Ultrastructure and fractal property of chromosomes in close-to-native yeast nuclei visualized using X-ray laser diffraction.

Genome compaction and activity in the nucleus depend on spatiotemporal changes in the organization of chromatins in chromosomes. However, the direct imaging of the chromosome structures in the nuclei has been difficult and challenging. Herein, we directly visualized the structure of chromosomes in frozen-hydrated nuclei of budding yeast in the interphase using X-ray laser diffraction. The reconstructed projection electron density maps revealed inhomogeneous distributions of chromosomes, such as a 300 nm assembly and fibrous substructures in the elliptic-circular shaped nuclei of approximately 800 nm. In addition, from the diffraction patterns, we confirmed the absence of regular arrangements of chromosomes and chromatins with 400-20 nm spacing, and demonstrated that chromosomes were composed of self-similarly assembled substructural domains with an average radius of gyration of 58 nm and smooth surfaces. Based on these analyses, we constructed putative models to discuss the organization of 16 chromosomes, carrying DNA of 4.1 mm in 800 nm ellipsoid of the nucleus at the interphase. We anticipate the structural parameters on the fractal property of chromosomes and the experimental images to be a starting point for constructing more sophisticated 3D structural models of the nucleus.

The photo-thermochemical properties and functions of Marchantia phototropin encoded by an unduplicated gene in land plant evolution.

Phototropin (phot) is a blue light photoreceptor in plants and possesses two photosensory light­oxygen-voltage (LOV1 and LOV2) domains with different photo-thermochemical properties. While liverworts contain a single copy of PHOT (e.g., MpPHOT in Marchantia polymorpha), many land plant species contain multicopy PHOT genes (e.g., AtPHOT1 and 2 in Arabidopsis thaliana) due to evolutionary gene duplication. The LOV domains of duplicated phot proteins have been studied in detail, but those of single-copy phot proteins remain to be characterized. As phot has not been duplicated in liverworts, we hypothesized that Mpphot may retain the ancestral function and photo-thermochemical properties. To learn more about the unduplicated phot proteins, we analyzed chloroplast relocation movement and the photo-thermochemical properties of LOV1 and LOV2 in Mpphot (Mpphot-LOV1 and Mpphot-LOV2, respectively). The function of Mpphot-LOV1, which induced a response to move chloroplasts to weak light (the accumulation response) in the absence of photoactive LOV2, differed from that of LOV1 of the duplicated phot proteins of A. thaliana (e.g., Atphot1-LOV1 preventing the accumulation response). On the other hand, the function of Mpphot-LOV2 was similar to that of LOV2 of the duplicated phots. The photo-thermochemical properties of Mpphot were a hybrid of those of the duplicated phots; the photochemical and thermochemical reactions of Mpphot were similar to those of the phot2- and phot1-type proteins, respectively. Our findings reveal conservation and diversification among LOV domains during phot duplication events in land plant evolution.

Common architectures in cyanobacteria Prochlorococcus cells visualized by X-ray diffraction imaging using X-ray free electron laser.

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.

Domain Organization in Plant Blue-Light Receptor Phototropin2 of Arabidopsis thaliana Studied by Small-Angle X-ray Scattering.

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.

Arabidopsis ROOT PHOTOTROPISM2 Is a Light-Dependent Dynamic Modulator of Phototropin1.

The Arabidopsis (Arabidopsis thaliana) blue light photoreceptor phototropin1 (phot1) is a blue light-activated Ser/Thr protein kinase that mediates various light responses, including phototropism. The function of phot1 in hypocotyl phototropism is dependent on the light induction of ROOT PHOTOTROPISM2 (RPT2) proteins within a broad range of blue light intensities. It is not yet known however how RPT2 contributes to the photosensory adaptation of phot1 to high intensity blue light and the phototropic responses under bright light conditions. We show that RPT2 suppresses the activity of phot1 and demonstrate that RPT2 binds to the PHOT1 light, oxygen or voltage sensing1 (LOV1) domain that is required for its high photosensitivity. Our biochemical analyses revealed that RPT2 inhibits autophosphorylation of phot1, suggesting that it suppresses the photosensitivity and/or kinase activity of phot1 through the inhibition of LOV1 function. We found that RPT2 proteins are degraded via a ubiquitin-proteasome pathway when phot1 is inactive and are stabilized under blue light in a phot1-dependent manner. We propose that RPT2 is a molecular rheostat that maintains a moderate activation level of phot1 under any light intensity conditions.

Methods and application of coherent X-ray diffraction imaging of noncrystalline particles.

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.

Molecular shape under far-red light and red light-induced association of Arabidopsis phytochrome B.

Phytochrome B (phyB) is a plant photoreceptor protein that regulates various photomorphogenic responses to optimize plant growth and development. PhyB exists in two photoconvertible forms: a red light-absorbing (Pr) and a far-red light-absorbing (Pfr) form. Therefore, to understand the mechanism of phototransformation, the structural characterization of full-length phyB in these two forms is necessary. Here, we report the molecular structure of Arabidopsis thaliana phyB in Pr form and the molecular properties of the Pfr form determined by small-angle X-ray scattering coupled with size-exclusion chromatography. In solution, the Pr form associated as a dimer with a radius of gyration of 50 Å. The molecular shape was a crossed shape, in which the orientation of the photosensory modules differed from that in the crystal structure of dimeric photosensory module. PhyB exhibited structural reversibility in the Pfr-to-Pr phototransformation and thermal reversion from Pfr to Pr in the dark. In addition, Pfr only exhibited nonspecific association, which distinguished molecular properties of Pfr form from those of the inactive Pr form.

Photoreaction Dynamics of Full-Length Phototropin from Chlamydomonas reinhardtii.

Phototropin (phot) is a blue light sensor involved in the light responses of several species from green algae to higher plants. Phot consists of two photoreceptive domains (LOV1 and LOV2) and a Ser/Thr kinase domain. These domains are connected by a hinge and a linker domain. So far, studies on the photochemical reaction dynamics of phot have been limited to short fragments, and the reactions of intact phot have not been well elucidated. Here, the photoreactions of full-length phot and of several mutants from Chlamydomonas reinhardtii (Cr) were investigated by the transient grating and circular dichroism (CD) methods. Full-length Cr phot is in monomeric form in both dark and light states and shows conformational changes upon photoexcitation. When LOV1 is excited, the hinge helix unfolds with a time constant of 77 ms. Upon excitation of LOV2, the linker helix unfolds initially followed by a tertiary structural change of the kinase domain with a time constant of 91 ms. The quantum yield of conformational change after adduct formation of LOV2 is much smaller than that of LOV1, indicating that reactive and nonreactive forms exist. The conformational changes associated with the excitations of LOV1 and LOV2 occur independently and additively, even when they are excited simultaneously. Hence, the role of LOV1 is not to enhance the kinase activity in addition to LOV2 function; we suggest LOV1 has different functions such as regulation of intermolecular interactions.

Measurements of Photoreaction and Kinase Activity of Phototropin, a Photoreceptor Protein for Tropic Response in Plants: Involvement of Kinase Activity in the Photosensitivity of Tropic Response.

Phototropin is a photoreceptor protein responsible for phototropic responses in plants. A phototropin molecule has two photoreceptive domains named LOV1 and LOV2 in the N-terminal region. Blue light absorbed by a chromophore in these domains triggers conformational changes in the protein moiety. The C-terminal region of phototropin forms a Ser/Thr kinase that is activated by these conformational changes. The activated phototropin kinase transmits signals downstream leading to tropic responses. The lifetime of the activated state may concern the sensitivity of the tropic responses to light. Thus, spectrophotometric and kinase activity analyses of phototropin are important to understand the light signaling processes related to the photosensitivity. The preparation of polypeptide samples of Arabidopsis phototropin and the methods of spectroscopic measurements and kinase assay of these samples are shown in this chapter.

Diffraction apparatus and procedure in tomography X-ray diffraction imaging for biological cells at cryogenic temperature using synchrotron X-ray radiation.

X-ray diffraction imaging is a technique for visualizing the structure of biological cells. In X-ray diffraction imaging experiments using synchrotron radiation, cryogenic conditions are necessary in order to reduce radiation damage in the biological cells. Frozen-hydrated biological specimens kept at cryogenic temperatures are also free from drying and bubbling, which occurs in wet specimens under vacuum conditions. In a previous study, the diffraction apparatus KOTOBUKI-1 [Nakasako et al. (2013), Rev. Sci. Instrum. 84, 093705] was constructed for X-ray diffraction imaging at cryogenic temperatures by utilizing a cryogenic pot, which is a cooling device developed in low-temperature physics. In this study a new cryogenic pot, suitable for tomography experiments, has been developed. The pot can rotate a biological cell over an angular range of ±170° against the direction of the incident X-ray beam. Herein, the details and the performance of the pot and miscellaneous devices are reported, along with established experimental procedures including specimen preparation. The apparatus has been used in tomography experiments for visualizing the three-dimensional structure of a Cyanidioschyzon merolae cell with an approximate size of 5 µm at a resolution of 136 nm. Based on the experimental results, the necessary improvements for future experiments and the resolution limit achievable under experimental conditions within a maximum tolerable dose are discussed.

Classification of ab initio models of proteins restored from small-angle X-ray scattering.

In structure analyses of proteins in solution by using small-angle X-ray scattering (SAXS), the molecular models are restored by using ab initio molecular modeling algorithms. There can be variation among restored models owing to the loss of phase information in the scattering profiles, averaging with regard to the orientation of proteins against the direction of the incident X-ray beam, and also conformational fluctuations. In many cases, a representative molecular model is obtained by averaging models restored in a number of ab initio calculations, which possibly provide nonrealistic models inconsistent with the biological and structural information about the target protein. Here, a protocol for classifying predicted models by multivariate analysis to select probable and realistic models is proposed. In the protocol, each structure model is represented as a point in a hyper-dimensional space describing the shape of the model. Principal component analysis followed by the clustering method is applied to visualize the distribution of the points in the hyper-dimensional space. Then, the classification provides an opportunity to exclude nonrealistic models. The feasibility of the protocol was examined through the application to the SAXS profiles of four proteins.

Photoreaction Dynamics of LOV1 and LOV2 of Phototropin from Chlamydomonas reinhardtii.

Phototropin is a blue light sensor protein found in higher plants and green algae. Photochemical reactions of a variety of differently truncated constructs of a phototropin from Chlamydomonas reinhardtii (Cr) (LOV1, LOV1-hinge, LOV2, LOV2-linker, and hinge-LOV2) are investigated. In the dark state, LOV1 is in dynamic equilibrium between the monomer and dimer, and the main photochemical reaction is dimerization of the monomer and dissociation of the dimer. On the other hand, LOV1-hinge exists as the monomer and the photochemical reaction is the dimerization reaction associated with the unfolding of the helix of the hinge domain. LOV2 in the dark state is monomeric. The conformation changes after the photoexcitation of LOV2 and LOV2-linker are minor, which differs notably from the reaction of LOV2-Jα and LOV2-linker from Arabidopsis thaliana (At). The linker region, including the Jα helix, is rather stable upon photoexcitation. The helix of the hinge domain of hinge-LOV2 is slightly unfolded in the dark state, and the major photoreaction is the dimerization event. The dark recovery rate of LOV2 was found to decrease significantly in the presence of the hinge domain. These photochemical properties of Cr phot are considerably different from those of At phot regarding conformational changes and their kinetics, although Cr phot has been reported to rescue the phot function in At. The differences and the diversity of phots are discussed.

Blue light-excited LOV1 and LOV2 domains cooperatively regulate the kinase activity of full-length phototropin2 from Arabidopsis.

Phototropin2 (phot2) is a blue-light (BL) receptor that regulates BL-dependent activities for efficient photosynthesis in plants. phot2 comprises two BL-receiving light-oxygen-voltage-sensing domains (LOV1 and LOV2) and a kinase domain. BL-excited LOV2 is thought to be primarily responsible for the BL-dependent activation of the kinase. However, the molecular mechanisms by which small BL-induced conformational changes in the LOV2 domain are transmitted to the kinase remain unclear. Here, we used full-length wild-type and mutant phot2 proteins from Arabidopsis to study their molecular properties in the dark and under BL irradiation. Phosphorylation assays and absorption measurements indicated that the LOV1 domain assists the thermal relaxation of BL-excited LOV2 and vice versa. Using small-angle X-ray scattering and electron microscopy, we observed that phot2 forms a dimer and has a rod shape with a maximum length of 188 Å and a radius of gyration of 44 Å. Under BL, phot2 displayed large conformational changes that bent the rod shape. By superimposing the crystal structures of the LOV1 dimer, LOV2, and a homology model of the kinase to the observed changes, we inferred that the BL-dependent change consisted of positional shifts of both LOV2 and the kinase relative to LOV1. Furthermore, phot2 mutants lacking the photocycle in LOV1 or LOV2 still exhibited conformational changes under BL, suggesting that LOV1 and LOV2 cooperatively contribute to the conformational changes that activate the kinase. These results suggest that BL-activated LOV1 contributes to the kinase activity of phot2. We discuss the possible intramolecular interactions and signaling mechanisms in phot2.

Phototropin perceives temperature based on the lifetime of its photoactivated state.

Living organisms detect changes in temperature using thermosensory molecules. However, these molecules and/or their mechanisms for sensing temperature differ among organisms. To identify thermosensory molecules in plants, we investigated chloroplast positioning in response to temperature changes and identified a blue-light photoreceptor, phototropin, that is an essential regulator of chloroplast positioning. Based on the biochemical properties of phototropin during the cellular response to light and temperature changes, we found that phototropin perceives temperature based on the temperature-dependent lifetime of the photoactivated chromophore. Our findings indicate that phototropin perceives both blue light and temperature and uses this information to arrange the chloroplasts for optimal photosynthesis. Because the photoactivated chromophore of many photoreceptors has a temperature-dependent lifetime, a similar temperature-sensing mechanism likely exists in other organisms. Thus, photoreceptors may have the potential to function as thermoreceptors.

Light-Induced Conformational Changes of LOV2-Kinase and the Linker Region in Arabidopsis Phototropin2.

Phototropins (phots) are blue light sensors found in a variety of higher plants and algae. The photochemical reactions of this family of proteins have attracted much attention since their discovery. Phots have two light sensor domains called light-oxygen-voltage 1 (LOV1) and LOV2. After the formation of the characteristic adduct of the LOV domain, a conformational change of the C-terminal region of the LOV2 domain occurs, and characterizing this change is important for understanding biological function, that is, kinase activation. Here, the reaction dynamics of the Jα-helix and the extended region adjacent to the Jα-helix (connector) have been investigated. The conformation of the connector part and the Jα-helix were found to alter significantly in a two-state manner. Furthermore, the conformational change of the kinase domain was also successfully detected as a change in translational diffusion, although the CD intensity due to the kinase domain movement was almost silent. These observations indicate that the tertiary structure of the kinase domain changes. The rate of the kinase domain change is almost the same as that of the change for the LOV2-linker, suggesting that the conformational change of the linker is the rate-determining step for kinase activation.

Anomalous pressure effects on the photoreaction of a light-sensor protein from Synechocystis, PixD (Slr1694), and the compressibility change of its intermediates.

SyPixD (Slr1694) is a blue-light receptor that contains a BLUF (blue-light sensor using a flavin chromophore) domain for the function of phototaxis. The key reaction of this protein is a light-induced conformational change and subsequent dissociation reaction from the decamer to the dimer. In this study, anomalous effects of pressure on this reaction were discovered, and changes in the compressibility of its short-lived intermediates were investigated. While the absorption spectra of the dark and light states are not sensitive to pressure, the formation yield of the first intermediate decreases with pressure to about 40% at 150 MPa. Upon blue-light illumination with a sufficiently strong intensity, the transient grating signal, which represents the dissociation of the SyPixD decamer, was observed at 0.1 MPa, and the signal intensity significantly decreased with increasing pressure. This behavior shows that the dissociation of the decamer from the second intermediate state is suppressed by pressure. However, while the decamer undergoes no dissociation upon excitation of one monomer unit at 0.1 MPa, dissociation is gradually induced with increasing pressure. For solving this strange behavior, the compressibility changes of the intermediates were measured as a function of pressure at weak light intensity. Interestingly, the compressibility change was negative at low pressure, but became positive with increasing pressure. Because the compressibility is related to the volume fluctuation, this observation suggests that the driving force for this reaction is fluctuation of the protein. The relationship between the cavities at the interfaces of the monomer units and the reactivity was also discussed.

Blue Light-excited Light-Oxygen-Voltage-sensing Domain 2 (LOV2) Triggers a Rearrangement of the Kinase Domain to Induce Phosphorylation Activity in Arabidopsis Phototropin1.

Phototropin1 is a blue light (BL) receptor in plants and shows BL-dependent kinase activation. The BL-excited light-oxygen-voltage-sensing domain 2 (LOV2) is primarily responsible for the activation of the kinase domain; however, the molecular mechanism by which conformational changes in LOV2 are transmitted to the kinase domain remains unclear. Here, we investigated BL-induced structural changes of a minimum functional fragment of Arabidopsis phototropin1 composed of LOV2, the kinase domain, and a linker connecting the two domains using small-angle x-ray scattering (SAXS). The fragment existed as a dimer and displayed photoreversible SAXS changes reflected in the radii of gyration of 42.9 Å in the dark and 48.8 Å under BL irradiation. In the dark, the molecular shape reconstructed from the SAXS profiles appeared as two bean-shaped lobes in a twisted arrangement that was 170 Å long, 80 Å wide, and 50 Å thick. The molecular shape under BL became slightly elongated from that in the dark. By fitting the crystal structure of the LOV2 dimer and a homology model of the kinase domain to their inferred shapes, the BL-dependent change could be interpreted as the positional shift in the kinase domain relative to that of the LOV2 dimer. In addition, we found that lysine 475, a functionally important residue, in the N-terminal region of LOV2 plays a critical role in transmitting the structural changes in LOV2 to the kinase domain. The interface between the domains is critical for signaling, suitably changing the structure to activate the kinase in response to conformational changes in the adjoining LOV2.

Specimen preparation for cryogenic coherent X-ray diffraction imaging of biological cells and cellular organelles by using the X-ray free-electron laser at SACLA.

Coherent X-ray diffraction imaging (CXDI) allows internal structures of biological cells and cellular organelles to be analyzed. CXDI experiments have been conducted at 66 K for frozen-hydrated biological specimens at the SPring-8 Angstrom Compact Free-Electron Laser facility (SACLA). In these cryogenic CXDI experiments using X-ray free-electron laser (XFEL) pulses, specimen particles dispersed on thin membranes of specimen disks are transferred into the vacuum chamber of a diffraction apparatus. Because focused single XFEL pulses destroy specimen particles at the atomic level, diffraction patterns are collected through raster scanning the specimen disks to provide fresh specimen particles in the irradiation area. The efficiency of diffraction data collection in cryogenic experiments depends on the quality of the prepared specimens. Here, detailed procedures for preparing frozen-hydrated biological specimens, particularly thin membranes and devices developed in our laboratory, are reported. In addition, the quality of the frozen-hydrated specimens are evaluated by analyzing the characteristics of the collected diffraction patterns. Based on the experimental results, the internal structures of the frozen-hydrated specimens and the future development for efficient diffraction data collection are discussed.

Molecular mechanism of phototropin light signaling.

Phototropin (phot) is a blue light (BL) receptor kinase involved in the BL responses of several species, ranging from green algae to higher plants. Phot converts BL signals from the environment into biochemical signals that trigger cellular responses. In phot, the LOV1 and LOV2 domains of the N-terminal region utilize BL for cyclic photoreactions and regulate C-terminal serine/threonine kinase (STK) activity. LOV2-STK peptides are the smallest functional unit of phot and are useful for understanding regulation mechanisms. The combined analysis of spectroscopy and STK activity assay in Arabidopsis phots suggests that the decay speed of the photo-intermediate S390 in LOV2 is one of the factors contributing to light sensitive kinase activity. LOV2 and STK are thought to be adjacent to each other in LOV2-STK with small angle scattering (SAXS). BL irradiation induces LOV2-STK elongation, resulting in LOV2 shifting away from STK. The N- and C-terminal lateral regions of LOV2, A'α-helix, Jα-helix, and A'α/Aß gap are responsible for the propagation of the BL signal to STK via conformational changes. The comparison between LOV2-STK and full-length phot from Chlamydomonas suggests that LOV1 is directly adjacent to LOV2 in LOV2-STK; therefore, LOV1 may indirectly regulate STK. The molecular mechanism of phot is discussed.