Published papers

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Phototropin2 (phot2) is a blue-light (BL) receptor protein that regulates the BL-dependent activities of plants for efficient photosynthesis. Phot2 is composed of two light-oxygen-voltage sensing domains (LOV1 and LOV2) to absorb BL, and a kinase domain. Photo-activated LOV domains, especially LOV2, play a major role in photo-dependent increase in the phosphorylation activity of the kinase domain. The atomic details of the overall structure of phot2 and the intramolecular mechanism to convert BL energy to a phosphorylation signal remain unknown. We performed structural studies on the LOV fragments LOV1, LOV2, LOV2-linker, and LOV2-kinase, and full-length phot2, using small-angle X-ray scattering (SAXS). The aim of the study was to understand structural changes under BL irradiation and discuss the molecular mechanism that enhance the phosphorylation activity under BL. SAXS is a suitable technique for visualizing molecular structures of proteins in solution at low resolution and is advantageous for monitoring their structural changes in the presence of external physical and/or chemical stimuli. Structural parameters and molecular models of the recombinant specimens were obtained from SAXS profiles in the dark, under BL irradiation, and after dark reversion. LOV1, LOV2, and LOV2-linker fragments displayed minimal structural changes. However, BL-induced rearrangements of functional domains were noted for LOV2-kinase and full-length phot2. Based on the molecular model together with the absorption measurements and biochemical assays, we discuss the intramolecular interactions and domain motions necessary for BL-enhanced phosphorylation activity of phot2.

© 2020 Federation of European Biochemical Societies Analysis of the conformational changes of protein is important to elucidate the mechanisms of protein motions correlating with their function. Here, we studied the spontaneous domain motion of unliganded glutamate dehydrogenase from Thermococcus profundus using cryo-electron microscopy and proposed a novel method to construct free-energy landscape of protein conformations. Each subunit of the homo-hexameric enzyme comprises nucleotide-binding domain (NAD domain) and hexamer-forming core domain. A large active-site cleft is situated between the two domains and varies from open to close according to the motion of a NAD domain. A three-dimensional map reconstructed from all cryo-electron microscopy images displayed disordered volumes of NAD domains, suggesting that NAD domains in the collected images adopted various conformations in domain motion. Focused classifications on NAD domain of subunits provided several maps of possible conformations in domain motion. To deduce what kinds of conformations appeared in EM images, we developed a novel analysis method that describe the EM maps as a linear combination of representative conformations appearing in a 200-ns molecular dynamics simulation as reference. The analysis enabled us to estimate the appearance frequencies of the representative conformations, which illustrated a free-energy landscape in domain motion. In the open/close domain motion, two free-energy basins hindered the direct transformation from open to closed state. Structure models constructed for representative EM maps in classifications demonstrated the correlation between the energy landscape and conformations in domain motion. Based on the results, the domain motion in glutamate dehydrogenase and the analysis method to visualize conformational changes and free-energy landscape were discussed. Database: The EM maps of the four conformations were deposited to Electron Microscopy Data Bank (EMDB) as accession codes EMD-9845 (open), EMD-9846 (half-open1), EMD-9847 (half-open2), and EMD-9848 (closed), respectively. In addition, the structural models built for the four conformations were deposited to the Protein Data Bank (PDB) as accession codes 6JN9 (open), 6JNA (half-open1), 6JNC (half-open2), and 6JND (closed), respectively.

© 2020, International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature. Microscopic imaging techniques have been developed to visualize events occurring in biological cells. Coherent X-ray diffraction imaging is one of the techniques applicable to structural analyses of cells and organelles, which have never been crystallized. In the experiment, a single noncrystalline particle is illuminated by an X-ray beam with almost complete spatial coherence. The structure of the particle projected along the direction of the beam is, in principle, retrieved from a finely recorded diffraction pattern alone by using iterative phase-retrieval algorithms. Here, we describe fundamental theory and experimental methods of coherent X-ray diffraction imaging and the recent application in structural studies of noncrystalline specimens by using X-rays available at Super Photon Ring of 8-Gev and SPring-8 Angstrom Compact Free Electron Laser in Japan.

Phytochrome A (phyA) is a photoreceptor protein of plants that regulates the red/far-red light photomorphogenic responses of plants essential for growth and development. PhyA, composed of approximately 1100 amino acid residues, folds into photosensory and output signaling modules. The photosensory module covalently binds phytochromobilin as a chromophore for photoreversible interconversion between inactive red light-absorbing (Pr) and active far-red light-absorbing (Pfr) forms to act as a light-driven phosphorylation enzyme. To understand the molecular mechanism in the initial process of photomorphogenic response, we studied the molecular structures of large phyA (LphyA) from Pisum sativum, which lacks the 52 residues in the N-terminal, by small-angle X-ray scattering combined with multivariate analyses applied to molecular models predicted from the scattering profiles. According to our analyses, Pr was in a dimer and had a four-leaf shape, and the subunit was approximated as a bent rod of 175 × 50 Å. The scattering profile of Pfr was calculated from that recorded for a mixture of Pr and Pfr under red-light irradiation by using their population determined from the absorption spectrum. The Pfr dimer exhibited a butterfly shape composed of subunits with a straight rod of 175 × 50 Å. The shape differences between Pr and Pfr indicated conformational changes in the Pr/Pfr interconversion which would be essential to the interaction with protein molecules involved in transcriptional control.

Visualization of intracellular structures and their spatial organization inside cells without any modification is essential to understand the mechanisms underlying the biological functions of cells. Here, we investigated the intracellular structure of cyanobacteria Prochlorococcus in the interphase by X-ray diffraction imaging using X-ray free-electron laser. A number of diffraction patterns from single cells smaller than 1 µm in size were collected with high signal-to-noise ratio with a resolution of up to 30 nm. From diffraction patterns, a set of electron density maps projected along the direction of the incident X-ray were retrieved with high reliability. The most characteristic structure found to be common among the cells was a C-shaped arrangement of 100-nm sized high-density spots, which surrounded a low-density area of 100 nm. Furthermore, a three-dimensional map reconstructed from the projection maps of individual cells was non-uniform, indicating the presence of common structures among cyanobacteria cells in the interphase. By referring to the fluorescent images for distributions of thylakoid membranes, nucleoids, and carboxysomes, we inferred and represented their spatial arrangements in the three-dimensional map. The arrangement allowed us to discuss the relevance of the intracellular organization to the biological functions of cyanobacteria.

© Springer Nature Singapore Pte Ltd. 2017. Coherent X-ray diffraction imaging (CXDI) is a lensless imaging technique for visualizing the structures of noncrystalline particles at a resolution of several tens of nanometers. The targets are particles with dimensions in the sub-micrometer to micrometer range. We carried out CXDI experiments at 66 K to investigate the internal structures of a whole frozen-hydrated cell and a chloroplast of Cyanidioschyzon merolae and a cyanobacteria cell by using coherent X-ray light sources, such as synchrotron and X-ray free-electron laser facilities. Owing to the short wavelength of the X-rays used (0.225 nm), the absorption and multiple scattering of X-rays inside the specimens were negligible. Diffraction patterns from each specimen particle adsorbed onto a thin membrane were collected at resolutions better than 50 μm -1 in reciprocal space. Therefore, structures of specimen particles can be illustrated at a resolution higher than 200 nm in real space. The most probable electron density map was retrieved from each diffraction pattern. Theinternal structures of the specimens are described here, particularly the structural correlation of the chloroplast of C. merolae and the cyanobacteria cell. Based on the experimental results, we discuss the feasibility of CXDI in the structural analyses of biological cells and cellular organelles.