柴山 修哉

X-ray fluorescence holography (XFH) is a powerful atomic resolution technique capable of directly imaging the local atomic structure around atoms of a target element within a material. Although it is theoretically possible to use XFH to study the local structures of metal clusters in large protein crystals, the experiment has proven difficult to perform, especially on radiation-sensitive proteins. Here, the development of serial X-ray fluorescence holography to allow the direct recording of hologram patterns before the onset of radiation damage is reported. By combining a 2D hybrid detector and the serial data collection used in serial protein crystallography, the X-ray fluorescence hologram can be directly recorded in a fraction of the measurement time needed for conventional XFH measurements. This approach was demonstrated by obtaining the Mn Kα hologram pattern from the protein crystal Photosystem II without any X-ray-induced reduction of the Mn clusters. Furthermore, a method to interpret the fluorescence patterns as real-space projections of the atoms surrounding the Mn emitters has been developed, where the surrounding atoms produce large dark dips along the emitter-scatterer bond directions. This new technique paves the way for future experiments on protein crystals that aim to clarify the local atomic structures of their functional metal clusters, and for other related XFH experiments such as valence-selective XFH or time-resolved XFH.

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.

Hydration water plays a crucial role for activating the protein dynamics required for functional expression. Yet, the details are not understood about how hydration water couples with protein dynamics. A temperature hysteresis of the ice formation of hydration water is a key phenomenon to understand which type of hydration water, unfreezable or freezable hydration water, is crucial for the activation of protein dynamics. Using neutron scattering, we observed a temperature-hysteresis phenomenon in the diffraction peaks of the ice of freezable hydration water, whereas protein dynamics did not show any temperature hysteresis. These results show that the protein dynamics is not coupled with freezable hydration water dynamics, and unfreezable hydration water is essential for the activation of protein dynamics. Decoupling of the dynamics between unfreezable and freezable hydration water could be the cause of the distinct contributions of hydration water to protein dynamics.

Hemoglobin is one of the best-characterized proteins with respect to structure and function, but the internal ligand diffusion pathways remain obscure and controversial. Here we captured the CO migration processes in the tense (T), relaxed (R), and second relaxed (R2) quaternary structures of human hemoglobin by crystallography using a high-repetition pulsed laser technique at cryogenic temperatures. We found that in each quaternary structure, the photodissociated CO molecules migrate along distinct pathways in the α and β subunits by hopping between the internal cavities with correlated side chain motions of large nonpolar residues, such as α14Trp(A12), α105Leu(G12), β15Trp(A12), and β71Phe(E15). We also observe electron density evidence for the distal histidine [α58/β63His(E7)] swing-out motion regardless of the quaternary structure, although less evident in α subunits than in β subunits, suggesting that some CO molecules have escaped directly through the E7 gate. Remarkably, in T-state Fe(II)-Ni(II) hybrid hemoglobins in which either the α or β subunits contain Ni(II) heme that cannot bind CO, the photodissociated CO molecules not only dock at the cavities in the original Fe(II) subunit, but also escape from the protein matrix and enter the cavities in the adjacent Ni(II) subunit even at 95 K, demonstrating the high gas permeability and porosity of the hemoglobin molecule. Our results provide a comprehensive picture of ligand movements in hemoglobin and highlight the relevance of cavities, nonpolar residues, and distal histidines in facilitating the ligand migration.

A newly identified microbial rhodopsin, NM-R3, from the marine flavobacterium<italic>Nonlabens marinus</italic>, was recently shown to drive chloride ion uptake, extending our understanding of the diversity of mechanisms for biological energy conversion. To clarify the mechanism underlying its function, we characterized the crystal structures of NM-R3 in both the dark state and early intermediate photoexcited states produced by laser pulses of different intensities and temperatures. The displacement of chloride ions at five different locations in the model reflected the detailed anion-conduction pathway, and the activity-related key residues—Cys¹⁰⁵, Ser⁶⁰, Gln²²⁴, and Phe⁹⁰—were identified by mutation assays and spectroscopy. Comparisons with other proteins, including a closely related outward sodium ion pump, revealed key motifs and provided structural insights into light-driven ion transport across membranes by the NQ subfamily of rhodopsins. Unexpectedly, the response of the retinal in NM-R3 to photostimulation appears to be substantially different from that seen in bacteriorhodopsin.

Today, we can determine the structural basis of a biological molecule by X-ray crystallography or X-ray solution scattering. However, static structures do not say a lot for the realtime transition of the functional dynamics. Here we introduce the pump-probe method using combined single bunch X-ray from synchrotron source and pulsed laser system in sub nanosecond time resolution. Time-resolved X-ray solution scattering measurement of dimeric hemoglobin revealed a spiral motion of two subunits and three intermediate states. The pump-probe method is even powerful technique in the other methods of measurement for the protein dynamics.

The binding of ligands by proteins is accompanied by rapid structural changes that are essential to function. A recent crystallographic study has revealed the ligation-linked protein motions in an allosteric protein, human hemoglobin, in both allosteric forms (T and R) upon photolysis of bound CO at cryogenic temperatures. The results show how differently the α and β subunits, within each allosteric form, respond to loss of ligand, and where the free ligand lies, establishing that the mechanism of protein control of ligand binding is radically different between the subunits.