佐藤 文菜

Certain cyanobacteria alter their photosynthetic light absorption between green and red, a phenomenon called complementary chromatic acclimation. The acclimation is regulated by a cyanobacteriochrome-class photosensor that reversibly photoconverts between green-absorbing (Pg) and red-absorbing (Pr) states. Here, we elucidated the structural basis of the green/red photocycle. In the Pg state, the bilin chromophore adopted the extended C15- Z , anti structure within a hydrophobic pocket. Upon photoconversion to the Pr state, the bilin is isomerized to the cyclic C15- E , syn structure, forming a water channel in the pocket. The solvation/desolvation of the bilin causes changes in the protonation state and the stability of π-conjugation at the B ring, leading to a large absorption shift. These results advance our understanding of the enormous spectral diversity of the phytochrome superfamily.

Molecular motion in the solid state is typically precluded by the highly dense environment, and only molecules with a limited range of sizes show such dynamics. Here, we demonstrate the solid-state rotational motion of two giant molecules, i.e., triptycene and pentiptycene, by encapsulating a bulky N-heterocyclic carbene (NHC) Au(I) complex in the crystalline media. To date, triptycene is the largest molecule (surface area: 245 Å2 ; volume: 219 Å3 ) for which rotation has been reported in the solid state, with the largest rotational diameter among reported solid-state molecular rotors (9.5 Å). However, the pentiptycene rotator that is the subject of this study (surface area: 392 Å2 ; volume: 361 Å3 ; rotational diameter: 13.0 Å) surpasses this record. Single-crystal X-ray diffraction analyses of both the developed rotors revealed that these possess sufficient free volume around the rotator. The molecular motion in the solid state was confirmed using variable-temperature solid-state 2 H spin-echo NMR studies. The triptycene rotor exhibited three-fold rotation, while temperature-dependent changes of the rotational angle were observed for the pentiptycene rotor.

Controlled self‐assembly of Pt(II) complexes is key to the development of optical and stimuli‐responsive materials, but designing and precisely controlling them is still difficult owing to weak intermolecular interactions. Herein, we report the successful water‐vapor‐induced assembly of an anionic Pt(II) complex [Pt(CN)2(ppy)]– (Hppy = 2‐phenylpyridine) electrostatically loaded on cationically charged layered double hydroxide (LDH) nanoparticles consisting of Mg2+ and Al3+ ions. When the Pt(II) complexes were densely loaded onto the LDH nanoparticles, the assembly was maintained, even in dilute aqueous media. In the case of sparse loading, the Pt(II) complexes were loaded discretely in the dry state; however, when water vapor was adsorbed, the increased mobility of the Pt(II) complexes led to their assembly on the LDH nanoparticles. This resulted in a drastic change in luminescence from green to orange owing to the presence of water vapor.

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.

Crystalline-state chemiluminescence (CL) reactions of 1,2-dioxetane isomers with two fluorophore side chains showed isomeric differences in CL properties and reaction behavior, reflecting solid-to-solid phase transitions.

κ-(BEDT-TTF)2Cu[N(CN)2]Br (κ-Br) is an organic superconductor that changes to an Anderson-type insulator due to the random defects introduced by x-ray irradiation. In this study, we directly investigated the effects of irradiation on the local structures in the anion layer of κ-Br using x-ray fluorescence holography. The local structures around the Cu atoms in the anion layer were directly reconstructed from holograms. Prior to radiation damage, the reconstruction clearly shows the atomic images of the Br and N atoms that are directly coordinated to Cu. In the irradiated samples, the N images in the reconstructions were strongly suppressed as a result of the positional fluctuations of N introduced by the defects induced by irradiation. Using the previously proposed "bond-shifted"model and the thermal vibration amplitudes calculated from molecular dynamics simulations, we simulated the holograms and the atomic reconstructions of the anion layer. The simulations show that the "bond-shifted"model, along with thermal vibrations at 100 K, suppresses the N atomic images, as was observed in the experimental reconstructions.

<p>The nickel(<sc>ii</sc>)–chloranilato complex shows reversible vapochromism with changes in dimensionality upon exposure to various vapours and subsequent drying by heating.</p>

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.

X-ray fluorescence holography (XFH) is a technique that can directly image the 3D arrangement of atoms around an element in a sample. The holograms contain both intensity and phase information, allowing atomic reconstruction without needing prior structural information or a tentative structural model. XFH has already been used to reveal the local structures of various inorganic samples, and recently, work has begun on XFH for soft matter. In this paper, we review the progress of XFH on soft materials. First, we review the fundamental principles of XFH. Second, we review inverse mode XFH on soft materials, and the results of the experiments on hemoglobin, myoglobin, and κ-(BEDT-TTF)2Cu[N(CN)2]Br crystals. In the last section, we report the progress of the development of normal mode holography for soft materials. The new apparatus and scanning method is described, and results of the initial tests on the protein Photosystem II are discussed.

<p>Crystalline-state chemiluminescence of 1,2-dioxetane isomers showed contrastive luminescence and reaction properties depending on the crystal-structural characteristics.</p>

A hybrid vapoluminescent system exhibiting fast and repeatable response was constructed using periodic mesoporous organosilica with bipyridine moieties (BPy-PMO) and a Pt(II) complex bearing a potentially luminescent 2-phenylpyridinato (ppy) ligand. An intense red luminescence appeared when the Pt(II)-complex immobilised BPy-PMO was exposed to methanol vapour and disappeared on exposure to pyridine vapour. The ON-OFF vapochromic behaviour occurred repeatedly in a methanol/pyridine/heating cycle. Interestingly, a rapid response was achieved in the second cycle and cycles thereafter. Scanning and transmission electron microscopies (SEM/TEM), absorption and emission, and nuclear magnetic resonance spectroscopies, mass spectrometry, and powder X-ray diffraction indicated that methanol vapour induced Si-C cleavage and thus liberated [Pt(ppy)(bpy)]Cl (bpy = 2,2'-bipyridine) from the BPy-PMO framework. Furthermore, the self-assembling properties of the Pt(II) complex resulted in the formation of highly luminescent micro/nanocrystals that were homogeneously dispersed on the porous support. The unique vapoluminescence triggered by the unprecedented protodesilylation on exposure to protic solvent vapour at room temperature is attributable to BPy-PMO being a giant ligand and an effective vapour condenser. Consequently, this hybrid system presents a new strategy for developing sensors using bulk powdery materials.

Objective - Inflammation provoked by the imbalance of fatty acid composition, such as excess saturated fatty acids (SFAs), is implicated in the development of metabolic diseases. Recent investigations suggest the possible role of the NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3) inflammasome, which regulates IL-1β (interleukin 1β) release and leads to inflammation, in this process. Therefore, we investigated the underlying mechanism by which SFAs trigger NLRP3 inflammasome activation. Approach and Results - The treatment with SFAs, such as palmitic acid and stearic acid, promoted IL-1β release in murine primary macrophages while treatment with oleic acid inhibited SFA-induced IL-1β release in a dose-dependent manner. Analyses using polarized light microscopy revealed that intracellular crystallization was provoked in SFA-treated macrophages. As well as IL-1β release, the intracellular crystallization and lysosomal dysfunction were inhibited in the presence of oleic acid. These results suggest that SFAs activate NLRP3 inflammasome through intracellular crystallization. Indeed, SFA-derived crystals activated NLRP3 inflammasome and subsequent IL-1β release via lysosomal dysfunction. Excess SFAs also induced crystallization and IL-1β release in vivo. Furthermore, SFA-derived crystals provoked acute inflammation, which was impaired in IL-1β-deficient mice. Conclusions - These findings demonstrate that excess SFAs cause intracellular crystallization and subsequent lysosomal dysfunction, leading to the activation of the NLRP3 inflammasome, and provide novel insights into the pathogenesis of metabolic diseases.

© 2018 The Physical Society of Japan. Atomic-resolution holography makes it possible to obtain the three-dimensional (3D) structure around a target atomic site. Translational symmetry of the atomic arrangement of the sample is not necessary, and the 3D atomic image can be measured when the local structure of the target atomic site is oriented. Therefore, 3D local atomic structures such as dopants and adsorbates are observable. Here, the atomic-resolution holography comprising photoelectron holography, X-ray fluorescence holography, neutron holography, and their inverse modes are treated. Although the measurement methods are different, they can be handled with a unified theory. The algorithm for reconstructing 3D atomic images from holograms plays an important role. Although Fourier transform-based methods have been proposed, they require the multiple-energy holograms. In addition, they cannot be directly applied to photoelectron holography because of the phase shift problem. We have developed methods based on the fitting method for reconstructing from single-energy and photoelectron holograms. The developed methods are applicable to all types of atomic-resolution holography.

New measurement methodologies have begun to be proposed with the recent progress in the life sciences. Here, we introduce two new methodologies, X-ray fluorescence holography for protein structural analysis and diffracted X-ray tracking (DXT), to observe the dynamic behaviors of individual single molecules.

While high-throughput screening for protein crystallization conditions have rapidly evolved in the last few decades, it is also becoming increasingly necessary for the control of crystal size and shape as increasing diversity of protein crystallographic experiments. For example, X-ray crystallography (XRC) combined with photoexcitation and/or spectrophotometry requires optically thin but well diffracting crystals. By contrast, large-volume crystals are needed for weak signal experiments, such as neutron crystallography (NC) or recently developed X-ray fluorescent holography (XFH). In this article, we present, using hemoglobin as an example protein, some techniques for obtaining the crystals of controlled size, shape, and adequate quality. Furthermore, we describe a few case studies of applications of the optimized hemoglobin crystals for implementing the above mentioned crystallographic experiments, providing some hints and tips for the further progress of advanced protein crystallography.

Hemoglobin, the vital O-2 carrier in red blood cells, has long served as a classic example of an allosteric protein. Although high-resolution X-ray structural models are currently available for both the deoxy tense (T) and fully liganded relaxed (R) states of hemoglobin, much less is known about their dynamics, especially on the picosecond to subnanosecond time scales. Here, we investigate the picosecond dynamics of the deoxy and CO forms of human hemoglobin using quasielastic neutron scattering under near physiological conditions in order to extract the dynamics changes upon ligation. From the analysis of the global motions, we found that whereas the apparent diffusion coefficients of the deoxy form can be described by assuming translational and rotational diffusion of a rigid body, those of the CO form need to involve an additional contribution of internal large-scale motions. We also found that the local dynamics in the deoxy and CO forms are very similar in amplitude but are slightly lower in frequency in the former than in the latter. Our results reveal the presence of rapid large-scale motions in hemoglobin and further demonstrate that this internal mobility is governed allosterically by the ligation state of the heme group.

The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light-and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.

The protein crystallography beamline BL2S1, constructed at one of the 5 T superconducting bending-magnet ports of the Aichi synchrotron, is available to users associated with academic and industrial organizations. The beamline is mainly intended for use in X-ray diffraction measurements of single-crystals of macromolecules such as proteins and nucleic acids. Diffraction measurements for crystals of other materials are also possible, such as inorganic and organic compounds. BL2S1 covers the energy range 7-17 keV (1.8-0.7 angstrom) with an asymmetric-cut curved single-crystal monochromator [Ge(111) or Ge(220)], and a platinum-coated Si mirror is used for vertical focusing and as a higher-order cutoff filter. The beamline is equipped with a single-axis goniometer, a CCD detector, and an open-flow cryogenic sample cooler. High-pressure protein crystallography with a diamond anvil cell can also be performed using this beamline.

Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metal-loprotein samples are described. Human hemoglobin, an alpha(2)beta(2) tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A chi-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 x 6 x 3 mm(3)) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0 degrees-70 degrees. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules. Published by AIP Publishing.

Picosecond time-resolved X-ray diffraction has been used to study the nanoscale thermal transportation dynamics of bare gold nanocrystals and thiol-based self-assembled monolayer (SAM)-coated integrated gold nanocrystals on a SiO2 glass substrate. A temporal lattice expansion of 0.30-0.33% was observed in the bare and SAM-coated nanocrystals on the glass substrate; the thermal energy inside the gold nanocrystals was transported to the contacted substrate through the gold-SiO2 interface. The interfacial thermal conductivity between the single-layered gold nanocrystal film and the SiO2 substrate is estimated to be 45 MW m(-2) K-1 from the decay of the Au 111 peak shift, which was linearly dependent on the transient temperature. For the SAM-coated gold nanocrystals, the thermal dissipation was faster than that of the bare gold nanocrystal film. The thermal flow from the nanocrystals to the SAM-coated molecules promotes heat dissipation from the laser-heated SAM-coated gold nanocrystals. The thermal transportation of the laser-heated SAM-coated gold nanocrystal film was analyzed using the bidirectional thermal dissipation model.

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.

Trans-cis photoisomerization in an azo compound containing azobenzene chromophores and long alkyl chains leads to a photoinduced crystal-melt transition (PCMT). X-ray structure analysis of this crystal clarifies the characteristic coexistence of the structurally ordered chromophores through their pi center dot center dot center dot pi interactions and disordered alkyl chains around room temperature. These structural features reveal that the PCMT starts near the surface of the crystal and propagates into the depth, sacrificing the pi center dot center dot center dot pi interactions. A temporal change of the powder X-ray diffraction pattern under light irradiation and a two-component phase diagram allow qualitative analysis of the PCMT and the following reconstructive crystallization of the cis isomer as a function of product accumulation. This is the first structural characterization of a compound showing the PCMT, overcoming the low periodicity that makes X-ray crystal structure analysis difficult.

Singular value decomposition (SVD) analysis has important applications for time-dependent crystallographic data, extracting significant information. Herein, a successful application of SVD analysis of time-resolved powder diffraction data over the course of an in-situ photodimerization reaction of anthracene derivatives is introduced. SVD revealed significant results in the case of 9-methylanthracene and 1-chloroanthracene. The results support the formation of the 9-methylanthracene stable dimer phase and suggest the existence of an excimer state.

Ionic species often play important roles in chemical reactions occurring in water and other solvents, but it has been elusive to determine the solvent-dependent molecular structure with atomic resolution. The triiodide ion has a molecular structure that sensitively changes depending on the type of solvent and its symmetry can be broken by strong solute-solvent interaction. Here, by applying pump-probe x-ray solution scattering, we characterize the exact molecular structure of I-3(-) ion in water, methanol, and acetonitrile with subangstrom accuracy. The data reveal that I-3(-) ion has an asymmetric and bent structure in water. In contrast, the ion keeps its symmetry in acetonitrile, while the symmetry breaking occurs to a lesser extent in methanol than in water. The symmetry breaking of I-3(-) ion is reproduced by density functional theory calculations using 34 explicit water molecules, confirming that the origin of the symmetry breaking is the hydrogen-bonding interaction between the solute and solvent molecules. DOI: 10.1103/PhysRevLett.110.165505

We study shock compressed fused quartz in the nonlinear elastic region using single-shot time-resolved x-ray scattering measurements. The first sharp diffraction peak (FSDP) of fused quartz shifts to the high Q region under shock compression. In contrast, the short-range order structure does not change around 3.5 GPa. The nanosecond FSDP shift provides clear evidence of intermediate-range order structural changes in the nonlinear elastic region. Because the intermediate-order structure is too short to produce the final structural state in the nonlinear elastic region, the FSDP shift is lower compared with hydrostatic experiments. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4764526]

The [4 pi + 4 pi] photodimerization process of the 9-substituted anthracene derivative crystalline 9-methylanthracene (9-MA) was investigated using time-resolved X-ray powder diffraction. The study was carried out in-situ using a CCD area detector. Sequential and parametric Rietveld refinement was applied for quantitative phase analysis. The results of traditional sequential Rietveld refinement showed that the evolution of the dimerization process can be described using the Johnson-Mehl-Avrami-Kolmogorov (JMAK) model. The parameters of the JMAK equation were obtained successfully by parametric Rietveld refinement and suggest that the reaction follows heterogeneous nucleation and one-dimensional growth with a decreasing nucleation rate.

Time-resolved X-ray absorption spectra of photoexcited nithenium(H)-tris-2,2'-bipyridine ([Ru-II(bpy)(3)](2+) in the triplet metal-to-ligand charge transfer ((MLCT)-M-3) state are measured and analyzed to investigate transient structural changes directly related to the photophysical properties of the complex. The results from visible (400 rim) and UV (267 nm) excitation indicate that electrostatic interaction between the oxidized Ru atom and the reduced bipyridine ligand is the dominant factor affecting the Ru-N bond contraction. This thus leads to two groups of Ru ligand distances, one exhibiting the ground-state Ru-N distance and another yielding a slightly decreased Ru-N distance due to the localized MLCT excited state. The EXAFS analysis of the photoexcited complex was analyzed toward one single Ru-N distance., yielding a contraction of 0.04 (0.01) angstrom with an increased DW factor (corresponding to a 0.05 angstrom mean increase).

A ligand-migration mechanism of myoglobin was studied by a multidisciplinary approach that used x-ray crystallography and molecular dynamics simulation. The former revealed the structural changes of the protein along with the ligand migration, and the latter provided the statistical ensemble of protein conformations around the thermal average. We developed a novel computational method, homogeneous ensemble displacement, and generated the conformational ensemble of ligand-detached species from that of ligand-bound species. The thermally averaged ligand-protein interaction was illustrated in terms of the potential of mean force. Although the structural changes were small, the presence of the ligand molecule in the protein matrix significantly affected the 3D scalar field of the potential of mean force, in accordance with the self-opening model proposed in the previous x-ray study. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4704586]

Extensive efforts have been devoted to developing electron donor acceptor systems that mimic the utilization of solar energy that occurs in photosynthesis. X-ray crystallographic analysis shows how absorbed photon energy is stabilized in those compounds by structural changes upon photoinduced electron transfer (ET). In this study, structural changes of a simple electron donor acceptor dyad, 9-mesityl-10-methylacridinium cation (Acr(+)-Mes), upon photoinduced ET were directly observed by laser pump and X-ray probe crystallographic analysis. The N-methyl group in Acr(+) was bent, and a weak electrostatic interaction between Mes and a counteranion in the crystal (ClO4) was generated by photoinduced ET. These structural changes correspond to reduction and oxidation due to photoinduced ET and directly elucidate the mechanism in Acr(+)-Mes for mimicking photosynthesis efficiently.