Yojiro Oba

Distinct Features of the Histone Core Structure in Nucleosomes Containing the Histone H2A.B Variant

Nucleosomes containing a human histone variant, H2A.B, in an aqueous solution were analyzed by small-angle neutron scattering utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome are detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails are compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Therefore, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.

Conformational characterization of a protein complex involving intrinsically disordered protein by small-angle neutron scattering using the inverse contrast matching method: a case study of interaction between α-synuclein and PbaB tetramer as a model chaperone

Cumulative genomics and proteomics data have now highlighted the presence of intrinsically disordered proteins (IDPs), which are devoid of stable secondary or tertiary structures under physiological conditions. While the flexible nature of IDPs precludes their study by crystallographic methods, IDP interactions with their cognate proteins, during which the IDPs often form their secondary structures, have been characterized by nuclear magnetic resonance (NMR) spectroscopy. In view of this, a complementary small-angle neutron scattering (SANS) technique has been developed for probing IDP conformations in larger protein complexes. As a model interaction system, α-synuclein (αSN) bound to an archaeal homotetrameric chaperone, PbaB, was analyzed. To selectively observe the SANS profile of αSN in the complex, the bacterially produced PbaB was fractionally (75%) deuterated using D2O and deuterated glucose for contrast matching to approximately 100% D2O solvent. By employing 75%-deuterated PbaB, the conformational changes of αSN upon capture by this tetrameric chaperone were successfully observed with minimal background scattering. Together with the present NMR data, the SANS data reveal that the PbaB tetramer grasps the N-terminal segments of αSN, disrupting the residual ordered structure in this region, while leaving the remaining regions flexible within a slightly reduced conformational space.

Structural characterization of the circadian clock protein complex composed of KaiB and KaiC by inverse contrast-matching small-angle neutron scattering

The molecular machinery of the cyanobacterial circadian clock consists of three proteins: KaiA, KaiB, and KaiC. Through interactions among the three Kai proteins, the phosphorylation states of KaiC generate circadian oscillations in vitro in the presence of ATP. Here, we characterized the complex formation between KaiB and KaiC using a phospho-mimicking mutant of KaiC, which had an aspartate substitution at the Ser431 phosphorylation site and exhibited optimal binding to KaiB. Mass-spectrometric titration data showed that the proteins formed a complex exclusively in a 6:6 stoichiometry, indicating that KaiB bound to the KaiC hexamer with strong positive cooperativity. The inverse contrast-matching technique of small-angle neutron scattering enabled selective observation of KaiB in complex with the KaiC mutant with partial deuteration. It revealed a disk-shaped arrangement of the KaiB subunits on the outer surface of the KaiC C1 ring, which also serves as the interaction site for SasA, a histidine kinase that operates as a clock-output protein in the regulation of circadian transcription. These data suggest that cooperatively binding KaiB competes with SasA with respect to interaction with KaiC, thereby promoting the synergistic release of this clock-output protein from the circadian oscillator complex.

Molecular Assembly of Wheat Gliadins into Nanostructures: A Small-Angle X-ray Scattering Study of Gliadins in Distilled Water over a Wide Concentration Range

Gliadin, one of the major proteins together with glutenin composing gluten, affects the physical properties of wheat flour dough. In this study, nanoscale structures of hydrated gliadins extracted into distilled water were investigated primarily by small-angle X-ray scattering (SAXS) over a wide range of concentrations. Gliadins are soluble in distilled water below 10 wt %. Guinier analyses of SAXS profiles indicate that gliadins are present as monomers together with small amounts of dimers and oligomers in a very dilute solution. The SAXS profiles also indicate that interparticle interference appears above 0.5 wt % because of electrostatic repulsion among gliadin assemblies. Above 15 wt %, gliadins form gel-like hydrated solids. At greater concentrations, a steep upturn appears in the low-q region owing to the formation of large aggregates, and a broad shoulder appears in the middle-q region showing density fluctuation inside. This study demonstrates that SAXS can effectively disclose the nanostructure of hydrated gliadin assemblies.

Magnetic scattering in the simultaneous measurement of small-angle neutron scattering and Bragg edge transmission from steel

A technique for the analysis of magnetic scattering has been developed, where small-angle neutron scattering and Bragg edge transmission measurements are performed simultaneously. This technique is shown to provide crystallographic information for ferrite crystallites and nanostructural information for precipitates in steel.

Solution structure of variant H2A.Z.1 nucleosome investigated by small-angle X-ray and neutron scatterings

Solution structures of nucleosomes containing a human histone variant, H2A.Z.1, were measured by small-angle X-ray and neutron scatterings (SAXS and SANS). SAXS revealed that the outer shape, reflecting the DNA shape, of the H2A.Z.1 nucleosome is almost the same as that of the canonical H2A nucleosome. In contrast, SANS employing a contrast variation technique revealed that the histone octamer of the H2A.Z.1 nucleosome is smaller than that of the canonical nucleosome. The DNA within the H2A.Z.1 nucleosome was more susceptible to micrococcal nuclease than that within the canonical nucleosome. These results suggested that the DNA is loosely wrapped around the histone core in the H2A.Z.1 nucleosome.

Magnetic Intraparticle Structure in Ferromagnetic Pd Nanoparticle

The small-angle neutron scattering measurement of Pd nanoparticles was performed to investigate their intraparticle magnetic structures. The magnetic scattering from the magnetization of the samples was observed using the polarized neutron. The nuclear scattering indicated that the Pd nanoparticles can be regarded as spherical particles, and the magnetic scattering is explained based on the shell model that the particle is composed of magnetic core and shell. The magnetic moment of the core was larger than that of the shell. These results suggest that the spontaneous magnetization of the Pd nanoparticles mainly exists in the core region.

New insight into the dynamical system of αB-crystallin oligomers

α-Crystallin possesses a dynamic quaternary structure mediated by its subunit dynamics. Elucidation of a mechanism of subunit dynamics in homo-oligomers of αB-crystallin was tackled through deuteration-assisted small-angle neutron scattering (DA-SANS) and electrospray ionization (ESI) native mass spectrometry (nMS). The existence of subunit exchange was confirmed with DA-SANS, and monomers liberated from the oligomers were observed with nMS. With increasing temperature, an increase in both the exchange rate and monomer population was observed despite the absence of oligomer collapse. It is proposed that transiently liberated subunits, namely, “traveling subunits,” play a role in subunit exchange. Moreover, we propose that protein function is regulated by these traveling subunits.

Temperature dependence of nanostructure in PbSe–ZnSe composite thin film

The nanostructure of PbSe–ZnSe composite thin films prepared by the hot-wall deposition (HWD) method was investigated using small-angle x-ray (SAXS) scattering. The SAXS profiles indicate the formation of two kinds of nanoparticles: large nanoparticles that vanish and small particles that increase in size with increasing temperature. At high substrate temperatures, the volume fraction of all the nanoparticles estimated from SAXS is consistent with that of PbSe obtained by chemical analysis. This shows that PbSe forms nanoparticles at high substrate temperatures. On the other hand, the same analysis for the volume fraction at low substrate temperatures reveals that the chemical composition of the nanoparticles differs from PbSe. Pb nanoparticles are probably formed at low substrate temperatures and disappear with increasing substrate temperature.

Energy-resolved small-angle neutron scattering from steel

Recent progress of pulsed neutron sources has enabled energy-resolved analysis of neutron attenuation spectra, which include information on neutron scattering. In this study, a new analysis technique for small-angle neutron scattering (SANS) is demonstrated. A clear difference is observed in the neutron attenuation spectra between steels with different nanostructures; this difference can be understood as arising from attenuation due to SANS. The neutron attenuation spectra calculated from the corresponding SANS profiles agree well with the experimentally observed attenuation spectra. This result indicates that measurement of neutron attenuation spectra may enable the development of a novel experimental technique, i.e. energy-resolved SANS.