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WaterLOGSY and STD experiments are widely used as NMR-based screening techniques in drug research. In the present study, an improved STD pulse sequence was developed, and its efficiency and applicability of observing the ligand signals were evaluated compared with the WaterLOGSY experiment. A combination of presaturation, a WET sequence and subsequent repeated Z-filters can provide the most effective water suppression, which is incorporated into the STD pulse sequence. In a sample solution of tryptophan and glucose in the presence of human serum albumin, the improved STD experiment only succeeded in selective detections of the bound ligand signals, even resonating close to water.

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An efficient pulse sequence for observing the ligand signals resonating close to the water signal has been developed by incorporating the WET technique into the saturation transfer difference pulse sequence. Although several pulse sequences have been developed for observing a ligand binding with a protein receptor, the ligand signals resonating close to the water were undetectable owing to the interference of the huge water signal in the samples containing 95% (1)H(2)O. On the point of sample preparation, it is preferable to avoid the solvent exchange in the protein samples. In the proposed pulse sequence, a WET sequence is incorporated for the selective suppression of the water resonance. The efficient water suppression and the clear observation of the bound ligand signals close to the water have been demonstrated using the lysozyme-glucose complex.

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An efficient pulse sequence for observing a ligand binding with a receptor has been developed by incorporating the WATERGATE W5 sequence. In the conventional water ligand observed via gradient spectroscopy (WaterLOGSY) techniques, the water resonance is selectively excited using, e.g. the double-pulsed field gradient spin-echo (DPFGSE) sequence at the initial portion of pulse sequence. In the current version, the modified WATERGATE W5 sequence is incorporated at the initial portion of the pulse sequence, and the resonance at the water frequency can be selectively reserved by the modified WATERGATE W5 sequence. The efficiency of ligand-observed NMR screening techniques has been demonstrated using the human serum albumin (HSA)-tryptophan complex.

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alpha-Synuclein is the major component of the filamentous Lewy bodies and Lewy-related neurites, neuropathological hallmarks of Parkinson's disease. Although numerous studies on alpha-synuclein fibrillation have been reported, the molecular mechanisms of aggregation and fibrillation at the initial stage are still unclear. In the present study, structural properties and propensities to form fibrils of alpha-synuclein at the initial stage were investigated using 2D (1)H-(15)N NMR spectroscopy, electron microscope, and small angle X-ray scattering (SAXS). Observation of the 2D (1)H-(15)N HSQC spectra indicated significant attenuation of many cross peak intensities in the regions of KTKEGV-type repeats and the non-Abeta component of Alzheimer's disease amyloid (NAC), suggesting that these regions contributed fibril formation. Oligomerization comprising heptamer was successfully monitored at the initial stage using the time-dependent SAXS measurements.

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Phospholipase C (PLC) plays an important role in intracellular signal transduction by hydrolyzing phosphatidylinositol 4,5-bis-phosphate, a membrane phospholipid. Currently, thirteen mammalian PLC isozymes have been identified, which are divided into six classes on the basis of structure and mechanisms. All the PLC isozymes share common domains including catalytic X and Y domains, protein kinase C conserved region 2 (C2) domain, EF-hand motif and pleckstrin homology (PH) domain. In this study, the PLC-eta1 PH domain has been over-expressed and purified. The most undesirable feature of the protein was instability, resulting in precipitation during the purification process. With the aim of structural characterization, a solution condition was optimized using SDS-PAGE and NMR spectroscopy. A circular dichroism spectrum indicated that the PLC-eta1 PH domain mainly comprised beta-strands, which was also suggested by the 2D 1H-15N HSQC spectrum.

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Translational motions of water molecules in various systems equilibrated at room temperature are thought to be diffusive and nondirectional. We performed molecular dynamics simulations of a protein system and showed that the water molecules collectively move around the protein. The motions of two water molecules, which were about 12 A away from each other, are correlated to each other. Such collective motions of water can be regarded as flows around the protein, and the flows exhibited various coherent patterns: fair currents, vortices, and divergent flows. The patterns were highly fluctuating: a set of patterns changed to a different set of patterns within a time scale of 10 ps. Thus, the water motions observed in a scale of length smaller than 12 A and a time scale shorter than 10 ps were nondiffusive, and the motions above these scales were diffusive, where the flows disappeared. The flows near the protein surface had an orientational propensity to be highly parallel to the protein surface, and this propensity gradually vanished with an increment of distance from the protein surface. The divergent patterns of flows, which frequently emerge during the fluctuations of flows, may temporarily cause solvent drying in the vicinity of solutes. The current simulation is supportive of a molecular interaction mechanism that the fluctuations of hydration structure induce attractive interactions between solutes.

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One of the most well known characteristics for Parkinson's disease (PD) is a polymerization of wild-type or mutant alpha-synuclein into aggregates and fibrils, commonly observed as Lewy bodies and Lewy neuritis in PD patients. Although numerous studies on alpha-synuclein fibrillation have been reported, the molecular mechanisms of aggregation and fibrillation are not well understood yet. In the present study, structural properties and propensities to form fibrils of wild-type, A30P, E46K, and A53T alpha-synucleins were investigated using fluorescence and circular dichroism (CD) methods. The results from these studies were analyzed using singular value decomposition (SVD) method which estimates a number of conformationally independent species for a given process. The time-dependent CD spectra of the wild-type alpha-synuclein indicated a multi-step process in the fibril formation, and SVD analysis using the time-dependent CD spectra revealed that five or nine intermediates were formed at the early stage of fibrillation.

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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.

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Two new triterpenes named 7-oxodihydrokarounitriol (1) and 7,11-dioxodihydrokarounidiol (2), and one known triterpene, 7-oxodihydrokarounidiol (3), were isolated from the unsaponifiable matter of the seeds of Trichosanthes cucumeroides. The structures of 1 and 2 were elucidated as (3alpha, 11beta, 13alpha, 14beta, 20alpha)-3,11,29-trihydroxy-13-methyl-26-norolean-8-ene-7-one, and (3alpha,13alpha,14beta,20alpha)-3,29-dihydroxy-13-methyl-26-norolean-8-ene-7,11-dione on the basis of extensive NMR (1H, 13C, 1H-1H COSY, DEPT, HMQC, HMBC and NOESY) and MS studies.

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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.

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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.

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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.

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A member of the PIAS (protein inhibitor of activated STAT) family of proteins, PIAS1, have been reported to serve as an E3-type SUMO ligase for tumor suppressor p53 and its own. It also was proposed that the N-terminal domain of PIAS1 interacts with DNA as well as p53. Extensive biochemical studies have been devoted recently to understand sumoylations and its biological implications, whereas the structural aspects of the PIAS family and the mechanism of its interactions with various factors are less well known to date. In this study, the three-dimensional structure of the N-terminal domain (residues 1-65) of SUMO ligase PIAS1 was determined by NMR spectroscopy. The structure revealed a unique four-helix bundle with a topology of an up-down-extended loop-down-up, a part of which the helix-extended loop-helix represented the SAP (SAF-A/B, Acinus, and PIAS) motif. Thus, this N-terminal domain may be referred to as a four-helix SAP domain. The glutathione S-transferase pull-down assay demonstrated that this domain possesses a binding ability to tumor suppressor p53, a target protein for sumoylation by PIAS1, whereas gel mobility assays showed that it has a strong affinity toward A/T-rich DNA. An NMR analysis of the four-helix SAP domain complexed with the 16-bp-long DNA demonstrated that one end of the four-helix bundle is the binding site and may fit into the minor groove of DNA. The three-dimensional structure and its binding duality are discussed in conjunction with the biological functions of PIAS1 as a SUMO ligase.

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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.

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Hho1p is assumed to serve as a linker histone in Saccharomyces cerevisiae and, notably, it possesses two putative globular domains, designated HD1 (residues 41-118) and HD2 (residues 171-252), that are homologous to histone H5 from chicken erythrocytes. We have determined the three-dimensional structure of globular domain HD1 with high precision by heteronuclear magnetic resonance spectroscopy. The structure had a winged helix-turn-helix motif composed of an alphabetaalphaalphabetabeta fold and closely resembled the structure of the globular domain of histone H5. Interestingly, the second globular domain, HD2, in Hho1p was unstructured under physiological conditions. Gel mobility assay demonstrated that Hho1p preferentially binds to supercoiled DNA over linearized DNA. Furthermore, NMR analysis of the complex of a deletion mutant protein (residues 1-118) of Hho1p with a linear DNA duplex revealed that four regions within the globular domain HD1 are involved in the DNA binding. The above results suggested that Hho1p possesses properties similar to those of linker histones in higher eukaryotes in terms of the structure and binding preference towards supercoiled DNA.

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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.

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