Structural and energetic analysis of metastable intermediate states in the E1P-E2P transition of Ca2+-ATPase.

Sarcoplasmic reticulum (SR) Ca2+-ATPase transports two Ca2+ ions from the cytoplasm to the SR lumen against a large concentration gradient. X-ray crystallography has revealed the atomic structures of the protein before and after the dissociation of Ca2+, while biochemical studies have suggested the existence of intermediate states in the transition between E1P⋅ADP⋅2Ca2+ and E2P. Here, we explore the pathway and free energy profile of the transition using atomistic molecular dynamics simulations with the mean-force string method and umbrella sampling. The simulations suggest that a series of structural changes accompany the ordered dissociation of ADP, the A-domain rotation, and the rearrangement of the transmembrane (TM) helices. The luminal gate then opens to release Ca2+ ions toward the SR lumen. Intermediate structures on the pathway are stabilized by transient sidechain interactions between the A- and P-domains. Lipid molecules between TM helices play a key role in the stabilization. Free energy profiles of the transition assuming different protonation states suggest rapid exchanges between Ca2+ ions and protons when the Ca2+ ions are released toward the SR lumen.

Acceleration of generalized replica exchange with solute tempering simulations of large biological systems on massively parallel supercomputer.

Generalized replica exchange with solute tempering (gREST) is one of the enhanced sampling algorithms for proteins or other systems with rugged energy landscapes. Unlike the replica-exchange molecular dynamics (REMD) method, solvent temperatures are the same in all replicas, while solute temperatures are different and are exchanged frequently between replicas for exploring various solute structures. Here, we apply the gREST scheme to large biological systems containing over one million atoms using a large number of processors in a supercomputer. First, communication time on a multi-dimensional torus network is reduced by matching each replica to MPI processors optimally. This is applicable not only to gREST but also to other multi-copy algorithms. Second, energy evaluations, which are necessary for the multistate bennet acceptance ratio (MBAR) method for free energy estimations, are performed on-the-fly during the gREST simulations. Using these two advanced schemes, we observed 57.72 ns/day performance in 128-replica gREST calculations with 1.5 million atoms system using 16,384 nodes in Fugaku. These schemes implemented in the latest version of GENESIS software could open new possibilities to answer unresolved questions on large biomolecular complex systems with slow conformational dynamics.

Implementation of residue-level coarse-grained models in GENESIS for large-scale molecular dynamics simulations.

Residue-level coarse-grained (CG) models have become one of the most popular tools in biomolecular simulations in the trade-off between modeling accuracy and computational efficiency. To investigate large-scale biological phenomena in molecular dynamics (MD) simulations with CG models, unified treatments of proteins and nucleic acids, as well as efficient parallel computations, are indispensable. In the GENESIS MD software, we implement several residue-level CG models, covering structure-based and context-based potentials for both well-folded biomolecules and intrinsically disordered regions. An amino acid residue in protein is represented as a single CG particle centered at the Cα atom position, while a nucleotide in RNA or DNA is modeled with three beads. Then, a single CG particle represents around ten heavy atoms in both proteins and nucleic acids. The input data in CG MD simulations are treated as GROMACS-style input files generated from a newly developed toolbox, GENESIS-CG-tool. To optimize the performance in CG MD simulations, we utilize multiple neighbor lists, each of which is attached to a different nonbonded interaction potential in the cell-linked list method. We found that random number generations for Gaussian distributions in the Langevin thermostat are one of the bottlenecks in CG MD simulations. Therefore, we parallelize the computations with message-passing-interface (MPI) to improve the performance on PC clusters or supercomputers. We simulate Herpes simplex virus (HSV) type 2 B-capsid and chromatin models containing more than 1,000 nucleosomes in GENESIS as examples of large-scale biomolecular simulations with residue-level CG models. This framework extends accessible spatial and temporal scales by multi-scale simulations to study biologically relevant phenomena, such as genome-scale chromatin folding or phase-separated membrane-less condensations.

[Storage Stability of Nitrite in Nitrite Analysis of Meat Products].

For the analysis of nitrite ions in food, the stabilities of nitrite ions in meat products and their standard solutions were evaluated. Nitrite is easily oxidized or reduced; hence, products with standard solutions or colour retention agent must be carefully handled. To assess the stability and decreasing trend of nitrite, we examined the storage stability of standard solutions using calibration curves, the time course of nitrite in chopped meat products stored under different conditions, and the time course of nitrite in the sample solutions. Regarding calibration curves, the storage stability was determined for standard solutions that were prepared with ultrapure water at concentrations of 0.025 and 0.4 µg/mL and were stored at 5℃ for one year. The results revealed no changes in concentration of any solution over time, suggesting that no readjustments to the standard solution concentration were necessary before testing until one year after their preparation. Time course of nitrite in chopped meat products stored under different conditions showed a significant decrease in nitrite in refrigerated storage (5℃), whereas stability of nitrite was maintained for up to 1 day in frozen storage (-20℃) and for 14 days in frozen storage (-40℃). The time course of nitrite in the sample solutions showed that the quantitative values of nitrite in the extract remained unchanged within one week of extraction for the meat products tested in the study.

[Improvement of Nitrite Analysis Method in Food Products].

The conventional analysis method has problems with extraction efficiency, operability, and reproducibility. In this study, we attempted to solve these problems and improve the analytical method to obtain sufficient extraction efficiency and good operability and accuracy. The conventional method was able to get sufficient extraction in dried meat products, where the extraction efficiency of the conventional method was low, by increasing the concentration of sodium hydroxide solution at the time of homogenization. Suction filtration after adding the defoaming agent was added allowed for accurate volume adjustment. The turbidity of the extract caused by insufficient addition of zinc acetate solution was removed by increasing the amount of zinc acetate solution that was added. Turbidity caused by starch was removed by adding pancreatin. The RSD of the quantitative values was improved by adding sodium hydroxide solution and 80-90℃ water and immediately homogenizing. Furthermore, by changing the dilution factor of the extract solution in the colorimetric method, the inhibition of coloration by reducing substances was suppressed, and more accurate quantitative values could be obtained than with the conventional method. The recovery rate was 78.5-105% (RSD 0.7-5.8%), which was a good result. This method was considered to be a useful analytical method that can contribute to improving the inspection accuracy of nitrite ion analysis.

[Study on Migrants Found in Migration Solutions from Commercially Available Polyethylene Products].

Migrants found in migration solutions obtained from commercially available polyethylene products that may contain food were studied and analysed via liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-QTOF) for non-target screening and LC-MS/MS for quantifying 14 substances in migration solutions. Furthermore, an analytical approach based on the retention gap was developed for accurate separation techniques using LC-MS/MS. Irganox 1076 was detected at a maximum of 1.5 mg/kg, which was 1/4 of the Specific Migration Limit in the EU, in nine commercially available plastic bags tested. This is in accordance with European Regulation No 10/2011/EU. Furthermore, migration of Erucamide and Irgafos 168-oxide was confirmed.

[Surveillance of the Naturally Derived Benzoic Acid Levels in Fruits and Fruit Products, and Comparison of Analytical Methods].

Benzoic acid (BA) is typically found in natural food; therefore, naturally occurring BA must be distinguished from added BA preservatives. In this study, we investigated BA levels in 100 samples of fruit products and their fresh fruits as raw materials using dialysis and steam distillation approaches. BA was detected in the range (minimum-maximum) of 2.1-1380 µg/g and 2.2-1950 µg/g in dialysis and steam distillation, respectively. Steam distillation indicated higher BA levels than dialysis.

[Study of Rapid HPLC Method for the Determination of Antioxidants in Foods, and Standard Solution Stability during Storage].

In this study, we simultaneously determined three antioxidants, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tert-butylhydroquinone (TBHQ), using HPLC equipped both with a photodiode array detector and a fluorescence detector in 25 minutes per sample. Due to the combined use of the two detectors, we could achieve improved target selectivity. Further, quantification at the specific wavelengths for each target substance particularly increased BHT detection sensitivity. This approach enabled us to avoid repeated measurements during daily inspections. Furthermore, detections were performed using LC-MS/MS instead of GC-MS to overcome the problem of helium gas shortage.In addition, we investigated antioxidant stability in standard solutions during storage. Although TBHQ was stable in methanol with ascorbic acid at -20℃, ascorbic acid storage has possibility to lead to decrease in BHT and BHA concentrations. We recognized that the mixture of BHT and BHA dissolved in methanol at 4℃ and that of BHT, BHA and TBHQ dissolved in methanol with ascorbic acid at -20℃ were suitable for about one year.

Elucidation of interactions regulating conformational stability and dynamics of SARS-CoV-2 S-protein.

The ongoing COVID-19 pandemic caused by the new coronavirus, SARS-CoV-2, calls for urgent developments of vaccines and antiviral drugs. The spike protein of SARS-CoV-2 (S-protein), which consists of trimeric polypeptide chains with glycosylated residues on the surface, triggers the virus entry into a host cell. Extensive structural and functional studies on this protein have rapidly advanced our understanding of the S-protein structure at atomic resolutions, although most of these structural studies overlook the effect of glycans attached to the S-protein on the conformational stability and functional motions between the inactive down and active up forms. Here, we performed all-atom molecular dynamics simulations of both down and up forms of a fully glycosylated S-protein in solution as well as targeted molecular dynamics simulations between them to elucidate key interdomain interactions for stabilizing each form and inducing the large-scale conformational transitions. The residue-level interaction analysis of the simulation trajectories detects distinct amino acid residues and N-glycans as determinants on conformational stability of each form. During the conformational transitions between them, interdomain interactions mediated by glycosylated residues are switched to play key roles on the stabilization of another form. Electrostatic interactions, as well as hydrogen bonds between the three receptor binding domains, work as driving forces to initiate the conformational transitions toward the active form. This study sheds light on the mechanisms underlying conformational stability and functional motions of the S-protein, which are relevant for vaccine and antiviral drug developments.

New parallel computing algorithm of molecular dynamics for extremely huge scale biological systems.

In this paper, we address high performance extreme-scale molecular dynamics (MD) algorithm in the GENESIS software to perform cellular-scale molecular dynamics (MD) simulations with more than 100,000 CPU cores. It includes (1) the new algorithm of real-space nonbonded interactions maximizing the performance on ARM CPU architecture, (2) reciprocal-space nonbonded interactions minimizing communicational cost, (3) accurate temperature/pressure evaluations that allows a large time step, and (4) effective parallel file inputs/outputs (I/O) for MD simulations of extremely huge systems. The largest system that contains 1.6 billion atoms was simulated using MD with a performance of 8.30 ns/day on Fugaku supercomputer. It extends the available size and time of MD simulations to answer unresolved questions of biomacromolecules in a living cell.

Coarse-Grained Modeling of Multiple Pathways in Conformational Transitions of Multi-Domain Proteins.

Large-scale conformational transitions in multi-domain proteins are often essential for their functions. To investigate the transitions, it is necessary to explore multiple potential pathways, which involve different intermediate structures. Here, we present a multi-basin (MB) coarse-grained (CG) structure-based Go̅ model for describing transitions in proteins with more than two moving domains. This model is an extension of our dual-basin Go̅ model in which system-dependent parameters are determined systematically using the multistate Bennett acceptance ratio method. In the MB Go̅ model for multi-domain proteins, we assume that intermediate structures may have partial inter-domain native contacts. This approach allows us to search multiple transition pathways that involve distinct intermediate structures using the CG molecular dynamics (MD) simulations. We apply this scheme to an enzyme, adenylate kinase (AdK), which has three major domains and can move along two different pathways. Using the optimized mixing parameters for each pathway, AdK shows frequent transitions between the Open, Closed, and the intermediate basins and samples a wide variety of conformations within each basin. The explored multiple transition pathways could be compared with experimental data and examined in more detail by atomistic MD simulations.

[Attempt to Calculate the Efficiency of Methylation Using 1H-NMR for Analyzing Brominated Vegetable Oil].

We studied the efficiency of methylation for analyzing brominated vegetable oil (BVO). In this report, we investigated whether 1H-NMR is an applicable method for assessing the efficiency of methylation to analyze BVO. 1H-NMR sufficiently calculated the efficiency of methylation using each integral and the numbers of protons derived from the methyl group, which is characteristic in products, and the methine group, which is characteristic in unreacted substances. Additionally, the efficiency of methylation calculated via 1H-NMR was in good agreement with changes in the peak area of BVO fatty acid methyl esters (BVOFAMEs) after various heating times obtained from GC-FID analysis. Therefore, 1H-NMR is applicable for calculating the efficiency of methylation to analyze BVO.

A singularity-free torsion angle potential for coarse-grained molecular dynamics simulations.

Conventional torsion angle potentials used in molecular dynamics (MD) have a singularity problem when three bonded particles are collinearly aligned. This problem is often encountered in coarse-grained (CG) simulations. Here, we propose a new form of the torsion angle potential, which introduces an angle-dependent modulating function. By carefully tuning the parameters for this modulating function, our method can eliminate the problematic angle-dependent singularity while being combined with existing models. As an example, we optimized the modulating function of the torsion angle potential for popular CG models of biomolecules based on the statistics over experimental structures deposited in the Protein Data Bank. By applying our method to designed and natural biomolecules, we show that the new torsion angle potential is able to eliminate the singularity problem while maintaining the structural features in the original models. Furthermore, by comparing our design with previous methods, we found that our new potential has advantages in computational efficiency and numerical stability. We strongly recommend the usage of our new potential in the CG simulations of flexible molecules.

[Determination of Nitrate Ion in Cheese and Sake].

The Japanese official analysis method for determination of nitrate ions in food products used as food additives is associated with various challenges. In some kinds of cheese, the extract becomes suspended. The volume of extracted solution is often not accurate owing to the presence of residues in the solution. Moreover, the determination with liquid chromatography-ultraviolet detection (HPLC-UV) is difficult owing to the influence of impurities. Sake usually does not contain lipids or proteins ; therefore, its analysis can be simplified by omitting the co-precipitation steps to remove them. In the present study, for cheese, the amount of sodium hydroxide solution that causes suspension was reduced, and the influence of residues was removed by adjusting the volume after suction filtration. Whereas, sake was diluted with water and centrifuged. Furthermore, solid-phase extraction (SPE) method using cartridge containing carbon molecular sieve to remove the influence of impurities on the chromatogram was successfully established. The recoveries of the nitrate ions were good outcomes of 91.3-99.6% (CV 0.9-4.5%) (n=5). The analysis range was 0.010-0.20 g/kg for cheese, 0.010-0.20 g/L for milk, and 0.010-0.10 g/kg for sake. The developed analysis methods are considered useful, because various challenges of the official analysis method can be solved and the operation are efficient.

Scaling molecular dynamics beyond 100,000 processor cores for large-scale biophysical simulations.

The growing interest in the complexity of biological interactions is continuously driving the need to increase system size in biophysical simulations, requiring not only powerful and advanced hardware but adaptable software that can accommodate a large number of atoms interacting through complex forcefields. To address this, we developed and implemented strategies in the GENESIS molecular dynamics package designed for large numbers of processors. Long-range electrostatic interactions were parallelized by minimizing the number of processes involved in communication. A novel algorithm was implemented for nonbonded interactions to increase single instruction multiple data (SIMD) performance, reducing memory usage for ultra large systems. Memory usage for neighbor searches in real-space nonbonded interactions was reduced by approximately 80%, leading to significant speedup. Using experimental data describing physical 3D chromatin interactions, we constructed the first atomistic model of an entire gene locus (GATA4). Taken together, these developments enabled the first billion-atom simulation of an intact biomolecular complex, achieving scaling to 65,000 processes (130,000 processor cores) with 1 ns/day performance. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

[Determination of Aspartame, Acesulfame Potassium, and Sucralose in Chewing Gum by Dialysis Extraction].

The three sweeteners, aspartame, acesulfame potassium, and sucralose, in chewing gum were determined by using dialysis and direct extraction methods. The results revealed that the previously reported dialysis method tended to show poor extraction of aspartame in comparison with the direct-extraction method. The direct extraction also caused operational problems, such as the gum base adhering to the instruments. Therefore, we attempted to improve the dialysis method by changing the dialysate, to which the three sweeteners were extracted while the sample stayed inside the dialysis tube. By changing the dialysate to 60% methanol and dialyzing for 24 hr at room temperature or 2 hr with shaking while heating at 50℃, all three sweeteners were extracted as good as those with the direct-extraction method.

Kinetic energy definition in velocity Verlet integration for accurate pressure evaluation.

In molecular dynamics (MD) simulations, a proper definition of kinetic energy is essential for controlling pressure as well as temperature in the isothermal-isobaric condition. The virial theorem provides an equation that connects the average kinetic energy with the product of particle coordinate and force. In this paper, we show that the theorem is satisfied in MD simulations with a larger time step and holonomic constraints of bonds, only when a proper definition of kinetic energy is used. We provide a novel definition of kinetic energy, which is calculated from velocities at the half-time steps (t - Δt/2 and t + Δt/2) in the velocity Verlet integration method. MD simulations of a 1,2-dispalmitoyl-sn-phosphatidylcholine (DPPC) lipid bilayer and a water box using the kinetic energy definition could reproduce the physical properties in the isothermal-isobaric condition properly. We also develop a multiple time step (MTS) integration scheme with the kinetic energy definition. MD simulations with the MTS integration for the DPPC and water box systems provided the same quantities as the velocity Verlet integration method, even when the thermostat and barostat are updated less frequently.

[Method of Quantitative Analysis by HPLC and Confirmation by LC-MS/MS of Erythritol, Maltitol, Lactitol and Trehalose in Foods].

A quantitative determination method of erythritol, maltitol, lactitol and trehalose in foods by HPLC, and confirmation method by LC-MS/MS were developed. HPLC analysis was performed on a separation column packed with amino group-binding polymer with acetonitrile-water (80 : 20) as the mobile phase. The column was operated at room temperature, and the three sugar alcohols and trehalose were quantified. LC-MS/MS confirmation was performed on an amino group-bound column with acetonitrile-ammonium acetate solution as the mobile phase, with detection in the SRM mode. At low sample dilution ratios, the analysis may be affected by matrix derived from the sample, but this can be suppressed by 1,000-fold or greater dilution. Recoveries of the three sugar alcohols and trehalose spiked into food samples, such as tea, jelly, tablets (ramune candy), and chocolate, exceeded 90% (CV≦6.1%) in HPLC and 94% (CV≦4.8%) in LC-MS/MS.

[Survey of Aluminium Content of Processed Food Using Baking Powder (2015-2016)].

The aluminium (Al) content of Japanese confectionery and foods containing flour was investigated. Some of these items were investigated in previous studies, which examined foods that made use of baking powder containing aluminium potassium sulfate (Alum). Al was detected in 41 of the 123 samples at levels ranging from 0.01 (limit of quantitation) to 0.40 mg/g. The detection rate of Al in almost all confectionery (except Japanese confectionery) was decreased as compared with previous studies. However, the detection rate of Al in Japanese confectionery and foods containing flour was high. For 4 of the 41 samples tested, consuming one serving once a week would result in an Al intake exceeding the PTWI for young children (body weight=16 kg).

Flexible fitting to cryo-EM density map using ensemble molecular dynamics simulations.

Flexible fitting is a computational algorithm to derive a new conformational model that conforms to low-resolution experimental data by transforming a known structure. A common application is against data from cryo-electron microscopy to obtain conformational models in new functional states. The conventional flexible fitting algorithms cannot derive correct structures in some cases due to the complexity of conformational transitions. In this study, we show the importance of conformational ensemble in the refinement process by performing multiple fittings trials using a variety of different force constants. Application to simulated maps of Ca2+ ATPase and diphtheria toxin as well as experimental data of release factor 2 revealed that for these systems, multiple conformations with similar agreement with the density map exist and a large number of fitting trials are necessary to generate good models. Clustering analysis can be an effective approach to avoid over-fitting models. In addition, we show that an automatic adjustment of the biasing force constants during the fitting process, implemented as replica-exchange scheme, can improve the success rate. © 2017 Wiley Periodicals, Inc.