Sakurako Shimotakahara

Characterization of fibrillation process of α-synuclein at the initial stage

alpha-Synuclein is the major component of the filamentous Lewy bodies and Lewy-related neurites, neuropathological hallmarks of Parkinsons 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 Alzheimers 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.

An efficient use of the WATERGATE W5 sequence for observing a ligand binding with a protein receptor

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. Copyright

Thermal unfolding of ribonuclease T1 studied by multi- dimensional NMR spectroscopy

Abstract 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 α-helix and two β-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°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 β-strands (B3, B4, and B5 in β-sheet B). These β-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°C. Thus, the two-state transition of RNase T1 appears to involve locally different conformational changes.

Application of WET sequence for the detection of the ligand signals resonating close to water

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% 1H2O. 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. Copyright

Two new triterpenes from the seeds of Trichosanthes cucumeroides

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 (3α,11β,13α,14β,20α)-3,11,29-trihydroxy-13-methyl-26-norolean-8-ene-7-one, and (3α,13α,14β,20α)-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.