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Can current simulations quantitatively predict the stability of ribonucleic acids (RNAs)? In this research, we apply a free-energy perturbation simulation of RNAs containing inosine, a modified ribonucleic base, to the derivation of RNA nearest-neighbor parameters. A parameter set derived solely from 30 simulations was used to predict the free-energy difference of the RNA duplex with a mean unbiased error of 0.70 kcal/mol, which is a level of accuracy comparable to that obtained with parameters derived from 25 experiments. We further show that the error can be lowered to 0.60 kcal/mol by combining the simulation-derived free-energy differences with experimentally measured differences. This protocol can be used as a versatile method for deriving nearest-neighbor parameters of RNAs with various modified bases.

Two types of implementation of the Hartree-Fock exchange energy were developed with the real-space grid approach for the purpose to achieve a high efficiency in the parallel execution of the hybrid exchange functional in the density functional theory (DFT). First, a parallel implementation of the three-dimensional (3D) fast Fourier transform (FFT), referred to as PFFT, was adapted to solve the Poisson equations for the electrostatic potentials of the densities of the orbital pairs. In the other approach, the Poisson equations were solved iteratively through the conjugate gradient (CG) procedures where the operation of Laplacian was parallelized by the domain decomposition scheme. The comparison of the parallel performances for the exchange energy calculation were made between these two approaches and it was revealed that the calculation with the FFT method is faster that that with CG. The method with FFT is more advantageous than CG because the larger band width can be available in the collective MPI communication associated with the parallel execution of FFT. We also implemented the projection operator to circumvent the laborious calculation of the exchange energy at every SCF step, which made a significant contribution to expedite the convergence.

Eukaryotic genome is packaged in a nucleus in the form of chromatin. The fundamental structural unit of the chromatin is the protein-DNA complex, nucleosome, where DNA of about 150 bp is wrapped around a histone core almost twice. In cellular processes such as gene expression, DNA repair and duplication, the nucleosomal DNA has to be unwrapped. Histone proteins have their variants, indicating there are a variety of constitutions of nucleosomes. These different constitutions are observed in different cellular processes. To investigate differences among nucleosomes, we calculated free energy profiles for unwrapping the outer superhelical turn of CENP-A nucleosome and compared them with those of the canonical nucleosome. The free energy profiles for CENP-A nucleosome suggest that CENP-A nucleosome is most stable when 16 to 22 bps are unwrapped in total whereas the canonical nucleosome is most stable when it is fully wrapped. This indicates that the flexible conformation of CENP-A nucleosome is ready to provide binding sites for the structural integrity of the centromere.

The Manganese cluster (CaMn4O5) in a photosystem II (PSII) is the reaction center of the light-driven oxidation reaction which generates the molecular oxygen. In the present paper we address the issue of the effect of the environment on the free energy associated with the oxidation of the Mn cluster in S1 state by conducting the large-scale QM/MM-ER simulations which involves the whole of the PSII monomer. It was found by the simulations at the level of B3LYP functional that the environment surrounding the Mn cluster reduces the vertical oxidation free energy ∆µvrt by 64.8 kcal/mol. A decomposition analysis of the free energy ∆µvrt revealed that the system composed of peptide chains, ligands, lipids, and potassium ions contributes to lower ∆µvrt by −98.0 kcal/mol, while the solvent water makes an opposite contribution of 38.9 kcal/mol. Reduction of the vertical oxidation free energy directly leads to the lowering of the activation free energy ∆Gac for the electron transfer reaction from the Mn cluster in S1 state to the neighboring Tyr+z. Consequently, the electron transfer rate was found to be enhanced by a factor of 10^12 by virtue of the influence of the environment.

酵素反応は生体発動分子の化学・力学エネルギー変換を司る。酵素では反応中心の周辺の残基が巧みに配置されることで中間体となる構造を安定化し、活性化自由エネルギーを低下させることで反応性を向上させている。経験的原子価結合法(empirical valence bond method) はこのような酵素サイズの分子について、その活性化自由エネルギーなどを低コストで計算し予測できる手法である。現在、経験的原子価結合法は例えば酵素反応中心の周囲の残基への変異による反応への影響を予測できる。しかしながら、これまで経験的原子価結合法は初期構造依存性が高い手法と考えられており、モデリングに基づく構造を元にした計算はほとんど試みられてこなかった。 本発表では、モデリング構造を用いて経験的原子価結合法を実施した場合に起きる問題を説明し、そのシンプルな解決策を提示する。, 発動分子科学 第五回領域会議

生物は生存に必要な様々な化学反応を温和な環境で実現させるため、触媒として働くタンパク質である酵素を用いている。これまでに酵素の挙動を原子レベルで解析するため、種々の理論的手法が考案されてきた。これまでの試みでは、大きく分けて量子化学的効果を考慮して分子運動を解析する量子化学計算(QM)と、系を完全に古典的な力学系として捉える古典分子力学(MM)の2つが良く用いられている。前者は電子をあらわに扱うことで高精度な計算を可能とするが、生体分子サイズの大きな構造変化を扱うことや、長時間の力学的挙動を追うことは計算量の観点から難しい。一方で後者は高速な計算が可能なことからより大きな構造変化を捉える計算に向くが、電子移動の関わる現象、特に化学反応に対し解析を行うことは困難である。この利点を組み合わせ、活性中心付近をQMで計算し他をMMで計算することで精度と構造変化の解析の両立を狙ったQM/MM法などが精力的に開発されているが、MMを用いて分子運動を解析する分子動力学法(MD)と比べ計算量が多く、大規模な生体分子の構造変化を追跡するにはまだ課題が多い手法となっている。 経験的原子価結合法(Empirical Valence Bond; EVB)は、このQM/MMとMMのさらに中間に位置する手法であり、低コストで反応過程におけるエネルギー面や反応に伴う構造変化を見積もることができる手法である。EVB法の生体分子への応用では、これまでにpH値の推定や酵素を対象とした反応活性化エネルギーの推定が行われ、実験的に得られたpH値や反応速度から推定される活性化エネルギーをよく再現することが報告されてきた。EVB法はMDと同程度のオーダーの計算速度を持ち、さらに活性中心付近に変異を入れた際の酵素反応の活性予測が可能など、特に活性中心の再設計などの発動分子の作成に威力を発揮することが期待される手法である。本研究では、これまで難しいと考えられてきた、対象の酵素の結晶構造がまだ得られておらずホモログ（起源を同じくする遺伝子）の結晶構造しか得られていない場合に、活性化自由エネルギーを精度良く計算する方法を報告する。, 量子生命科学会第２回大会

酵素反応は生体発動分子の化学・力学エネルギー変換を司る。酵素では反応中心の周辺の残基が巧みに配置されることで中間体となる構造を安定化し、活性化自由エネルギーを低下させることで反応性を向上させている。経験的原子価結合法(empirical valence bond method) はこのような酵素サイズの分子について、その活性化自由エネルギーなどを低コストで計算し予測できる手法である。現在、経験的原子価結合法は例えば酵素反応中心の周囲の残基への変異による反応への影響を予測できる。しかしながら、これまで経験的原子価結合法は初期構造依存性が高い手法と考えられており、モデリングに基づく構造を元にした計算はほとんど試みられてこなかった。 本発表では、モデリング構造を用いて経験的原子価結合法を実施した場合に起きる問題を説明し、そのシンプルな解決策を提示する。, 発動分子科学 第四回領域会議

The free-energy difference of two physicochemical states is an essential value describing the stability of the molecules. In the context of protein engineering, the free-energy changes upon mutations can answer whether the protein is stabilized or destabilized upon mutations.Developments in molecular dynamics simulation, combined with the growth of the computational power in the recent computer hardware,provides us a way to predict the free-energy difference of proteins upon mutations at a cost of moderate computational requirements.We reimplemented a state-of-the-art free-energy estimation method for protein mutations, FEP/REST (free energy perturbation /replica exchange with solute tempering) and its improvements on a molecular dynamics software GROMACS. We evaluated its prediction capability by comparing it with experimental measurements. Accuracies, parameter choices and extensions will be discussed., 2020 Biophysical Society Annual Meeting

Free-energy perturbation (FEP)は分子の一部構造を変更した場合の自由エネルギー差を求める事ができる手法であり、サンプリング法であるReplica-exchange with solute tempering(REST)法と組み合わせることで広く水中の分子の構造をサンプルし精度の良い自由エネルギー差を求めることが可能となる。本発表ではFEP/REST法をタンパク質変異体の自由エネルギー計算への応用について述べる。, 第33回分子シミュレーション討論会

How enzymes accelerate the electron transfer? We studied the photosystem II (PSII), a key enzyme in the photosynthesis to generate molecular oxygen, to elucidate the role of enzymatic environments. The energetics of the enzymatic reactions was studied with QM/MM simulations of the manganese cluster (CaMn4O5) in the PSII. A B3LYP level calculation coupled with the free energy estimation using the energy representation method revealed that the Mn cluster reduces the vertical oxidation free energy by 64.8 kcal/mol. A decomposition analysis of the free energy revealed that the enzymatic complex (proteins, ligands, lipids and ions) lowers the oxidation free energy, whereas the solvent water makes a net positive free energy contribution to the overall oxidation free energy., 3rd QST International Symposium

X-ray crystallographic analysis and single molecule analysis of electron microscopy provide us precise images of biomolecules, but the information is basically static. X-ray or neutron scattering give us structural information of the molecules in solution, but they are low resolution. We integrate such information with aids of computer modeling and simulation to build the 3D structures in solution and investigate the dynamics of mono- or di-nucleosome which are fundamental structural units of chromatin. We show that histone tails, which are difficult to experimentally see because of their nature of being in intrinsically disordered, are a key to stabilize various conformations., 第57回日本生物物理学会年会

Nucleosomes create the diverse conformations of chromatin by the intra- and inter-nucleosome interactions. The key factors are the unwrapping of DNA and the unstacking of nucleosomes. We investigated the energetics using all-atom molecular dynamics simulations with an enhanced sampling method on a large scale. As for the intra-nucleosomal interaction, free energy for unwrapping the outer superhelical turn of the nucleosomal DNA is about 11.5 kcal/mol, which agrees well with values obtained in single molecule experiments. At one end of the DNA, the first five bps unwrap, after which the next five bps unwrap at the same end with little increase in the free energy. The unwrapping then starts at the other end of the DNA, where 10 bps are unwrapped. At this point, further 15 bps unwrap at either end. The unwrapping of the outer superhelical turn is completed when the other end of the DNA unwraps. A detailed analysis of the free energy profile revealed a variety of conformational states, indicating there are many potential paths to outer superhelical turn unwrapping, but the dominant path is likely to be asymmetric. … 略, Biophysical Society Thematic Meeting, Les Houches 2019

Recent advances in the RNA sequencing technology revealed that not only tRNAs but many mRNAs are chemically modified, e.g. deaminated, methylated or hydroxymethylated, in living cells. Furthermore, several evidences indicate that the RNA secondary structure change induced by the modification is deeply connected to the gene function. However, the structure prediction of the modified RNAs is difficult because the fundamental parameters to predict the structure are undetermined. Current RNA structure prediction model is built upon the nearest-neighbor model, which represents the score (energy) of the secondary structure as the sum of energies ("nearest-neighbor parameters") per two neighboring base pairs. These nearest-neighbor parameters are derived from the free-energy differences upon RNA duplex formation, which are typically determined from the UV adsorption measurements. Determining nearest neighbor parameters containing one modified RNA requires ca. 20 UV adsorption measurements. The cost for the synthesis of modified RNA sequences makes this approach difficult to scale with various RNA modifications. We developed a method that estimates the nearest neighbor parameters by combining the molecular dynamics (MD) simulation with the UV adsorption experiments. The free-energy differences were estimated in MD through free-energy perturbation method. The results were combined with the UV adsorption measurements to compensate the biases between the simulation and the experiment. With the new method, the required number of experiments to determine the parameters can be greatly reduced. We applied the method to estimate the nearest neighbor parameter containing inosine-cytosine pairs. The derived free-energy parameters were consistent to the parameters of canonical RNAs. Comparison to the recently reported inosine-containing nearest neighbor parameters from experiments (Wright et al., 2018) will also be discussed., Annual Meeting of the Biophysical Society

Acetylation of histone H3K23 has emerged as an essential posttranslational modification associated with cancer and learning and memory impairment, yet our understanding of this epigenetic mark remains very limited. Here, we identified the native MORF complex as a histone H3K23-specific acetyltransferase and elucidated its mechanism of action. The acetyltransferase function of the catalytic MORF subunit is positively regulated by the DPF domain of MORF (MORFDPF). The crystal structure of MORFDPF in complex with crotonylated H3K14 peptide provides mechanistic insight into selectivity of this epigenetic reader and its ability to recognize both histone and DNA. Mass spectrometry, biochemical and genomic analyses show co-existence of the H3K23ac and H3K14ac modifications in vitro and co-occupancy of the MORF subunits, H3K23ac, and H3K14ac at promoters of MORF target genes in vivo. Together, our findings reveal a high correlation between two acetylation sites, H3K23 and H3K14, and suggest a model in which interaction of MORFDPF with acylated H3K14 promotes acetylation of H3K23 by the native MORF complex to activate gene transcription.