山本 直樹

Incoherent inelastic and quasi-elastic neutron scattering (INS) and terahertz time-domain spectroscopy (THz-TDS) are spectroscopy methods that directly detect molecular dynamics, with an overlap in the measured energy regions of each method. Due to the different characteristics of their probes (i.e., neutron and light), the information obtained and the sample conditions suitable for each method differ. In this review, we introduce the differences in the quantum beam properties of the two methods and their associated advantages and disadvantages in molecular spectroscopy. Neutrons are scattered via interaction with nuclei; one characteristic of neutron scattering is a large incoherent scattering cross-section of a hydrogen atom. INS records the auto-correlation functions of atomic positions. By using the difference in neutron scattering cross-sections of isotopes in multi-component systems, some molecules can be selectively observed. In contrast, THz-TDS observes the cross-correlation function of dipole moments. In water-containing biomolecular samples, the absorption of water molecules is particularly large. While INS requires large-scale experimental facilities, such as accelerators and nuclear reactors, THz-TDS can be performed at the laboratory level. In the analysis of water molecule dynamics, INS is primarily sensitive to translational diffusion motion, while THz-TDS observes rotational motion in the spectrum. The two techniques are complementary in many respects, and a combination of the two is very useful in analyzing the dynamics of biomolecules and hydration water.

Amyloid fibrils have been an important subject as they are involved in the development of many amyloidoses and neurodegenerative diseases. The formation of amyloid fibrils is typically initiated by nucleation, whereas its exact mechanisms are largely unknown. With this situation, we have previously identified prefibrillar aggregates in the formation of insulin B chain amyloid fibrils, which have provided an insight into the mechanisms of protein assembly involved in nucleation. Here, we have investigated the formation of insulin B chain amyloid fibrils under different pH conditions to better understand amyloid nucleation mediated by prefibrillar aggregates. The B chain showed strong propensity to form amyloid fibrils over a wide pH range, and prefibrillar aggregates were formed under all examined conditions. In particular, different structures of amyloid fibrils were found at pH 5.2 and pH 8.7, making it possible to compare different pathways. Detailed investigations at pH 5.2 in comparison with those at pH 8.7 have suggested that the evolution of protofibril-like aggregates is a common mechanism. In addition, different processes of evolution of the prefibrillar aggregates have also been identified, suggesting that the nucleation processes diversify depending on the polymorphism of amyloid fibrils.

We report expression and purification of a FLT3 protein with ITD mutation (FLT3-ITD) with a steady tyrosine kinase activity using a silkworm-baculovirus system, and its application as a fast screening system of tyrosine kinase inhibitors. The FLT3-ITD protein was expressed in Bombyx mori L. pupae infected by gene-modified nucleopolyhedrovirus, and was purified as an active state. We performed an inhibition assay using 17 kinase inhibitors, and succeeded in screening two inhibitors for FLT3-ITD. The result has paved the way for screening FLT3-ITD inhibitors in a fast and easy manner, and also for structural studies.