Biomedical subjects
Publications and source records attributed to Mitsuru Tashiro.
The very specific binding of the HgII ion unexpectedly and significantly stabilizes naturally occurring thymine-thymine base mispairing in DNA duplexes. Following this finding, we prepared DNA duplexes containing metal-mediated base pairs at the desired sites, as well as novel double helical architectures consisting only of thymine-HgII-thymine pairs.
Spectroscopic elucidation of a 2-isopropylmalic acid (2-iPMA)-aluminum(III) complex has been carried out using (1)H, (13)C and (27)Al NMR spectroscopy, diffusion-ordered NMR spectroscopy (DOSY) and electrospray ionization mass spectrometry (ESI-MS). 2-iPMA is secreted by Saccharomyces cerevisiae and can dissolve Al(III) in the culture medium. The (1)H chemical shift perturbation and (1)H DOSY clearly indicated the formation of the 2-iPMA-Al(III) complex. The measurements of (13)C and (27)Al NMR spectroscopy and ESI-MS demonstrated that the major form of a complex is comprised four 2-iPMA and two Al(III) species. This compound is expected to possess strong Al(III)-detoxification capability.
A structural characterization of bound water molecules in the cyclic tetrasaccharide, cyclo-{-->6}-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->), was carried out by NMR spectroscopy. H-1', 2'-OH, H-3', and 4'-OH of the 3-O-glycosylated residue and H-1 of the 6-O-glycosylated residue were found to cross-relax with protons of bound waters using the double-pulsed field-gradient spin-echo ROESY experiment. In the crystal structure, one water molecule is located in the center of the plate, and its temperature factor is very low, indicating that this water molecule is an intrinsic component.
Water ligand observed via gradient spectroscopy (WaterLOGSY), saturation transfer difference and NOE pumping NMR techniques were used to identify ligand binding with a receptor. Although these experiments were originally designed to observe ligands in complexes, their application is limited by the affinity of ligands towards target molecules. Here the improved WaterLOGSY pulse sequence was developed by incorporating the double pulsed field gradient spin-echo and gradient-tailored excitation WATERGATE sequences. The efficiency of these ligand-observed NMR screening techniques was investigated using the ribonuclease T1-inhibitor system.
ROESY pulse sequences are presented and evaluated to identify bound waters in the cyclic tetrasaccharide. The first experiment incorporated the double-pulsed field gradient spin-echo (DPFGSE) for selective water excitation at the initial portion of the pulse sequence. Although long, shaped pulses were used in DPFGSE to achieve the highly selective excitation of water resonance that is very close to resonances of the cyclic tetrasaccharide, the approach was not effective because of the loss of sensitivity. Concomitant use of long delays and moderate length of shaped pulses in the portion of DPFGSE gained more sensitivity. A simple approach incorporating spin-echo with long delays instead of DPFGSE also afforded a sensitive spectrum. Practical aspects of these ROESY experiments are illustrated using the cyclic tetrasaccharide cyclo-{-->6}-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->).
A complex of 1,6-anhydro-beta-maltose with rubidium and that of 1,6-anhydro-beta-D-glucopyranose with rubidium were characterized using 87Rb NMR spectroscopy, diffusion-ordered NMR spectroscopy (DOSY) and electrospray ionization mass spectrometry (ESI-MS). Although subtle differences were observed in the 1H chemical shifts of 1,6-anhydro-beta-maltose in between the presence and absence of rubidium in deuterium oxide, measurements of the spin-lattice relaxation time (T1) of the 87Rb nucleus, the diffusion coefficients of 1,6-anhydro-beta-maltose using 1H DOSY and ESI-MS indicated the complex formation of 1,6-anhydro-beta-maltose with rubidium. The complex formation with rubidium was also identified for 1,6-anhydro-beta-D-glucopyranose using NMR and ESI-MS techniques.
The characterization of water molecules bound to ribonuclease T1 (RNase T1) was carried out using cold-spray ionization mass spectrometry (CSI-MS). CSI-MS is a variant of electrospray ionization mass spectrometry (ESI-MS) operating at low temperature, and is particularly suitable for investigating the weaker molecular associations, since the temperature at the spray interface is much lower than that in the conventional ESI-MS. In this approach, ion peaks due to the addition of nine water molecules were identified at a spray temperature of 48 degrees C. This result showed good agreement with that inferred by the combinational analysis of NMR and X-ray crystallography, indicating that CSI-MS is capable of rapidly providing reliable information to characterize the number of water molecules bound to a macromolecule.
Thermal unfolding of ribonculease (RNase) T1 was studied by 1H nuclear Overhauser enhancement spectroscopy (NOESY) and 1H- 15N heteronuclear single-quantum coherence (HSQC) NMR spectroscopy at various temperatures. Native RNase T1 is a single-chain molecule of 104 amino acid residues, and has a single alpha-helix and two beta-sheets, A and B, which consist of two and five strands, respectively. Singular value decomposition analysis based on temperature-dependent HSQC spectra revealed that the thermal unfolding of RNase T1 can be described by a two-state transition model. The midpoint temperature and the change in enthalpy were determined as 54.0 degrees C and 696 kJ/mol, respectively, which are consistent with results obtained by other methods. To analyze the transition profile in more detail, we investigated local structural changes using temperature-dependent NOE intensities. The results indicate that the helical region starts to unfold at lower temperature than some beta-strands (B3, B4, and B5 in beta-sheet B). These beta-strands correspond to the hydrophobic cluster region, which had been expected to be a folding core. This was confirmed by structure calculations using the residual NOEs observed at 56 degrees C. Thus, the two-state transition of RNase T1 appears to involve locally different conformational changes.
A difference diffusion-based NMR technique and cold-spray ionization mass spectrometry were employed as a solution-based approach for identifying a ligand binding with a protein receptor. The difference diffusion-based NMR technique, called difference NOE-pumping, can directly detect the ligand interacting with a protein receptor. This technique uses a simple pulse sequence and the diffusion filter can easily be optimized. The cold-spray ionization mass spectrometry (CSI-MS), a variant of electrospray ionization mass spectrometry (ESI-MS) operating at low temperature, has been applied to detect the ligand-receptor complex. The efficiency of these techniques for identifying binding ligands is demonstrated with the human serum albumin (HSA)-drug system.
A structural characterization of bound water molecules in ribonuclease T1 (RNase T1) was carried out by nuclear magnetic resonance spectroscopy and molecular dynamics simulation. Amide protons of residues Trp59, Leu62, Tyr68 and Phe100 were found to cross-relax with protons of bound waters. Molecular dynamics simulations of the 120 water molecules observed in the free form of the crystal structure indicate that these amide protons donate hydrogen bonds to the less mobile water molecules. Hydrogen-bonded chains of the water molecules that are identified in the simulation study are located in the hairpin-like loop of RNase T1, comprising residues 62 to 76. The temperature factors of the observed water molecules in the crystal structure are very low, indicating that these bound waters are intrinsic components of RNase T1.
19F NMR techniques were employed to characterize the binding property of the widely used general anesthetic halothane with human serum albumin (HSA). It was found that 19F(1H) NOE and 2D 1H-19F HOESY experiments detected intermolecular NOEs between halothane 19F and HSA protons. Measurements of the diffusion coefficients for halothane were also carried out by 1H and 19F NMR, indicating the interaction of halothane with HSA. The present results indicate that these techniques are very suitable to identify a fluorine-containing ligand binding with a protein receptor in the drug-discovery process.
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Multidimensional T1rho-, diffusion-filtered and diffusion-ordered NOESY techniques were applied to identify segments of a ligand binding with a protein receptor. These experiments can easily provide intermolecular NOEs of the complex, which are of great significance for characterizing the binding epitopes of a ligand. This information cannot be obtained by high-throughput 1D NMR experiments, used for determining the binding affinity, although multidimensional NMR experiments require more experiment time. The present results indicate that the current experiments using T1rho- and diffusion-edited techniques are very suitable for identifying segments of a ligand binding with a protein receptor in the drug discovery process.
The 1H-magnetic resonance imaging technique was applied for monitoring the extent of the heat coagulation in the shell egg. It is demonstrated that spin-spin relaxation time (T2) is an effective marker to observe the extent of coagulation in egg white and yolk and that the T2 value image is quite useful to recognize non-destructively the extent and status of coagulation of the heated eggs. This technique can also be applied to the material science as well as food science for observation of the inner status of the objects.