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Biological polypeptides are known to containcis-linkage in their main chain as a minor but important feature. Such anomalous connection of amino acids has different structural and functional effects on proteins. Experimental evidence ofcis-bonds in proteins is mainly obtained using X-ray crystallography and other methods in the field of structural biology. To date, extensive analyses have been carried out on the experimentally foundcis-bonds using the Protein Data Bank entry bases and/or residue bases; however, their consistency in each protein has not been examined on a global scale. Data accumulation and advances in methodology enable the use of new approaches from a proteomic point of view. Here, we sought to describe a simple procedure for the detection and confirmation ofcis-bonds from a set of experimental coordinates for a protein to discriminate this type of bond from isomerizable and/or misassigned bonds. The resulting set of consistentcisbonds provides unprecedented insights into the trend of “highciscontent” proteins and the upper limit of consistentcisbonds per polypeptide length. Recognizing such limit would not only be important for a practical check of upcoming structures, but also for the design of novel protein folds beyond the evolutionally-acquired repertoire.

All living organisms have evolved to contain a set of proteins with variable physical and chemical properties. Efforts in the field of structural biology have contributed to uncovering the shape and the variability of each component. However, quantification of the variability has been performed mostly by multiple pair-wise comparisons. A set of experimental coordinates for a given protein can be used to define the “morphness/unmorphness”. To understand the evolved repertoire in an organism, here we show the results of global analysis of more than a thousand Escherichia coli proteins, by the recently introduced method, distance scoring analysis (DSA). By collecting a new index “UnMorphness Factor” (UMF), proposed in this study and determined from DSA for each of the proteins, the lowest and the highest boundaries of the experimentally observable structural variation are comprehensibly defined. The distribution plot of UMFs obtained for E. coli represents the first view of a substantial fraction of non-redundant proteome set of an organism, demonstrating how rigid and flexible components are balanced. The present analysis extends to evaluate the growing data from single particle cryo-electron microscopy, providing valuable information on effective interpretation to structural changes of proteins and the supramolecular complexes.

In high-level radioactive waste disposal repositories, there are long-term complex thermal, hydraulic, and mechanical (T–H–M) phenomena that involve the generation of heat from the waste, the infiltration of ground water, and swelling of the bentonite buffer. The ability to model such coupled phenomena is of particular importance to the repository design and assessments of its safety. We have developed a T–H–M-coupled analysis program that evaluates the long-term behavior around the repository (called “near-field”). We have also conducted centrifugal model tests that model the long-term T–H–M-coupled behavior in the near-field. In this study, we conduct H–M-coupled numerical simulations of the centrifugal near-field model tests. We compare numerical results with each other and with results obtained from the centrifugal model tests. From the comparison, we deduce that: (1) in the numerical simulation, water infiltration in the rock mass was in agreement with the experimental observation. (2) The constant-stress boundary condition in the centrifugal model tests may cause a larger expansion of the rock mass than in the in situ condition, but the mechanical boundary condition did not affect the buffer behavior in the deposition hole. (3) The numerical simulation broadly reproduced the measured bentonite pressure and the overpack displacement, but did not reproduce the decreasing trend of the bentonite pressure after 100 equivalent years. This indicates the effect of the time-dependent characteristics of the surrounding rock mass. Further investigations are needed to determine the effect of initial heterogeneity in the deposition hole and the time-dependent behavior of the surrounding rock mass.

The objective of this study is to evaluate the long-term geomechanical behavior of a geological repository for high-level radioactive waste disposal, using the centrifugal near-field model test. The model consisted of a sedimentary rock mass, bentonite buffer, and model overpack, and was enclosed within a pressure vessel. Tests were conducted with a centrifugal force field of 30 G under isotropic stress-constraint conditions with confining pressures of 5–10 MPa and injection of pore water up through a time period equivalent to about 165 years in the field. Our results showed that the measured values and the temporal changes in the displacement of the overpack, the soil pressure of the bentonite, and the strain of the rock mass were clearly dependent on the confining pressure. These data were not convergent during the test. Our data experimentally revealed that long-term behavior in the near field was changed by the geomechanical interaction between the deformation stress of the bedrock/disposal hole and the swelling behavior of the bentonite buffer.

Systematic analysis of statistical and dynamical properties of proteins is critical to understanding cellular events. Extraction of biologically relevant information from a set of high-resolution structures is important because it can provide mechanistic details behind the functional properties of protein families, enabling rational comparison between families. Most of the current structure comparisons are pairwise-based, which hampers the global analysis of increasing contents in the Protein Data Bank. Additionally, pairing of protein structures introduces uncertainty with respect to reproducibility because it frequently accompanies other settings for superimposition. This study introduces intramolecular distance scoring, for the analysis of human proteins, for each of which at least several high-resolution are available. We show that the results are comprehensively used to overview advances at the atomic level exploration of each protein and protein family. This method, and the interpretation based on model calculations, provide new criteria for understanding specific and non-specific structure variation in a protein, enabling global comparison of the dynamics among a vast variety of proteins from different species.

Visual opsins bind 11-cis retinal at an orthosteric site to form rhodopsins but increasing evidence suggests that at least some are capable of binding an additional retinoid(s) at a separate, allosteric site(s). Microspectrophotometric measurements on isolated, dark-adapted, salamander photoreceptors indicated that the truncated retinal analog, β-ionone, partitioned into the membranes of green-sensitive rods; however, in blue-sensitive rod outer segments, there was an enhanced uptake of four or more β-ionones per rhodopsin. X-ray crystallography revealed binding of one β-ionone to bovine green-sensitive rod rhodopsin. Cocrystallization only succeeded with extremely high concentrations of β-ionone and binding did not alter the structure of rhodopsin from the inactive state. Salamander green-sensitive rod rhodopsin is also expected to bind β-ionone at sufficiently high concentrations because the binding site is present on its surface. Therefore, both blue- and green-sensitive rod rhodopsins have at least one allosteric binding site for retinoid, but β-ionone binds to the latter type of rhodopsin with low affinity and low efficacy.

Most eukaryotic organisms sense a majority of extracellular signals such as light, odorants and neurotransmitters via heptahelical membrane receptors called GPCRs, which are structurally distinct from microbial retinal proteins (photoreceptors) . After several years from the first high-resolution structure determination of a prototypical GPCR visual rhodopsin in its inactive state, there has been significant progress over the past few years in the X-ray crystallographic study on this large family of receptors. Although the number of available structure is still limited, some valuable insights are obtained with regard to the structure and function of rhodopsin-like GPCRs.

Geological disposal in soft rocks is expected as one of the most practicable methods for isolation of high-level radioactive wastes. Since the country rock around the deep tunnels tends to be heated for a long term due to continuous collapse of nuclides, creep behavior of the soft rocks of impermeable nature under high temperature should be studied. Under different temperatures from 24°C to 95°C, a series of unconfined compression tests was conducted on a tuffaceous rock, Ohya stone, at creep stress ratios ranging from 0.6 to 1.0. The results show that the creep behavior is accelerated by high temperatures over 60°C. That is, for higher temperatures, the time to failure is decreased, while the minimum strain rate is increased. A creep model is then presented, in which attention is paid to the change in strain rate with time. Unique relationships between the minimum strain rate and various creep parameters are used, that appear to be dependent on neither creep stress ratio nor temperature. According to the proposed model, the creep failure will not take place if the creep stress ratio is lower than 0.44.

Intramolecular remote coupling within the polypeptide backbones of membrane proteins is difficult to analyze owing to the limited structural information available at the atomic level. Nonetheless, recent progress in the crystallographic study of G protein-coupled receptors (GPCRs) has provided an unprecedented opportunity for understanding the sophisticated architecture of heptahelical transmembrane (7TM) bundles. These 7TM bundles can respond to a wide range of extracellular stimuli while retaining the common function of binding trimeric G proteins. Here we have systematically analyzed select sets of inactive-like 7TM bundles to highlight the structural conservation of the receptors, in terms of intramolecular Cα-Cα distances. Distances with the highest scores were found to be dominated by the intrahelical distances of helix III, regardless of the choice of bundles in the set, indicating that the intracellular half of this helix is highly conserved. Unexpectedly, the distances between the cytoplasmic side of helix I and the extracellular region of helix VI provided the largest contribution to the high score populations among the interhelical pairs in most of the selected sets, including class B, C and frizzled receptors. These findings are expected to be valuable in further studies of GPCRs with unknown structure and of other protein families.

高レベル放射性廃棄物処分事業において，施設を構成する岩盤・岩石に対して，化石地下水や海水起源の塩類，空気供給による環境変化など化学的要因による劣化の検討をすることが必要と考えられる．しかし，現行の岩石劣化に関する試験，特にスレーキング試験では，これらの化学的な条件は想定されておらず計測項目もスレーキング率や含水比等であり，岩石劣化に伴う変化が予想される浸漬溶液の水質分析なども規定されていない．そこで本研究では，地下水質や空気供給状況を考慮できるスレーキング試験方法を提案するとともに，新たな評価項目を提示した．この方法を3種類の堆積性岩石に適用した結果，黄鉄鉱を含有する岩石では，空気供給状況の違いによって黄鉄鉱の溶脱挙動が異なること等が明らかになった．

Structural studies on retinal proteins have advanced significantly in recent years. Among the proteins whose structure has been solved by X-ray crystallography, rhodopsin is the only one from eukaryotic organisms having visual function. The structural model of rhodopsin also represents the first atomic template for a much larger superfamily of G protein-coupled receptors. Its natural abundance in vertebrate retinal rod cells and a novel single-step purification method made it possible to obtain three-dimensional crystals for X-ray diffraction study. The ground state structure has been refined so far to 2.6 A resolution, by which the details of the hepta-helical transmembrane region are resolved including functional internal water molecules. Possible structural change upon photo-excitation is likely to involve interaction changes between retinal chromophore and the surrounding residues. Further studies with microspectroscopy and X-ray diffraction with improved crystals that diffract to 2 A resolution will ...

A program on the structural genomics of membrane proteins has started at the BIRC, AIST, involving other academic institutions and industrial companies. Emphasis is being put on the development of techniques for the structural determination of membrane proteins of biological importance and ligand-receptor interactions by means of electron microscopy, X-ray diffraction, NMR, and computer simulation. Most efforts at the present stage, however, are being directed to finding suitable expression and purification systems and crystallization conditions for such proteins. The program is expected to be linked with the human full-length cDNA project and should lead to medical and industrial uses.

Activation of G protein-coupled receptors (GPCRs) is triggered and regulated by structural rearrangement of the transmembrane heptahelical bundle containing a number of highly conserved residues. In rhodopsin, a prototypical GPCR, the helical bundle accommodates an intrinsic inverse-agonist 11- cis -retinal, which undergoes photo-isomerization to the all-trans form upon light absorption. Such a trigger by the chromophore corresponds to binding of a diffusible ligand to other GPCRs. Here we have explored the functional role of water molecules in the transmembrane region of bovine rhodopsin by using x-ray diffraction to 2.6 Å. The structural model suggests that water molecules, which were observed in the vicinity of highly conserved residues and in the retinal pocket, regulate the activity of rhodopsin-like GPCRs and spectral tuning in visual pigments, respectively. To confirm the physiological relevance of the structural findings, we conducted single-crystal microspectrophotometry on rhodopsin packed in our three-dimensional crystals and show that its spectroscopic properties are similar to those previously found by using bovine rhodopsin in suspension or membrane environment.

Direct tension tests are indispensable for obtaining an accurate understanding of the deformation and failure characteristics of rocks in tension. For this technical note, the authors collected original papers published in international journals and surveyed the previously reported methods and results of direct tension tests on rocks. Some important factors affecting the strength and stress–strain curve are discussed, such as the shape, size, anisotropy, and water content of the specimen; the loading rate; and the confining pressure. The relationship between the results of the direct tension tests and those of other tests also is discussed. Although various types of testing apparatuses have been adopted in direct tension tests on rocks, the dependence of test results on the apparatuses has not been investigated. In this study, direct tension tests were conducted using various testing apparatuses in seven laboratories. The results of the direct tension tests showed that direct tensile strengths were not dependent on the type of test apparatus used or whether a flexible linkage system was used. The results of the tuff in this study showed that the coefficient of variation of the direct tensile strengths is smaller than that of the Brazilian tensile strengths. Moreover, the results revealed that the coefficient of variation of the direct tensile strengths is comparable to that of the uniaxial compressive strengths of the tuff. The authors noted that to obtain the stress–strain curve from the pre-failure region to the post-failure region, it is necessary to conduct a test with a high-stiffness machine and without a flexible linkage system. This technical note summarized the results of the standardization activity by the authors in the Japanese Geotechnical Society.

Purpose. To investigate genetic and clinical features of patients with rhodopsin (RHO) mutations in two Japanese families with autosomal dominant retinitis pigmentosa (adRP).Methods. Whole-exome sequence analysis was performed in ten adRP families. IdentifiedRHOmutations for the cosegregation analysis were confirmed by Sanger sequencing. Ophthalmic examinations were performed to evaluate the RP phenotypes. The impact of theRHOmutation on the rhodopsin conformation was examined by molecular modeling analysis.Results. In two adRP families, we identified twoRHOmutations (c.377G>T (p.W126L) and c.1036G>C (p.A346P)), one of which was novel. Complete cosegregation was confirmed for each mutation exhibiting the RP phenotype in both families. Molecular modeling predicted that the novel mutation (p.W126L) might impair rhodopsin function by affecting its conformational transition in the light-adapted form. Clinical phenotypes showed that patients with p.W126L exhibited sector RP, whereas patients with p.A346P exhibited classic RP.Conclusions. Our findings demonstrated that the novel mutation (p.W126L) may be associated with the phenotype of sector RP. Identification ofRHOmutations is a very useful tool for predicting disease severity and providing precise genetic counseling.

G-protein-coupled receptors (GPCRs) are involved in a vast variety of cellular signal transduction processes from visual, taste and odor perceptions to sensing the levels of many hormones and neurotransmitters. As a result of agonist-induced conformation changes, GPCRs become activated and catalyze nucleotide exchange within the G proteins, thus detecting and amplifying the signal. GPCRs share a common heptahelical transmembrane structure as well as many conserved key residues and regions. Rhodopsins are prototypical GPCRs that detect photons in retinal photoreceptor cells and trigger a phototransduction cascade that culminates in neuronal signaling. Biophysical and biochemical studies of rhodopsin activation, and the recent crystal structure determination of bovine rhodopsin, have provided new information that enables a more complete mechanism of vertebrate rhodopsin activation to be proposed. In many aspects, rhodopsin might provide a structural and functional template for other members of the GPCR family.

Membrane proteins, encoded by ~20% of genes in almost all organisms, including humans, are critical for cellular communication, electrical and ion balances, structural integrity of the cells and their adhesions, and other functions. Atomic-resolution structures of these proteins furnish important information for understanding their molecular organization and constitute major breakthroughs in our understanding of how they participate in physiological processes. However, obtaining structural information about these proteins has progressed slowly (1, 2), mostly because of technical difficulties in the purification and handling of integral membrane proteins. Instability of the proteins in environments lacking phospholipids, the tendency for them to aggregate and precipitate, and/or difficulties with highly heterogeneous preparations of these proteins isolated from heterologous expression systems have hindered application of standard structure determination techniques to these molecules. Among membrane proteins, G-protein-coupled receptors (GPCRs)1 are of special importance because they form one of the largest and most diverse groups of receptor proteins. More than 400 nonsensory receptors identified in the human genome are involved in the regulation of virtually all physiological processes. Drug addiction, mood control, and memory (via 5-HT6 or neuropeptide receptors) are just a short list of processes in which GPCRs are critically implicated. Another even larger group of GPCRs consist of sensory receptors involved in the fundamental process of translation of light energy (rhodopsin and cone pigments), the detection of chemoattractant molecules, or the detection of compounds stimulating the taste buds (3, 4). The activity of GPCRs comes about when binding of diffusable extracellular ligands causes them to switch from quiescent forms to an active conformation capable of interaction with hundreds of G-proteins. Their roles as extracellular ligand-binding proteins make them attractive targets for drug design. GPCRs account for ~40% of all therapeutic intervention, and major GPCR research projects are found throughout the pharmaceutical industry (5, 6). A paucity of structural data is available for GPCRs. The crystal structure of a member of the largest subgroup (I) of GPCRs, rhodopsin (7), and a ligand-binding domain of the metabotropic glutamate receptor with and without the ligand (8) have been determined recently. The data allow models, firmly based on the atomic-resolution structural information, to be further tested as to the conformational changes that these receptors undergo in going from the quiescent to the signaling state. In this article, we describe the further refinement of rhodopsin (7) and provide some clues about how the receptor could be activated by light.

In the purple membrane of Halobacterium salinarium, bacteriorhodopsin trimers are arranged in a hexagonal lattice. When purple membrane sheets are incubated at high temperature with neutral detergent, membrane vesicularization takes place, yielding inside-out vesicles with a diameter of 50 nm. The vesicular structure becomes unstable at low temperature, where successive fusion of the vesicles yields a crystal which is composed of stacked planar membranes. X-ray crystallographic analysis reveals that the bacteriorhodopsin trimers are arranged in a honeycomb lattice in each membrane layer and that neighbouring membranes orient in opposite directions. The native structure of the trimeric unit is preserved in the honeycomb lattice, irrespective of alterations in the in-plane orientation of the trimer. One phospholipid tightly bound to a crevice between monomers in the trimeric unit is suggested to act as a glue in the formation of the trimer.

The artificial isomeric 9-cis form of retinal has been investigated within the chromophore binding pocket of rhodopsin by x-ray crystallography and quantum-mechanics with particular emphasis on the geometry imposed on the chromophore by the protein. Comparison with the native 11-cis-rhodopsin structure reveals similarities and differences providing valuable insights into the photophysical properties of the visual pigment.

When the two-dimensional crystal of bacteriorhodopsin (bR), purple membrane, is incubated at high temperature (32 degreesC) with a small amount of the neutral detergent octylthioglucoside in the presence of the precipitant ammonium sulfate, a large fraction of the membrane fragments is converted into spherical vesicles with a diameter of 50 nm, which are able to assemble into optically isotropic hexagonal crystals when the precipitant concentration is increased. The vesicularization of purple membrane takes place under such a condition that the miscibility of the detergent to the aqueous phase becomes very low, and we suggest that it is initiated by insertion of the detergent molecules into the membrane. At low temperature, the transformation into the vesicular structure is inhibited and no large crystal is produced directly from membrane/detergent/precipitant mixtures. When a suspension of the spherical vesicles produced at the high temperature is cooled and concentrated below 15 degreesC, however, a birefringent hexagonal crystal is produced that diffracts X-rays beyond 2.5 A resolution. This new crystal belongs to the space group P622 with unit cell dimensions of a=b=104.7 A and c=114.1 A, and it is shown to be made up of stacked planar membranes, in each of which the bR trimers are arranged on a honeycomb lattice and the space among the proteins is filled with the detergent molecules and native lipids. These stacked membranes are suggested to be produced by successive fusion of the spherical vesicles. This implies that the crystallization is achieved without any step for complete solubilization of the protein. The present result offers a unique crystallization method that may be applicable to such membrane proteins that are liable to denature in the presence of an excess amount of detergent.

Detailed and systematic examination of high-resolution structural data is a rational strategy for understanding the function of biological macromolecules. G protein-coupled receptors (GPCRs) are an exceptionally valuable superfamily of proteins for such analysis. The most intriguing question is how a variety of extracellular stimuli evoke structural changes in the intracellular surface of the receptors. The recent active-like crystal structures of GPCRs provide information for uncovering common and distinct mechanisms of light-induced and ligand-induced activation. Based on systematic structural alignment, we have analyzed 3 receptors (rhodopsin, β2 adrenergic receptor, adenosine A2A receptor) and demonstrate that the extracellular movement of helix VI is significantly different between rhodopsin and the other 2 receptors, and that the extracellular side of helix III exhibits distinct features in the 3 receptors. These findings not only emphasize the specialization of rhodopsin as a photoreceptor but also provide insights into the mechanism leading to rearrangement of helix VI.

Recent accumulation of sequence and structural data, in conjunction with systematical classification into a set of families, has significantly advanced our understanding of diverse and specific protein functions. Analysis and interpretation of protein family data requires comprehensive sequence and structural alignments. Here, we present a simple scheme for analyzing a set of experimental structures of a given protein or family of proteins, using microbial rhodopsins as an example. For a data set comprised of around a dozen highly similar structures to each other (overall pairwise root-mean-squared deviation < 2.3 Å), intramolecular distance scoring analysis yielded valuable information with respect to structural properties, such as differences in the relative variability of transmembrane helices. Furthermore, a comparison with recent results for G protein-coupled receptors demonstrates how the results of the present analysis can be interpreted and effectively utilized for structural characterization of diverse protein families in general.

Through low-temperature absorption and circular dichroism (CD) spectroscopies, and G-protein (transducin) binding experiments, we have investigated molecular properties of the meta II intermediate of iodopsin, a cone visual pigment present in chicken red-sensitive cones. The meta II intermediate of iodopsin (metaiodopsin II, lambda max = 390 nm) displayed a positive CD band at about 390 nm and a large negative CD band below 300 nm. It dissociated into all-trans-retinal and the protein moiety. A long-lived intermediate corresponding to the meta III intermediate of rhodopsin was not observed in iodopsin, under our experimental conditions. Decay of metaiodopsin II was significantly suppressed in the presence of transducin, but not in the presence of both transducin and GTP, indicating that metaiodopsin II can interact with transducin and activate it. Both metaiodopsin II and metarhodopsin II displayed a large negative CD band below 300 nm. This fact suggested that during the formation of both meta II intermediates, some aromatic amino acid residues and/or a disulfide bond are rearranged, which may be important for expression of catalytic activity for exchange of GDP to GTP on transducin. On the other hand, metaiodopsin II decayed more than 10 times faster than metarhodopsin II. This fact may be one of the reasons why cones are less photosensitive than rods.

The photoactivation phenomena of the photosensitive enzyme nitrile hydratase (NHase) was studied by various spectroscopic methods. We have already shown that the photoactivation of NHase accompanies oxidation of an iron atom in the NHase [Honda et al. (1992) FEBS Lett. 301, 177-180]. From the results obtained in the present study by absorption, action, and fluorescence spectra, we show that the chromophore responsible for the photoactivation process is the iron complex, and the tryptophan residues in NHase induce the oxidation of the iron atom via an energy-transfer process. The nanosecond flash photolysis experiment revealed that this photoactivation process is completed within 50 ns, which suggests that the changes observable in the absorption spectra originate from an intramolecular electron transfer occurring from an electronically excited state. Also the role of a stabilizing reagent, namely, n-butyric acid (BA), was investigated using the above methods, which revealed that BA, besides its stabilizing effect, contributes to the increase in apparent photoactivation rate.