A protein molecule is a dielectric substance, so the binding of a ligand is expected to induce dielectric response in the protein molecule, considering that ligands are charged or polar in general. We previously reported that binding of adenosine triphosphate (ATP) to molecular motor myosin actually induces such a dielectric response in myosin due to the net negative charge of ATP. By this dielectric response, referred to as "dielectric allostery," spatially separated two regions in myosin, the ATP-binding region and the actin-binding region, are allosterically coupled. In this study, from the statistically stringent analyses of the extensive molecular dynamics simulation data obtained in the ATP-free and the ATP-bound states, we show that there exists the dielectric allostery that transmits the signal of ATP binding toward the distant lever-arm region. The ATP-binding-induced electrostatic potential change observed on the surface of the main domain induced a movement of the converter subdomain from which the lever arm extends. The dielectric response was found to be caused by an underlying large-scale concerted rearrangement of the electrostatic bond network, in which highly conserved charged/polar residues are involved. Our study suggests the importance of the dielectric property for molecular machines in exerting their function. Published by AIP Publishing.

Actin polymerization depends on the salt concentration, exhibiting a reentrant behavior: the polymerization is promoted by increasing KCl concentration up to 100 mM, and then depressed by further increase above 100 mM. We here investigated the physical mechanism of this reentrant behavior by calculating the polymerization energy, defined by the electrostatic energy change upon binding of an actin subunit to a filament, using an implicit solvent model based on the Poisson-Boltzmann (PB) equation. We found that the polymerization energy as a function of the salt concentration shows a non-monotonic reentrant-like behavior, with the minimum at about 100 mM (1:1 salt). By separately examining the salt concentration effect on the global electrostatic repulsion between the like-charged subunits and that on the local electrostatic attraction between the inter-subunit ionic-bond-forming residues in the filament, we clarified that the reentrant behavior is caused by the change in the balance between the two opposing electrostatic interactions. Our study showed that the non-specific nature of counterions, as described in the mean-field theory, plays an important role in the actin polymerization. We also discussed the endothermic nature of the actin polymerization and mentioned the effect of ATP hydrolysis on the G-F transformation, indicating that the electrostatic interaction is widely and intricately involved in the actin dynamics.

The generalize Born (GB) model is frequently used in MD simulations of biomolecular systems in aqueous solution. The GB model is usually based on the so-called Coulomb field approximation (CFA) for the energy density integration. In this study, we report that the GB model with CFA overdestabilizes the long-range electrostatic attraction between oppositely charged molecules (ionic bond forming two-helix system and kinesin-tubulin system) when the energy density integration cutoff, r(max), which is used to calculate the Born energy, is set to a large value. We show that employing large r(max), which is usually expected to make simulation results more accurate, worsens the accuracy so that the attraction is changed into repulsion. It is demonstrated that the overdestabilization is caused by the overestimation of the desolvation penalty upon binding that originates from CFA. We point out that the overdestabilization can be corrected by employing a relatively small cutoff (r(max) = 10-15 angstrom), affirming that the GB models, even with CFA, can be used as a powerful tool to theoretically study the protein-protein interaction, particularly on its dynamical aspect, such as binding and unbinding.

Protein uses allostery to execute biological function. The physical mechanism underlying the allostery has long been studied, with the focus on the mechanical response by ligand binding. Here, we highlight the electrostatic response, presenting an idea of "dielectric allostery". We conducted molecular dynamics simulations of myosin, a motor protein with allostery, and analyzed the response to ATP binding which is a crucial step in force-generating function, forcing myosin to unbind from the actin filament. We found that the net negative charge of ATP causes a large-scale, anisotropic dielectric response in myosin, altering the electrostatic potential in the distant actin-binding region and accordingly retracting a positively charged actin binding loop. A large-scale rearrangement of electrostatic bond network was found to occur upon ATP binding. Since proteins are dielectric and ligands are charged/polar in general, the dielectric allostery might underlie a wide spectrum of functions by proteins.

The intrinsically disordered protein (IDP) has distinct properties both physically and biologically: it often becomes folded when binding to the target and is frequently involved in signal transduction. The physical property seems to be compatible with the biological property where fast association and dissociation between IDP and the target are required. While fast association has been well studied, fueled by the fly-casting mechanism, the dissociation kinetics has received less attention. We here study how the intrinsic disorder affects the dissociation kinetics, as well as the association kinetics, paying attention to the interaction strength at the binding site (i.e., the quality of the "fly lure"). Coarse-grained molecular dynamics simulation of the pKID-KIX system, a well-studied IDP system, shows that the association rate becomes larger as the disorder-inducing flexibility that was imparted to the model is increased, but the acceleration is marginal and turns into deceleration as the quality of the fly lure is worsened. In contrast, the dissociation rate is greatly enhanced as the disorder is increased, indicating that intrinsic disorder serves for rapid signal switching more effectively through dissociation than association. (C) 2016 Wiley Periodicals, Inc.

© 2016 American Physical Society.Allostery is indispensable for a protein to work, where a locally applied stimulus is transmitted to a distant part of the molecule. While the allostery due to chemical stimuli such as ligand binding has long been studied, the growing interest in mechanobiology prompts the study of the mechanically stimulated allostery, the physical mechanism of which has not been established. By molecular dynamics simulation of a motor protein myosin, we found that a locally applied mechanical stimulus induces electrostatic potential change at distant regions, just like the piezoelectricity. This novel allosteric mechanism, "piezoelectric allostery", should be of particularly high value for mechanosensor/transducer proteins.

An important unresolved problem associated with actomyosin motors is the role of Brownian motion in the process of force generation. On the basis of structural observations of myosins and actins, the widely held lever-arm hypothesis has been proposed, in which proteins are assumed to show sequential structural changes among observed and hypothesized structures to exert mechanical force. An alternative hypothesis, the Brownian motion hypothesis, has been supported by single-molecule experiments and emphasizes more on the roles of fluctuating protein movement. In this study, we address the long-standing controversy between the lever-arm hypothesis and the Brownian motion hypothesis through in silico observations of an actomyosin system. We study a system composed of myosin II and actin filament by calculating free-energy landscapes of actin-myosin interactions using the molecular dynamics method and by simulating transitions among dynamically changing free-energy landscapes using the Monte Carlo method. The results obtained by this combined multi-scale calculation show that myosin with inorganic phosphate (P-i) and ADP weakly binds to actin and that after releasing P-i and ADP, myosin moves along the actin filament toward the strong-binding site by exhibiting the biased Brownian motion, a behavior consistent with the observed single-molecular behavior of myosin. Conformational flexibility of loops at the actin-interface of myosin and the N-terminus of actin subunit is necessary for the distinct bias in the Brownian motion. Both the 5.5-11 nm displacement due to the biased Brownian motion and the 3-5 nm displacement due to lever-arm swing contribute to the net displacement of myosin. The calculated results further suggest that the recovery stroke of the lever arm plays an important role in enhancing the displacement of myosin through multiple cycles of ATP hydrolysis, suggesting a unified movement mechanism for various members of the myosin family.

Association of protein molecules constitutes the basis for the interaction network in a cell. Despite its fundamental importance, the thermodynamic aspect of protein protein binding, particularly the issues relating to the entropy change upon binding, remains elusive. The binding of actin and myosin, which are vital proteins in motility, is a typical example, in which two different binding mechanisms have been argued: the binding affinity increases with increasing temperature and with decreasing salt-concentration, indicating the entropy-driven binding and the enthalpy-driven binding, respectively. How can these thermodynamically different binding mechanisms coexist? To address this question, which is of general importance in understanding protein protein bindings, we conducted an in silico titration of the actin myosin system by molecular dynamics simulation using a residue-level coarse-grained model, with particular focus on the role of the electrostatic interaction. We found a good agreement between in silico and in vitro experiments on the salt-concentration dependence and the temperature dependence of the binding affinity. We then figured out how the two binding mechanisms can coexist: the enthalpy (due to electrostatic interaction between actin and myosin) provides the basal binding affinity, and the entropy (due to the orientational disorder of water molecules) enhances it at higher temperatures. In addition, we analyzed the actin myosin complex structures observed during the simulation and obtained a variety of weak-binding complex structures, among which were found an unusual binding mode suggested by an earlier experiment and precursor structures of the strong-binding complex proposed by electron microscopy. These results collectively indicate the potential capability of a residue-level coarse-grained model to simulate the association dissociation dynamics (particularly for transient weak-bindings) exhibited by larger and more complicated systems, as in a cell.

The phosphorylated kinase-inducible activation domain (pKID) adopts a helix-loop-helix structure upon binding to its partner KIX, although it is unstructured in the unbound state. The N-terminal and C-terminal regions of pKID, which adopt helices in the complex, are called, respectively, αA and αB. We performed all-atom multicanonical molecular dynamics simulations of pKID with and without KIX in explicit solvents to generate conformational ensembles. Although the unbound pKID was disordered overall, αA and αB exhibited a nascent helix propensity
the propensity of αA was stronger than that of αB, which agrees with experimental results. In the bound state, the free-energy landscape of αB involved two low free-energy fractions: native-like and non-native fractions. This result suggests that αB folds according to the induced-fit mechanism. The αB-helix direction was well aligned as in the NMR complex structure, although the αA helix exhibited high flexibility. These results also agree quantitatively with experimental observations. We have detected that the αB helix can bind to another site of KIX, to which another protein MLL also binds with the adopting helix. Consequently, MLL can facilitate pKID binding to the pKID-binding site by blocking the MLL-binding site. This also supports experimentally obtained results. © 2012 by the authors
licensee MDPI, Basel, Switzerland.

The actomyosin molecular motor, the motor composed of myosin II and actin filament, is responsible for muscle contraction, converting chemical energy into mechanical work. Although recent single molecule and structural studies have shed new light on the energy-converting mechanism, the physical basis of the molecular-level mechanism remains unclear because of the experimental limitations. To provide a clue to resolve the controversy between the lever-arm mechanism and the Brownian ratchet-like mechanism, we here report an in silico single molecule experiment of an actomyosin motor. When we placed myosin on an actin filament and allowed myosin to move along the filament, we found that myosin exhibits a unidirectional Brownian motion along the filament. This unidirectionality was found to arise from the combination of a nonequilibrium condition realized by coupling to the ATP hydrolysis and a ratchet-like energy landscape inherent in the actin-myosin interaction along the filament, indicating that a Brownian ratchet-like mechanism contributes substantially to the energy conversion of this molecular motor.

DNA-binding sites for SYCRP1, which is a regulatory protein of the cyanobacterium Synechocystis sp. PCC6803, were predicted for the whole genome sequence by estimating changes in the binding free energy (Delta Delta G(total)(A)) for SYCRP1 for those sites. The Delta Delta G(total)(A) values were calculated by summing Delta Delta G values derived from systematic single base-pair substitution experiments ( symmetrical and cooperative binding model). Of the calculated binding sites, 23 sites with a Delta Delta G(total)(A) value < 3.9 kcal.mol(-1) located upstream or between the ORFs were selected as putative binding sites for SYCRP1. In order to confirm whether SYCRP1 actually binds to these binding sites or not, 11 sites with the lowest Delta Delta G(total)(A) values were tested experimentally, and we confirmed that SYCRP1 binds to ten of the 11 sites with a Delta Delta G(total) value < 3.9 kcal mol(-1). The best correlation coefficient between Delta Delta G(total)(A)and the observed Delta Delta G(total) for binding of SYCRP1 to those sites was 0.78. These results suggest that the Delta Delta G values derived from systematic single base-pair experiments may be used to screen for potential binding sites of a regulatory protein in the genome sequence.

To construct a new model of the propagation mechanism of infectious scrapie-type prion protein (PrPsc), here we conducted a disruption simulation of a PrPsc nonamer using structure-based molecular dynamics simulation method based on a hypothetical PrPsc model structure. The simulation results showed that the nonamer disrupted in cooperative manners into monomers via two significant intermediate states: (1) a nonamer with a partially unfolded surface trimer and (2) a hexamer and three monomers. Dimers and trimers were rarely observed. Then, we propose a new PrPsc propagation mechanism where a hexamer plays an essential role as a minimum infectious unit. (c) 2007 Elsevier Inc. All rights reserved.

We report the results of molecular dynamics simulations of the protein myosin carried out with an elastic network model. Quenching the system, we observe glassy behavior of a density correlation function and a density response function that are often investigated in structure glasses and spin glasses. In the equilibrium, the fluctuation-response relation, a representative relation of the fluctuation-dissipation theorem, holds that the ratio of the density correlation function to the density response function is equal to the temperature of the environment. Weshow that, in the quenched system that we study, this relation can be violated. In the case that this relation does not hold, this ratio can be regarded as an effective temperature. We find that this effective temperature of myosin is higher than the temperature of the environment. We discuss the relation between this effective temperature and energy transduction that occurs after ATP hydrolysis in the myosin molecule.

What granularity is needed to carry out computer simulations of biomolecular reactions/motions? This is one of the central issues of the in silico biomolecular computing. In this paper, we addressed this issue by studying model granularity dependence of the native structure dynamics of protein molecules. We conducted molecular dynamics simulations employing three different protein models: the model with full atomic details and two coarse - grained models in which only C α atoms interacting with each other through simple potentials are considered. In addition to the observed agreement among the three models in terms of isotropic thermal fluctuation, principal component analysis showed that the coarse-grained models can also reproduce the anisotropy (or directionality) of the fluctuation, particularly of collective modes having relevance to molecular function. This indicates that the dependence of the essential dynamics of a protein molecule on the model granularity is weak, although it was also shown that incorporation of the Lennard - Jones-type potential into the harmonic-potential-based coarse-grained model improves the reproducibility to some degree, and that a plastic nature of structural dynamics observed in the full atomic model transforms into an elastic one in the coarse-grained models. The coarse-grained model can be applied to a molecular motor system, which may lead to a new view of biomolecular computing in the context of biological physics. © 2004 Kluwer Academic Publishers.

The cAMP receptor protein SYCRP1 in cyanobacterium Synechoeystis sp. PCC 6803 is a regulatory protein that binds to the consensus DNA sequence (5'-AAATGTGATCTAGATCACATTT-3') for the cAMP receptor protein CRP in Escherichia coli. Here we examined the effects of systematic single base-pair substitutions at positions 4-8 (TGTGA) of the consensus sequence on the specific binding of SYCRP1. The consensus sequence exhibited the highest affinity, and the effects of base-pair substitutions at positions 5 and 7 were the most deleterious. The result is similar to that previously reported for CRP, whereas there were differences between SYCRP1 and CRP in the rank order of affinity for each substitution. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

An in vitro domainal shuffling strategy for protein evolution was proposed in (J. Kolkman and W. Stemmer, Nat. Biotech. 19 (423) 2001). Due to backhybridization, however this method appears unlikely to be an efficient means of iteratively generating massive libraries of combinatorially shuffled genes. Recombination at the domain level (30-300 residues) also appears too coarse to support the evolution of proteins with substantially new folds. In this work, the module (10-25 residues long) and pseudomodule are adopted as the fundamental units of protein structure. Each protein is modelled as an N to C-terminal tour of a digraph composed of pseudomodules. An in vitro method based on PNA-mediated Whiplash PCR (PWPCR), RNA-protein fusion, and restriction-based recombination, XWPCR is then presented for evolving proteins with a high affinity for a given motif, subject to the constraint that each corresponds to a walk on the pseudomodule digraph of interest. Simulations predict that PWPCR is an efficient method of producing massive, shuffled gene libraries encoding for proteins as long as roughly 600 residues.

The all-atom and the Ising-based models have both played their own roles to help our understanding of helix-coil transition. In this study, we address to what degree these two theoretical models can be consistent with each other in the nonstationary regime, complementing the preceding equilibrium study. We conducted molecular dynamics simulations of an all-atom model polyalanine chain and Monte Carlo simulations of a corresponding kinetic Ising chain. Nonstationary properties of each model were characterized through power spectrum, Allan variance, and autocorrelation analyses regarding the time course of a system order parameter. A clear difference was indicated between the two models: the Ising-based model showed a Lorentzian spectrum in the frequency domain and a single exponential form in the time domain, whereas the all-atom model showed a 1/f spectrum and a stretched exponential form. The observed stretched exponential form is in agreement with a very recent T-jump experiment. The effect of viscous damping on helix-coil dynamics was also studied. A possible source of the observed difference between the two models is discussed by considering the potential energy landscape, and the idea of dynamical disorder was introduced into the original Glauber model in the hope of bridging the gap between the two models. Other possible sources, e. g., the limitations of the Ising framework and the validity of the Markovian dynamics assumption, are also discussed. (C) 2003 American Institute of Physics.

To describe the polypeptide helix-coil transition, while the Ising-based theory has been playing the principal role for 40 years, we can now make use of computer simulation using the so-called "all-atom model" that is far more precise than the Ising-based model. In this study, by conducting molecular dynamics (MD) simulations of helix-coil transition exhibited by a short polyalanine chain, we investigated how the MD simulation results and the Ising-based theoretical values coincide with each other, placing a focus on their equilibrium statistical mechanical properties. Several important physical properties, such as temperature-dependent helix ratio, distribution of the helix-residue number, position-dependent helix ratio, and pair-correlation between residue states were taken up as the proving grounds on which we made a comparison between the all-atom model simulation and the Ising-based theory. As an overall trend, we realized that the Ising-based theoretical results agreed with the all-atom simulation results at least qualitatively, suggesting that the Ising-based model, though very simple, extracts the essence of the phenomenon with respect to the equilibrium properties. On the other hand we found some quantitative disagreements between them. The origins of the observed disagreements are discussed by going into details of the all-atom model. (C) 2002 American Institute of Physics.

We present a molecular dynamics study of the alpha-helix formation in a system consisting of a 15-residue poly(L-alanine) and surrounding water molecules. By applying a relatively high temperature, we observed the alpha-helix formation several times during a 17-ns run, and reversible helix-coil transitions were also observed. The alpha-helix formations were usually initiated by the beta-tum structures. A crank-shaft-like motion of the peptide was included in the folding process. In the formed alpha-helical domains, substantial 3(10)-helix formations were found especially at the termini, as observed by the NMR study. The folding time scale at room temperature estimated from our simulation was found to lie in the range of 100ns, which is in accord with the time scale of the T-jump experiments. The total energy of the whole system was lower in the alpha-helix state than in the random-coil state by 20.4 +/- 4.8 kcal/mol, which is consistent with the experimental value obtained by calorimetry. This energy decrease in forming the alpha-helix was mainly caused by the Coulombic energy and the torsional energy.

We report on molecular dynamics simulations of the helix-coil transition of a polypeptide. The simulated transition was two-state-like and similar to the solid-liquid-like transition that has been observed in computer simulations of an atomic cluster. At the transition temperature, the polypeptide chain fluctuated between a helical and a random-coil state, and we observed 1/f fluctuation through potential-energy fluctuations. The origin of the observed 1/f fluctuation is discussed, considering the underlying potential-energy landscape.

水中におけるミオシンの長時間分子動力学シミュレーションを行い、ATP結合によって誘起されるミオシンのアロステリック応答を研究した。表面に生じる分極電荷とアクチン結合領域近傍にあるループ領域の応答を解析により、ミオシンがアクチンとの結合親和性を制御する分子機構についての新しい描像をえることができた。また、ミオシン周囲の水の物性、およびATP結合による影響の解析から、アクチンとミオシンの結合に水が積極的に関与している様子がうかびあがってきた。一方、アンブレラサンプリングを用いたミオシンの自由エネルギー計算からは、ミオシンのレバーアーム部位の変化により分子内の電荷分布の変化が誘起されることが示された。この応答もアクチンとの結合親和性の制御に重要であると考えられた。レバーアーム部位の構造変化を反応座標にとって計算した自由エネルギープロファイルもふまえて、アクトミオシンのエネルギー変換機構（力発生機構）について静電相互作用を基軸にした全体像を構築することができた。25年度が最終年度であるため、記入しない。25年度が最終年度であるため、記入しない

これまでの研究で、Goモデルベースの粗視化モデルMD計算により蛋白質の遅い揺らぎをシミュレートし、結果の有効性・信頼性を検証してきた。そして、大幅な計算時間の軽減とともに高精度全原子モデルと同等の結果を得られることを示した(Nat.Comput)。本年度は、(1)これまで使:用してきたGoモデルの:Folding問題に対する応用(Chem.Phys)、そして、(2)モデルの拡張を行った。新たなモデルでは、結晶構造で頻繁にみられ、かつ遅い揺らぎ原因にもなる蛋白質の構造多型性を直接的に粗視化モデルに埋め込む工夫を行った。最も簡単な構造多型として、2つの構造が安定になる粗視化モデルを考案し、このモデルと従来のモデルとが質的な違いを見せるか否かを研究した。その結果、両者間には大きな相違は見られなかた。すなわち天然構造に本来的に備わったダイナミクスに既に構造多型問遷移の情報が埋め込まれていることが示唆された。換言すると、従来のGoタイプの粗視化モデルで蛋自質の構造多型間遷移のような遅い揺らぎも再現できる、ということである。この結果をふまえ、(3)応用として、昨年度来、研究を進めてきた分子モーター・アクトミオシン動作機構に関し、ミオシン側の構造多型間遷移が力発生の直接の原因とするレバーアーム仮説の可否を、Goタイプの粗視化モデルによるミオシンの揺らぎダイナミクス、および摂動に対する応答という観点から検証し、レバーアーム説を否定する結果が得られた。同様の手法で、DNA結合蛋白であるCRPのアロステリック制御機構について研究した(FEBSLett)。本研究の最終目標とした「遅い揺らぎ」とアロステリック構造変化および誘導適合との関わりについては、今後、これらの系の研究をさらに進展させた上で一定の答えに到達できると考える

DNA-based computing is a new computational paradigm that realizes an autonomous and parallel computation by employing DNA molecular reactions. In contrast to a computation on an electronic computer, DNAC can be used to solve biological problems such as gene expression profiling and gene network regulation by using the algorithm of molecular computing because DNAC has a direct interface with biological molecules. A hairpin DNA molecule, which forms a hairpin structure by intra-base pairing, is an effective DNA nano-device for DNAC. For instance, a whiplash PCR system, which is a promising architecture for DNAC, employs recursive and self-catalytic polymerization reactions of hairpin DNA molecules. The rate of hairpin structure formation does not depend on the concentration of hairpin DNA molecules because the hairpin structure formation is an intra-molecular reaction. In massively parallel DNAC, the concentration of DNA molecules that represent individual programs and data is very low. Therefore, the concentration-independent rate of hairpin formation is extremely effective in massively parallel DNAC. In this study the static and kinetic characteristics of structural transition of hairpin DNA molecules were investigated. New hairpin DNA nano devices for DNAC were then developed based on the characteristics of hairpin DNA molecules as well as optimization and extension of the whiplash PCR system. A new architecture for an autonomous DNAC using hairpin DNA molecules as effective computational components was also developed

Trough the 3-year project, we have developed new methods of computation of protein dynamics and investigated processes of protein functioning.(1) A coarse-grained method to simulate protein dynamics was newly developed and applied to the force-generation process of actomyosin system. Cooperative effects of the lever-arm swinging motion and the sliding motion of myosin head were found.(2) Folding process of proteins were investigated by using GO-like models. Complexity in the folding process of nearly symmetrical proteins was revealed by showing the co-existence of multiple pathways and the mechanism of selection of specific routes among them.(3) A novel concept of functional funnel was introduced by analyzing the functioning process of a photo-sensing protein.(4) 3-D structures of proteins were predicted from sequences by developing a new method to simulate the folding process. We attended the international contest for structure prediction, CASP7, and acquired fairly good scores in a category of new-fold prediction.(5) Sequence selection was simulated by computer. We found that by selecting sequences which have desired local configuration at the active-site, random sequences can evolve into the foldable protein-like sequences.(6) Hydrophobic hydration around the nano-meter size solutes were investigated by molecular dynamics simulation and the topology-sensitive hydration and hydrophobic interaction were found

アクチンフィラメントに沿ったミオシン分子の1方向的な滑り運動は,ミオシンとアクチンフィラメントとの間の相互作用エネルギー地形の特徴によって説明されることが示された(名古屋大・寺田,笹井氏との共同研究)。エネルギー地形はフィラメントに沿って非対称的であり,さらに,大局的にはファネル状になっていることがわかった。アクトミオシンの分子モーターとしての機能それ自体とカップルした,いわゆる"機能ファネル"がエネルギー地形に形成されているようである。また,分子間相互作用に関与すると推測されている一群のアミノ酸について置換の影響を調べたところ,過去のin vitro motility assayの実験結果と符合した。アクトミオシンの分子間相互作用の詳細に探りを入れるため,水分子をexplicitに取り入れたアクチン,ミオシンの全原子MD計算も本格的に開始した。まず,アクチン,ミオシンそれぞれ単体のアロステリーに注目した。現在のところ,結晶構造で示唆されているようなヌクレオチド結合状態の変化にともなう顕著な立体構造変化はみられない。また,アクチンの重合・脱重合過程の分子機構の解明にも取り組んだ。フィラメント構造の安定性には分子間の2種類の静電相互作用,および分子間の接触面の柔らかさが重要であることが分かった。関連研究として,プリオンの重合・脱重合過程についてMD計算による研究を行い,プリオンの脱重合過程のシミュレーション結果をもとに,プリオン重合の新たなメカニズムを議論した(岐阜大・中村,桑田氏との共同研究)。またアクトミオシンの滑り運動機構の研究成果をふまえ,キネシンー微小管系におけるキネシンの1方向的な滑り運動の計算機実験と理論解析を行った

ATP合成酵素はF1とFoの2つのドメインからなる。F1ではAIPの加水分解時に放出される自由エネルギーがγサブユニットの回転エネルギーに変換される。一方, Foは膜蛋白質であり, 膜内外のプロトンの電気化学ポテンシャルがc-ringの回転に変換される。しかし, エネルギー変換の分子機構はまだ謎につつまれているので, この解明を目標として昨年度に引き続き"1分子計算機実験"を行った。粗視化モデルを用いた1分子計算機実験によって(1)Fo内部でのプロトンの移動経路, (2)ラチェット機構によるFoのc-ring回転, およびF1のγサブユニット回転を調べた。(1)については, これまでの研究でperiplasm側からFo内部への入口経路を見出した一方, Fo内部からoytoplasm側への出口経路が全く観測されなかったため, その原因究明を行った。注目したのは, これまでの研究では考慮していなかった極性アミノ酸(Asn, Gln, Ser等)の寄与である。Fo分子内の極性残基の空間分布とそれによる局所的な比誘電率の違いはプロトン経路に大きく影響すると考えられる。そこで, これまでの粗視化MDプログラムに局所比誘電率とボルン・エネルギー項を実装し, プロトン経路の再調査のための準備を整えた。(2)については, Feymanラチェット機構/Rockingラチェット機構によってc-ring/γサブユニットに一方向性の回転ブラウン運動が生じ, また, 回転方向が外部パラメータに依存して逆転することを示した。これと平行して, ラチェット機構を可能にするタンパク質の柔らかいアーキテクチャーの性質を探るため, 立体構造内部のアミノ酸残基間ネットワークを解析し, タンパク質立体構造の普遍的な性質としてのフラクタル性と臨界パーコレーション性を見出した

ミオシンとアクチンは典型的なATP駆動蛋白質であり,生体運動の要であるが,肝心のATPエネルギーが力学エネルギーに変換される仕組みが未だによく分かっていない。特に,ミオシン・アクチンが水中でヌクレオチド依存的にどんな構造状態を示すのか,ヌクレオチド状態と構造状態・エネルギー状態との関係,さらに,蛋白質周囲の水和状態はどう関与しているのか,という点が判然としない。そこで,本研究では計算機シミュレーション(MD計算)によってこれらの難題に取り組んだ。特に,ミオシンの全原子MD計算に注力し,構造状態,エネルギー状態,水和状態のヌクレオチド依存性を調査した。得られたトラジェクトリー(200ns×80本)を解析したところ,アクチン結合領域にヌクレオチド依存的な構造状態(アロステリック応答)が見えてきた。さらに,ミオシン表面の水とイオンの分布にもアロステリック応答が観測された。自由エネルギー計算にも本格的に取り組み,先述のアロステリック応答の検証に加え,レバーアームのパワーストロークは自由エネルギー的に不利な構造変化であり,水-水間の相互作用も関与していることが見えてきた。さらに,粗視化モデルを用いたMD計算によってアクチン-ミオシン間の結合定数の塩濃度依存性・温度依存性を調べたところ,従来の理解(エントロピー駆動)とは異なり,結合にはエンタルピー項(静電相互作用)が寄与していることが示された。アクチンについても大規模計算を実行してヌクレオチド依存的な構造状態を探り,アクトミオシン複合体の全原子MD計算にも着手した。これらの計算機シミュレーションにより,エネルギー変換機構の解明に欠かせないアクチン-ミオシンの結合・解離機構の物理的な理解を前進させることができた

ATPはF1とFoの二つの回転モーターの協同的な働きによって合成されるが,それぞれのモーターの回転機構は十分に解明されていない。特にFoは不明な点が多いが,膜中の重要残基の静電(ボルン)エネルギーとcリングの回転ブラウン運動を必須要素に据えたモデルが今のところ有力視されている。このモデルの可否を調べるため,a-c複合体の全原子MD計算を行った。重要残基cGlu62をボルンエネルギー的に不利な状況におき,aサブユニットの動きを追った(cリングは空間に拘束)。200nsでは有意な動きは観測されなかったが,膜両側からcGlu62に通じる2本のプロトン半チャネル(これも上記モデルの必須要素)の存在が見えてきた。移動が見られなかった原因を探るためcリング単体(拘束なし)のMD計算を行ったところ,cリングの回転ブラウン運動を観測できた。aサブユニットの存在(および膜に面したcGlu62への水分子の出入り,脂質分子の種類の相違)がどう影響するかは今後の課題として残った。本研究で開発した粗視化ボルンエネルギー法の適用も同様である。F1については,βサブユニットの構造変化とβ-γ間の立体斥力が回転を駆動すると考えられてきたが,βと直に接する部分を失ったγ変異体も回転することから,従来と異なる回転機構の存在も示唆されていた。そこで,粗視化モデルを用いて計算機実験を行ったところ(α3β3複合体の協同的な構造変化を強制的かつ連続的に励起した),γ変異体の回転が観測された。β-γ間には静電引力が働いており,静電エネルギーの変化によってγに回転が生じたことが分かった。同じ粗視化モデルをリニアモーターであるKIF1Aに適用し,KIF1Aが微小管に結合する過程を観測したところ,KIFIAの微小管に沿った一方向的な動きにも静電相互作用が深く関与していること分かった

　細胞骨格フィラメントのダイナミクスはアクチン、チューブリン、線維状蛋白質の離合集散ダイナミクスである。本研究ではアクチンおよびチューブリンの多分子からなる系の分子動力学シミュレーションを行い、生命システム機能として興味深いアクチンフィラメントの「トレッドミル現象」や微小管の「動的不安定性」のメカニズムを分子動力学の立場から説明し、「分子動力学」と「細胞動力学」という生命システムの異なる階層の「動力学」の連関を理解することを目的とした。　本年度は特にアクチンの分子動力学計算において研究の進展があったのでそれを報告する。アクチンはモノマー状態（Ｇ状態）とフィラメント状態（Ｆ状態）の２状態をとる。Ｆ状態ではアクチンが重合して２重螺旋状のフィラメント構造が形成される。アクチンはATP加水分解酵素でもあり，Ｇ状態（Ｇアクチン）では分子中央部にあるクレフト基底部の核酸結合部位にATPが結合しているが，Ｆ状態（Ｆアクチン）になると加水分解が促進され，F状態のモノマー（プロトマー）の大部分はADP結合状態に変化している。興味深いことにＧアクチンがフィラメントに結合するときの親和性はATP結合状態とADP結合状態で異なり，ADP結合状態のＧアクチンの解離定数の方が大きい。このため，一方の端ではATPを結合したＧアクチンが次々に既存のフィラメントに結合してフィラメントが伸長し，他方の端ではATPが加水分解されてADP結合状態になったプロトマーが次々に解離していく，というような現象（トレッドミル現象）が生じる。　結合している核酸によって解離定数が異なることから，アクチンは核酸依存的な構造状態変化を示すことが推測できる。実際，ATP加水分解にともないＦ状態のプロトマーの構造状態が変化し，それによりフィラメントが不安定化することが電子顕微鏡観察によって示唆されてきた。特に，ATPからADP結合状態への変化にともない，分子中央部のクレフトの閉開状態，サブドメイン２のD-loopと呼ばれる領域の構造状態，そしてクレフトをはさんだ２つのドメイン間のプロペラ（ねじれ）角の捩れ状態の３つが顕著に変化することが示唆された。しかし，最近解明されたＧアクチンの結晶構造は，特にクレフト開閉状態とD-loopの構造状態に関して電顕の結果と一致しないことが明らかになってきた。電顕とX線による構造解析はそれぞれ一長一短があり，かつ両者が対立しているため，いまだにアクチンの核酸依存的な構造状態の解明には至っていない。　そこで，本研究では，異なる核酸結合状態のＧアクチンの分子動力学計算を多数行い，まず水中でのＧアクチンの構造状態とその核酸依存性を調べることにした。特に，争点になっているクレフト開閉状態，D-loopの構造状態，プロペラ角の捩れ状態を中心に調べた。クレフトに関しては，４０本のＭＤトラジェクトリーの平均は結晶構造からの有意なずれを示したものの，ATP結合状態とADP結合状態の間に有意な差はなく，ともに閉状態となった。しかし，個々のトラジェクトリーは開状態に達する大きな揺らぎを示し，さらに核酸がない状態では開状態になることが分かった。D-loopに関しては結晶構造ではATP結合状態でdisoder状態，ADP結合状態でαへリックスになることが示されていたが，これとは異なり，実際は動的なhelix-coil転移を示すことが分かった。プロペラ角については電顕結果と一致する結果が得られ，核酸依存的な構造状態変化はここに集約される可能性があることが分かった。これらのＧアクチンの構造状態の核酸依存性の結果，およびＦ状態のアクチン５量体のＭＤ計算結果からフィラメント不安定化機構について調べた。特に，Ｆ状態はプロトマー間の分子間相互作用によって安定化されるが，一方でプロトマーの内部には歪みが生じ（特にプロペラ角の自由度について），この歪みがＦ状態の不安定化を引き起こす，という機構が示唆された。

本研究課題「生体分子モーターの力学応答特性」に関して、以下の３つの研究を行い、それぞれに関して興味深い結果が得られた。１）ミオシン分子の力学的応答特性：ミオシン分子のような巨大分子を扱うため、これまでに開発してきた弾性体モデルベースの粗視化モデルを用い、ミオシン分子のATP結合部位周辺に局所的な力、すなわち「摂動」が与えたとき、ミオシン分子がどのような「応答」、すなわち構造変化を誘起するかを研究した。従来から提唱されている「レバーアーム」仮説によると、ミオシン分子の力発生は、ATP加水分解サイクルにおいて、ミオシン分子のATP結合部位の局所的な構造が変化し、この局所構造変化がミオシン分子尾部（レバーアーム）のアクチンフィラメントに沿ったスイングを誘起する、と考えられてきた。本研究結果は、非常に興味深いことに、この仮説と真っ向から対峙するものとなった。すなわち、ATP加水分解サイクルで生じうるATP結合部位周辺の局所的構造変化は、アクチンフィラメントに沿ったレバーアームのスイングを直接的には誘起しない、という結果になった。これは、仮にレバーアームのスイングがあるとしても、直接アクトミオシンの力発生の原因にはならない、ということを示唆する。２）計算結果のモデル依存性：前述の結果が、計算モデルの精度不足によるアーテファクトであるという可能性を払拭するため、１）で用いた計算モデルを拡張し、より現実的なモデル(Goモデル、Janus-Goモデル、全原子モデル）の分子動力学計算を行った。この場合、大規模な構造変化をシミュレートするため、Transition Path Sampling法と呼ばれる手法を用い、より少ない計算コストで構造変化ダイナミクスの統計性を高める工夫をした。１）の結果は3N-6次元での主要な平衡揺らぎが反映されたものであるから、この主要平衡揺らぎのモデル依存性を調べれば良い。その結果、期待通りに主要平衡揺らぎの頑健性が示され、計算結果の信頼性が示された。３）Ｇアクチンの全原子モデル分子動力学シミュレーション：Ｇアクチン（アクチンモノマー）を水溶液中に溶解させ、平衡揺らぎダイナミクスを解析した。Ｇアクチンには①クレフトの開閉、および②サブドメイン２と呼ばれる部位の構造形成崩壊、という顕著な２つのダイナミクスの存在が結晶構造解析から示されており、それらがアクチンの重合やミオシンとの相互作用に影響することが示唆されてきた。今回の水溶液中のアクチン単量体の分子動力学計算によって、これら２つのダイナミクスの兆候が実際に確認することができた。これらのダイナミクスが、アクチン分子内部のATP結合部位の局所構造変化、すなわち摂動によって、応答として誘起されるか、そして、アクチンフィラメント全体にかかる張力・応力に対して個々のアクチン分子がどのように応答すのか、次の研究課題としていく。