Subatomic structure of orthorhombic thaumatin at 0.89 Å reveals that highly flexible conformations are crucial for thaumatin sweetness.

Thaumatin is a sweet-tasting protein that elicits a sweet taste at a threshold of approximately 50 nM. Structure-sweetness relationships in thaumatin suggest that the basicity of two amino acids residues, Arg82 and Lys67, are particularly responsible for sweetness. Using tetragonal crystals, our structural analysis suggested that flexible sidechain conformations of these two residues play an important role in sweetness. However, in tetragonal crystals, Arg82 is adjacent to symmetry-related residues, and its flexibility is relatively restrained by the crystal packing. To reduce and diminish these symmetry-related effects, orthorhombic crystals were prepared, and their structures were successfully determined at a resolution of 0.89 Å. Within the orthorhombic lattice, two alternative conformations were more clearly visible at Lys67 than in a tetragonal system. Interestingly, for the first time, three alternative conformations at Arg82 were only found in an orthorhombic system. These results suggest the importance of flexible conformations in sweetness determinants. Such subtle structural variations might serve to adjust the complementarity of the electrostatic potentials of sweet receptors, thereby eliciting the potent sweet taste of thaumatin.

Oxygen-evolving photosystem II structures during S1-S2-S3 transitions.

Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.

Time-resolved serial crystallography to track the dynamics of carbon monoxide in the active site of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is part of the respiratory chain and contributes to the electrochemical membrane gradient in mitochondria as well as in many bacteria, as it uses the energy released in the reduction of oxygen to pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography to study the structural response of the active site upon flash photolysis of carbon monoxide (CO) from the reduced heme a3 of ba3-type CcO. In contrast with the aa3-type enzyme, our data show how CO is stabilized on CuB through interactions with a transiently ordered water molecule. These results offer a structural explanation for the extended lifetime of the CuB-CO complex in ba3-type CcO and, by extension, the extremely high oxygen affinity of the enzyme.

Serial femtosecond X-ray crystallography of an anaerobically formed catalytic intermediate of copper amine oxidase.

The mechanisms by which enzymes promote catalytic reactions efficiently through their structural changes remain to be fully elucidated. Recent progress in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers (XFELs) has made it possible to address these issues. In particular, mix-and-inject serial crystallography (MISC) is promising for the direct observation of structural changes associated with ongoing enzymic reactions. In this study, SFX measurements using a liquid-jet system were performed on microcrystals of bacterial copper amine oxidase anaerobically premixed with a substrate amine solution. The structure determined at 1.94 Šresolution indicated that the peptidyl quinone cofactor is in equilibrium between the aminoresorcinol and semiquinone radical intermediates, which accumulate only under anaerobic single-turnover conditions. These results show that anaerobic conditions were well maintained throughout the liquid-jet SFX measurements, preventing the catalytic intermediates from reacting with dioxygen. These results also provide a necessary framework for performing time-resolved MISC to study enzymic reaction mechanisms under anaerobic conditions.

Structure of thaumatin under acidic conditions: Structural insight into the conformations in lysine residues responsible for maintaining the sweetness after heat-treatment.

Thaumatin is an intensely sweet-tasting protein. Its sweetness persists when heated under acidic conditions, but disappears when heated at a pH above 7.0. To clarify how the structural features of thaumatin resist insoluble aggregation during heating under acidic conditions, we analysed its crystal structure obtained at pH 4.0, 6.0, and 8.0. Simultaneously, the melting temperature (Tm) at these pH levels was determined using differential scanning fluorimetry. At pH 4.0, the Tm of thaumatin was substantially lower and the overall B-factor value of its structure was higher than those at pH 6.0. Interestingly, the relative B-factor values for most lysine residues decreased as the pH reduced. These results suggest that the overall structure at pH 4.0 becomes flexible but the relative flexibility of some regions is lower than that at pH 6.0. Thus, the reduction in relative flexibility might play an important role in preventing thermal aggregation, thereby maintaining the sweetness.

Capturing structural changes of the S1 to S2 transition of photosystem II using time-resolved serial femtosecond crystallography.

Photosystem II (PSII) catalyzes light-induced water oxidation through an S i -state cycle, leading to the generation of di-oxygen, protons and electrons. Pump-probe time-resolved serial femtosecond crystallography (TR-SFX) has been used to capture structural dynamics of light-sensitive proteins. In this approach, it is crucial to avoid light contamination in the samples when analyzing a particular reaction intermediate. Here, a method for determining a condition that avoids light contamination of the PSII microcrystals while minimizing sample consumption in TR-SFX is described. By swapping the pump and probe pulses with a very short delay between them, the structural changes that occur during the S1-to-S2 transition were examined and a boundary of the excitation region was accurately determined. With the sample flow rate and concomitant illumination conditions determined, the S2-state structure of PSII could be analyzed at room temperature, revealing the structural changes that occur during the S1-to-S2 transition at ambient temperature. Though the structure of the manganese cluster was similar to previous studies, the behaviors of the water molecules in the two channels (O1 and O4 channels) were found to be different. By comparing with the previous studies performed at low temperature or with a different delay time, the possible channels for water inlet and structural changes important for the water-splitting reaction were revealed.

[Establishment of Simple and Quick Method to Confirm the Heating History of Milk and Dairy Products].

In Japan, the import quarantine procedure for dairy products was newly introduced in November 2017. The treatment such as 15 sec heating at 72℃ is required for virus inactivation when importing milk or dairy products from the area which is not free from foot and mouth disease. Moreover, the heating history of imported items is also inspected as import quality procedures. The IDF 63 method is known as one of the methods to confirm the heating history of milk by checking the alkaline phosphatase (ALP) activity. However, this procedure is complicated for daily quarantine inspection. Therefore, we attempted the ALP activity measurement based on the amount of fluorescent substance produced by the enzymatic reaction. Milk and dairy products derived from cow, sheep, and goat were tested after various heat treatment conditions. The ALP of heat-treated milk and dairy products derived from these species above were confirmed to be inactivated under substantially the same heat treatment for 15 sec at 72℃. The measurement method established in this study is simpler, faster, and requires smaller amount of sample compared to other methods. Additionally, the method was also applicable to confirm the heating history of various dairy products by making them into suspension.

Composition of uterine milk and its changes with gestational period in red stingrays (Hemitrygon akajei).

Uterine milk is secreted in the uterus for embryo nutrition in several elasmobranch species and may contribute to rapid embryonic growth, but the details of its composition and its functions are poorly understood. In this study, to explore the roles of uterine milk for embryos, its components throughout the gestational period were analysed in detail. Uterine milk was collected from pregnant red stingrays (Hemitrygon akajei) in the early, middle and late gestational periods, respectively (n= 3 for each period). The crude composition, constituent proteins and fatty acids in the milk were analysed. The uterine milk was rich in proteins throughout the gestational period, whereas lipids dramatically increased in the middle period and reduced slightly towards the late period. Some proteins potentially associated with nutrition, cartilage growth and embryonic immunity were found. Several enzymes related to central metabolism were also detected. The constituent fatty acids in the middle and late periods were similar to those in the egg yolks of elasmobranchs, except for C18:2, which was rich only in the uterine milk. The most abundant fatty acid in the milk was C16:1, which could function as a lipokine to promote lipid metabolism in the embryo. This study's data suggest that uterine milk may be secreted in addition to the egg yolk in elasmobranchs to support rapid and healthy embryonic growth.

Viscosity-adjustable grease matrices for serial nanocrystallography.

Serial femtosecond crystallography (SFX) has enabled determination of room temperature structures of proteins with minimum radiation damage. A highly viscous grease matrix acting as a crystal carrier for serial sample loading at a low flow rate of ~0.5 µl min-1 was introduced into the beam path of X-ray free-electron laser. This matrix makes it possible to determine the protein structure with a sample consumption of less than 1 mg of the protein. The viscosity of the matrix is an important factor in maintaining a continuous and stable sample column from a nozzle of a high viscosity micro-extrusion injector for serial sample loading. Using conventional commercial grease (an oil-based, viscous agent) with insufficient control of viscosity in a matrix often gives an unexpectedly low viscosity, providing an unstable sample stream, with effects such as curling of the stream. Adjustment of the grease viscosity is extremely difficult since the commercial grease contains unknown compounds, which may act as unexpected inhibitors of proteins. This study introduces two novel grease matrix carriers comprising known compounds with a viscosity higher than that of conventional greases, to determine the proteinase K structure from nano-/microcrystals.

A case of AL amyloidosis associated with follicular lymphoma with plasmacytic differentiation.

A 58-year-old woman underwent emergency surgical resection of the small intestine for intussusception as diagnosed at our hospital. Histopathological diagnosis of the resected specimen of the ileum was amyloid light chain (AL) amyloidosis. The colonoscopy after the surgical resection and following histopathological analysis of the biopsied specimens of the colon revealed follicular lymphoma (FL) grade 1 with plasmacytic differentiation. Histological findings of these ileal and colonic lesions were characteristic. In the ileum, CD10-positive lymphoid follicles and CD38-positive interfollicular plasma cell infiltration into villi were detected. The amyloid deposition was localized to the ileum and was adjacent to lymphoid follicles and interfollicular plasma cells. Furthermore, fluorescence in situ hybridization (FISH) for paraffin-embedded tissue sections (tissue-FISH) revealed that both the B cells in follicular lesions and the interfollicular plasma cells showed IGH/BCL2 fusion signals, which means the interfollicular plasma cells were originated from the differentiated neoplastic follicular B cells. The patient was treated with six courses of lymphoma chemotherapy and attained complete remission without any symptoms associated with amyloidosis. Further case analyses are needed to clarify the clinicopathological findings and to establish therapeutic strategy of AL amyloidosis associated with FL and FL with plasmacytic differentiation.

Homologous Expression and Characterization of Gassericin T and Gassericin S, a Novel Class IIb Bacteriocin Produced by Lactobacillus gasseri LA327.

Lactobacillus gasseri LA327, isolated from the large intestine tissue in humans, is a bacteriocinogenic strain with two kinds of class IIb bacteriocin structural genes, i.e., those for gassericin T (GT) and acidocin LF221A (Acd LF221A). In this study, DNA sequencing of the genes for GT and Acd LF221A from L. gasseri LA327 revealed that the amino acid sequences for GT corresponded with those for GT genes, except for GatK (histidine kinase). However, Acd LF221A genes had analogues which differed in at least one amino acid residue, to encode a class IIb bacteriocin designated gassericin S (GS). The LA327 strain retained antimicrobial activity after the deletion of the GT structural genes (gatAX); however, both GS and GT activities were lost by deletion of the putative ABC transporter gene (gatT). This indicates that the LA327 strain produces GS and GT and that GS secretion is performed via GT genes with the inclusion of gatT Homologous expression using deletion mutants of GS and GT, each containing a single peptide, elucidated that GS (GasAX) and GT (GatAX) showed synergistic activity as class IIb bacteriocins and that no synergistic activity was observed between GS and GT peptides. The molecular mass of GS was estimated to be theoretical ca. 5,400 Da by in situ activity assay after SDS-PAGE, clarifying that GS was actually expressed as an active class IIb bacteriocin. Furthermore, the stability of expressed GS to pH, heat, and protease was determined.IMPORTANCE Bacteriocins are regarded as potential alternatives for antibiotics in the absence of highly resistant bacteria. In particular, two-peptide (class IIb) bacteriocins exhibit the maximum activity through the synergy of two components, and their antimicrobial spectra are known to be relatively wide. However, there are few reports of synergistic activity of class IIb bacteriocins determined by isolation and purification of individual peptides. Our results clarified the interaction of each class IIb component peptide for GT and GS via the construction of homologous mutants, which were not dependent on the purification. These data may contribute to understanding the mechanisms of action by which class IIb bacteriocins exhibit wide antibacterial spectra.

Subatomic structure of hyper-sweet thaumatin D21N mutant reveals the importance of flexible conformations for enhanced sweetness.

One of the sweetest proteins found in tropical fruits (with a threshold of 50 nM), thaumatin, is also used commercially as a sweetener. Our previous study successfully produced the sweetest thaumatin mutant (D21N), designated hyper-sweet thaumatin, which decreases the sweetness threshold to 31 nM. To investigate why the D21N mutant is sweeter than wild-type thaumatin, we compared the structure of the D21N mutant solved at a subatomic resolution of 0.93 Šwith that of wild-type thaumatin determined at 0.90 Å. Although the overall structure of the D21N mutant resembles that of wild-type thaumatin, our subatomic resolution analysis successfully assigned and discriminated the detailed atomic positions of side-chains at position 21. The relative B-factor value of the side-chain at position 21 in the D21N mutant was higher than that of wild-type thaumatin, hinting at a greater flexibility of side-chain at 21 in the hyper-sweet D21N mutant. Furthermore, alternative conformations of Lys19, which is hydrogen-bonded to Asp21 in wild-type, were found only in the D21N mutant. Subatomic resolution analysis revealed that flexible conformations at the sites adjacent to positions 19 and 21 play a crucial role in enhancing sweet potency and may serve to enhance the complementarity of electrostatic potentials for interaction with the sweet taste receptor.

Positive Charges on the Surface of Thaumatin Are Crucial for the Multi-Point Interaction with the Sweet Receptor.

Thaumatin, an intensely sweet-tasting protein, elicits sweet taste with a threshold of only 50 nM. Previous studies from our laboratory suggested that the complex model between the T1R2-T1R3 sweet receptor and thaumatin depends critically on the complementarity of electrostatic potentials. In order to further validate this model, we focused on three lysine residues (Lys78, Lys106, and Lys137), which were expected to be part of the interaction sites. Three thaumatin mutants (K78A, K106A, and K137A) were prepared and their threshold values of sweetness were examined. The results showed that the sweetness of K106A was reduced by about three times and those of K78A and K137A were reduced by about five times when compared to wild-type thaumatin. The three-dimensional structures of these mutants were also determined by X-ray crystallographic analyses at atomic resolutions. The overall structures of mutant proteins were similar to that of wild-type but the electrostatic potentials around the mutated sites became more negative. Since the three lysine residues are located in 20-40 Å apart each other on the surface of thaumatin molecule, these results suggest the positive charges on the surface of thaumatin play a crucial role in the interaction with the sweet receptor, and are consistent with a large surface is required for interaction with the sweet receptor, as proposed by the multipoint interaction model named wedge model.

Experimental phase determination with selenomethionine or mercury-derivatization in serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) holds enormous potential for the structure determination of proteins for which it is difficult to produce large and high-quality crystals. SFX has been applied to various systems, but rarely to proteins that have previously unknown structures. Consequently, the majority of previously obtained SFX structures have been solved by the molecular replacement method. To facilitate protein structure determination by SFX, it is essential to establish phasing methods that work efficiently for SFX. Here, selenomethionine derivatization and mercury soaking have been investigated for SFX experiments using the high-energy XFEL at the SPring-8 Angstrom Compact Free-Electron Laser (SACLA), Hyogo, Japan. Three successful cases are reported of single-wavelength anomalous diffraction (SAD) phasing using X-rays of less than 1 Šwavelength with reasonable numbers of diffraction patterns (13 000, 60 000 and 11 000). It is demonstrated that the combination of high-energy X-rays from an XFEL and commonly used heavy-atom incorporation techniques will enable routine de novo structural determination of biomacromolecules.

XFEL structures of the influenza M2 proton channel: Room temperature water networks and insights into proton conduction.

The M2 proton channel of influenza A is a drug target that is essential for the reproduction of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across a membrane. Ordered water molecules arranged in "wires" inside the channel pore have been proposed to play a role in both the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. To visualize the solvent in the pore of the channel at room temperature while minimizing the effects of radiation damage, data were collected to a resolution of 1.4 Å using an X-ray free-electron laser (XFEL) at three different pH conditions: pH 5.5, pH 6.5, and pH 8.0. Data were collected on the Inwardopen state, which is an intermediate that accumulates at high protonation of the His37 tetrad. At pH 5.5, a continuous hydrogen-bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at pH 6.5 and 8.0, where the Inwardopen state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment.

Hydroxyethyl cellulose matrix applied to serial crystallography.

Serial femtosecond crystallography (SFX) allows structures of proteins to be determined at room temperature with minimal radiation damage. A highly viscous matrix acts as a crystal carrier for serial sample loading at a low flow rate that enables the determination of the structure, while requiring consumption of less than 1 mg of the sample. However, a reliable and versatile carrier matrix for a wide variety of protein samples is still elusive. Here we introduce a hydroxyethyl cellulose-matrix carrier, to determine the structure of three proteins. The de novo structure determination of proteinase K from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of the praseodymium atom was demonstrated using 3,000 diffraction images.

Atomic resolution structure of serine protease proteinase K at ambient temperature.

Atomic resolution structures (beyond 1.20 Å) at ambient temperature, which is usually hampered by the radiation damage in synchrotron X-ray crystallography (SRX), will add to our understanding of the structure-function relationships of enzymes. Serial femtosecond crystallography (SFX) has attracted surging interest by providing a route to bypass such challenges. Yet the progress on atomic resolution analysis with SFX has been rather slow. In this report, we describe the 1.20 Å resolution structure of proteinase K using 13 keV photon energy. Hydrogen atoms, water molecules, and a number of alternative side-chain conformations have been resolved. The increase in the value of B-factor in SFX suggests that the residues and water molecules adjacent to active sites were flexible and exhibited dynamic motions at specific substrate-recognition sites.

Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL.

Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique µ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously.

Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent.

The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.

Oil-free hyaluronic acid matrix for serial femtosecond crystallography.

The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins. However, the grease-based matrix has limited application for oil-sensitive proteins. Here we introduce a grease-free, water-based hyaluronic acid matrix. Applications for proteinase K and lysozyme proteins were able to produce electron density maps at 2.3-Å resolution.