Hiroyuki Hanzawa

1H nuclear magnetic resonance study of the solution conformation of an antibacterial protein, sapecin.

The solution conformation of an antibacterial protein sapecin has been determined by 1H nuclear magnetic resonance (NMR) and dynamical simulated annealing calculations. It has been shown that the polypeptide fold consists of one flexible loop (residues 4–12), one helix (residues 15–23), and two extended strands (residues 24–31 and 34–40). It was found that the tertiary structure of sapecin is completely different from that of rabbit neutrophil defensin NP‐5, which is homologous to sapecin in the amino acid sequences and also has the antibacterial activity. The three‐dimensional structure determination has revealed that a basic‐residue rich region and the hydrophobic surface face each other on the surface of sapecin.

Characterization of a functional domain of human calpastatin

Expression plasmids were constructed from the cDNA of human calpastatin to examine the contribution to the inhibition of calpain of highly conserved sequences in each of four repetitive domains. A series of deletion derivatives of domain 1 proteins, truncated at either the amino or carboxy terminus, were produced in E. coli. Deletion from the amino terminus past the amino terminal conserved sequence decreased the inhibition. When the middle conserved sequence, the M-sequence, was further deleted, no inhibition was detected, but deletion from the carboxy terminus past the carboxy terminal conserved sequence did not decrease the inhibition until the M-sequence was reached. Nuclear magnetic resonance and circular dichroism spectra showed that domain 1 has an unfolded structure. Peptides that contained the M-sequence and some neighboring sequences were synthesized to measure the minimum size of the inhibitory peptide, which was the M-sequence with the next six residues on the amino terminal side.

Synthesis of a tetrahydropyrano[2,3-d]oxazole analogue of trehazolin

Abstract Tetrahydropyrano[2,3- d ]oxazole 6 as an analogue of trehazolin was synthesized from compound 3 , which is a protected 1,3-bis(β- d -glucopyranosyl)thiourea. In contrast, compound 10 , a protected 1,3-bis(α- d -glucopyranosyl)thiourea, and 15 , a mixed species having one α- d -glucopyranosyl group and one β- d -glucopyranosyl group, did not yield the corresponding pyranooxazolines as anticipated, giving instead, respectively, a furo[2,3- d ]oxazole 14 and a complex mixture of unidentified products.

An Accurate Pharmacophore Mapping Method by NMR Spectroscopy

A wide variety of compound libraries are currently available to obtain active compounds for drug target proteins, but the affinities of initially screened compounds are usually too low and have to be improved by chemical modification of the compounds. In such cases, pharmacophore information of the compounds plays a key role for the next modification step. Structure determination of the protein–compound complex is too time-consuming to be applied for such situations. Therefore, many chemists would highly appreciate simple and accurate experimental procedures to obtain pharmacophore information. From this viewpoint, various ligand-observed NMR spectroscopy experiments have been proposed to characterize protein–ligand interactions. Among them, experiments exploiting nuclear Overhauser effects (NOEs; transferred NOE, saturation transfer difference (STD), pumped NOE, water-ligand observed by gradient spectroscopy (waterLOGSY), etc.) are widely used and are also utilized as a ligand pharmacophore (or epitope) mapping technique. However, it has recently been revealed that the difference of the longitudinal relaxation of each ligand proton severely interferes with the derived pharmacophore mapping result, and it is crucial to evaluate intermolecular cross-relaxation terms for accurate pharmacophore mapping. With this in mind, pharmacophore mapping by diffusion NMR spectroscopy, adiabatic fast passage NOESY (AFP-NOESY), and group epitope mapping considering relaxation of the ligand (GEM-CRL) quantitatively exploit the obtained intermolecular cross-relaxation effect. Herein, we propose a simple and rapid approach for pharmacophore mapping experiments, which utilizes the difference between the longitudinal relaxation rates of ligand protons with and without irradiation of the protons of the target protein. The longitudinal relaxation of ligand proton I is represented by the modified Bloch equation [Eq. (1)]

Proton nuclear magnetic resonance study of human interleukin 6: Chemical modifications and partial spectral assignments for the aromatic residues

Partial assignments for the 1H-NMR resonances of the aromatic residues in human interleukin 6 (IL-6) are reported. The homonuclear Hartmann-Hahn spectrum clearly shows all connectivities for the histidine, tyrosine and tryptophan residues that exist in IL-6. Using a deuterium exchange method, the imidazole proton resonances of His-16 and His-165 have been assigned. Iodination of the tyrosine residues led to the assignment of Tyr-32. Photo-chemically induced dynamic nuclear polarization data have shown that His-16, Tyr-32 and Trp-158 are exposed to solvent, whereas His-165, Tyr-98 and Tyr-101 are buried. Iodination of Tyr-32 gave no significant effect on IL-6 activity, suggesting that Tyr-32 is not responsible for IL-6 activity.

Crystallization and preliminary X-ray crystallographic studies on a Fab fragment of the mouse anti-human Fas monoclonal antibody HFE7A

The Fas-Fas ligand system is involved in apoptosis. The mouse anti-human Fas monoclonal antibody HFE7A (m-HFE7A) has a potential use in human therapy against autoimmune diseases such as rheumatoid arthritis. Information on the three-dimensional structure is essential for antibody humanization. Crystals of an antigen-binding fragment (Fab) of m-HFE7A were obtained by the hanging-drop vapour-diffusion method using sodium citrate as a precipitant and 2-methyl-2,4-pentanediol as an additive. Fast optimization to produce single crystals suitable for X-ray analysis was achieved by the streak-seeding technique. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 43.4, b = 74.0, c = 133.8 A. The crystals diffract at least to 2.5 A resolution.

The three dimensional structure of rat cytokine CINC/Gro in solution by homonuclear 3D NMR

The solution conformation of rat cytokine‐induced neutrophil chemoattractant (CINC/Gro), a small protein consisting of 72 amino acid residues with proinflammatory activities, and a member of the interleukin 8 family corresponding to a counterpart of human Gro, was investigated with homonuclear 2D and 3D NMR spectroscopy. At each phase of the structural analysis, the homonuclear 3D NOESY‐HOHAHA and HOHAHA‐NOESY spectra afforded valuable data, removing ambiguities intractable by conventional 2D NMR techniques. CINC/Gro exists as a dimer in solution and contains a triple stranded anti‐parallel β‐sheet and C‐terminal α‐helix in the monomer structure, as observed in human IL‐8, but non‐trivial differences are also observed.

Crystallization and preliminary x-ray studies of the Fab fragment from a humanized version of the mouse anti-human fas antibody HFE7a.

A humanized version of the apoptosis-inducing mouse anti-human Fas monoclonal antibody, HFE7A, is under further development for the treatment of autoimmune diseases such as rheumatoid arthritis. We have crystallized the antigen-binding fragment (Fab) of the humanized HFE7A. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 54.4 A, b = 82.7 A, c = 104.9 A and contain one Fab molecule in the asymmetric unit. X-ray diffraction data were collected to 2.8 A resolution.