of Citrobacter freundii GN346 is protonated and that when Tyr(150) loses a proton, a proton from Lys(67) would replace it. Consequently, Tyr(150) would be protonated during the entire titration.

Synthesis of Diazirinyl Photoprobe Carrying a Novel Cleavable Biotin

Photoaffinity labeling is a powerful tool for the identification of receptor proteins and their binding sites. The major drawback of this method, however, is the reliability of the labeling and the purity of labeled peptides after purification from a large number of unlabeled fragments. The recent development of diazirine-based biotinylated probes provides an efficient solution to this problem. In these probes, the diazirine photophore contributes to the formation of a stable cross-link that greatly improves the efficiency. The application of an N-acetylglucosamine photoprobe carrying biotinylated diazirine provided the first information regarding acceptor-site peptides of b-1,4-galactosyltransferase. We have also developed a novel method for the one-step introduction of a biotinylated diazirine photophore into unprotected carbohydrate ligands. Since biotin–avidin binding is essentially irreversible (Kd = 10 15 m), several approaches have been investigated to achieve efficient recovery of biotinylated products from an immobilized avidin matrix. Although the use of monomeric avidin contributes to the isolation of biotinylated products because of the lower affinity to biotin (Kd = 10 8 m), it requires a high

Alternative One-Pot Synthesis of (Trifluoromethyl)phenyldiazirines from Tosyloxime Derivatives: Application for New Synthesis of Optically Pure Diazirinylphenylalanines for Photoaffinity Labeling

Alternative one-pot synthesis of 3-(trifluoromethyl)-3-phenyldiazirine derivatives from corresponding tosyloximes is developed. The deprotonation of intermediate diaziridine by NH2(-) is a new approach for construction of diazirine. Moreover, a novel synthesis of optically pure (trifluoromethyl)diazirinylphenylalanine derivatives was attempted involving these methods.

Comprehensive synthesis of photoreactive (3-trifluoromethyl)diazirinyl indole derivatives from 5- and 6- trifluoroacetylindoles for photoaffinity labeling.

5- and 6-trifluoromethyldiazirinyl indoles were synthesized from corresponding bromoindole derivatives for the first time. They acted as mother skeletons for the comprehensive synthesis of various bioactive indole metabolites. These can be used in biological functional analysis as diazirine-based photoaffinity labels.

Photoactive ligands probing the sweet taste receptor. Design and synthesis of highly potent diazirinyl D-phenylalanine derivatives

Some D-amino acids such as d-tryptophan and D-phenylalanine are well known as naturally-occurring sweeteners. Photoreactive D-phenylalanine derivatives containing trifluoromethyldiazirinyl moiety at 3- or 4-position of phenylalanine, were designed as sweeteners for functional analysis with photoaffinity labeling. The trifluoromethyldiazirinyl D-phenylalanine derivatives were prepared effectively with chemo-enzymatic methods using L-amino acid oxidase and were found to have potent activity toward the human sweet taste receptor.