Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acs.langmuir.3c00968

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Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

We aim to develop a theory based on a concept other than the chemo-mechanical coupling (transduction of chemical free energy of ATP to mechanical work) for an ATP-driven protein complex. Experimental results conflicting with the chemo-mechanical coupling have recently emerged. We claim that the system comprises not only the protein complex but also the aqueous solution in which the protein complex is immersed and the system performs essentially no mechanical work. We perform statistical-mechanical analyses on V₁-ATPase (the A₃B₃DF complex) for which crystal structures in more different states are experimentally known than for F₁-ATPase (the α₃β₃γ complex). Molecular and atomistic models are employed for water and the structure of V₁-ATPase, respectively. The entropy originating from the translational displacement of water molecules in the system is treated as a pivotal factor. We find that the packing structure of the catalytic dwell state of V₁-ATPase is constructed by the interplay of ATP bindings to two of the A subunits and incorporation of the DF subunit. The packing structure represents the nonuniformity with respect to the closeness of packing of the atoms in constituent proteins and protein interfaces. The physical picture of rotation mechanism of F₁-ATPase recently constructed by Kinoshita is examined, and common points and differences between F₁- and V₁-ATPases are revealed. An ATP hydrolysis cycle comprises binding of ATP to the protein complex, hydrolysis of ATP into ADP and Pi in it, and dissociation of ADP and Pi from it. During each cycle, the chemical compounds bound to the three A or β subunits and the packing structure of the A₃B₃ or α₃β₃ complex are sequentially changed, which induces the unidirectional rotation of the central shaft for retaining the packing structure of the A₃B₃DF or α₃β₃γ complex stabilized for almost maximizing the water entropy. The torque driving the rotation is generated by water with no input of chemical free energy. The presence of ATP is indispensable as a trigger of the torque generation. The ATP hydrolysis or synthesis reaction is tightly coupled to the rotation of the central shaft in the normal or inverse direction through the water-entropy effect.

DOI： 10.3389/fmolb.2023.1159603

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Authorship：Corresponding author   Language：Japanese  

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Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/pro.4425

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/pro.4425

Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/pro.4404

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/pro.4404

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

We have developed a methodology for identifying further thermostabilizing mutations for an intrinsically thermostable membrane protein. The methodology comprises the following steps: (1) identifying thermostabilizing single mutations (TSSMs) for residues in the transmembrane region using our physics-based method; (2) identifying TSSMs for residues in the extracellular and intracellular regions, which are in aqueous environment, using an empirical force field FoldX; and (3) combining the TSSMs identified in steps (1) and (2) to construct multiple mutations. The methodology is illustrated for thermophilic rhodopsin whose apparent midpoint temperature of thermal denaturation Tm is ∼91.8 °C. The TSSMs previously identified in step (1) were F90K, F90R, and Y91I with ΔTm ∼5.6, ∼5.5, and ∼2.9 °C, respectively, and those in step (2) were V79K, T114D, A115P, and A116E with ΔTm ∼2.7, ∼4.2, ∼2.6, and ∼2.3 °C, respectively (ΔTm denotes the increase in Tm). In this study, we construct triple and quadruple mutants, F90K+Y91I+T114D and F90K+Y91I+V79K+T114D. The values of ΔTm for these multiple mutants are ∼11.4 and ∼13.5 °C, respectively. Tm of the quadruple mutant (∼105.3 °C) establishes a new record in a class of outward proton pumping rhodopsins. It is higher than Tm of Rubrobacter xylanophilus rhodopsin (∼100.8 °C) that was the most thermostable in the class before this study.

DOI： 10.1021/acs.jpcb.1c08684

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acsomega.1c06699

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.molliq.2020.114129

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-020-61966-4

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Language：English   Publishing type：Research paper (scientific journal)  

We develop a new methodology best suited to the identification of thermostabilizing mutations for an intrinsically stable membrane protein. The recently discovered thermophilic rhodopsin, whose apparent midpoint temperature of thermal denaturation Tm is measured to be ∼91.8 °C, is chosen as a paradigmatic target. In the methodology, we first regard the residues whose side chains are missing in the crystal structure of the wild type (WT) as the "residues with disordered side chains," which make no significant contributions to the stability, unlike the other essential residues. We then undertake mutating each of the residues with disordered side chains to another residue except Ala and Pro, and the resultant mutant structure is constructed by modifying only the local structure around the mutated residue. This construction is based on the postulation that the structure formed by the other essential residues, which is nearly optimized in such a highly stable protein, should not be modified. The stability changes arising from the mutations are then evaluated using our physics-based free-energy function (FEF). We choose the mutations for which the FEF is much lower than for the WT and test them by experiments. We successfully find three mutants that are significantly more stable than the WT. A double mutant whose Tm reaches ∼100 °C is also discovered.

DOI： 10.1021/acs.jcim.0c00063

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1063/1.5140499

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

We often encounter a case where two proteins, whose amino-acid sequences are similar, are quite different with regard to the thermostability. As a striking example, we consider the two seven-transmembrane proteins: recently discovered Rubrobacter xylanophilus rhodopsin (RxR) and long-known bacteriorhodopsin from Halobacterium salinarum (HsBR). They commonly function as a light-driven proton pump across the membrane. Though their sequence similarity and identity are ∼71 and ∼45%, respectively, RxR is much more thermostable than HsBR. In this study, we solve the three-dimensional structure of RxR using X-ray crystallography and find that the backbone structures of RxR and HsBR are surprisingly similar to each other: The root-mean-square deviation for the two structures calculated using the backbone Cα atoms of the seven helices is only 0.86 Å, which makes the large stability difference more puzzling. We calculate the thermostability measure and its energetic and entropic components for RxR and HsBR using our recently developed statistical-mechanical theory. The same type of calculation is independently performed for the portions playing essential roles in the proton-pumping function, helices 3 and 7, and their structural properties are related to the probable roles of water molecules in the proton-transporting mechanism. We succeed in elucidating how RxR realizes its exceptionally high stability with the original function being retained. This study provides an important first step toward the establishment of a method correlating microscopic, geometric characteristics of a protein with its thermodynamic properties and enhancing the thermostability through amino-acid mutations without vitiating the original function.

DOI： 10.1021/acs.jpcb.9b10700

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.molliq.2019.111374

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.jcim.9b00226

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Language：English   Publishing type：Research paper (scientific journal)  

A new method is developed for calculating hydration free energies (HFEs) of polyatomic solutes. The solute insertion is decomposed into the creation of a cavity in water matching the geometric characteristics of the solute at the atomic level (process 1) and the incorporation of solute-water van der Waals and electrostatic interactions (process 2). The angle-dependent integral equation theory combined with our morphometric approach and the three-dimensional interaction site model theory are applied to processes 1 and 2, respectively. Neither a stage of training nor parameterization is necessitated. For solutes with various sizes including proteins, the HFEs calculated by the new method are compared to those obtained using a molecular dynamics simulation based on solution theory in energy representation (the ER method developed by Matubayasi and co-workers), currently the most reliable tool. The agreement is very good especially for proteins. The new method is characterized by the following: The calculation can rapidly be finished; a solute possessing a significantly large total charge can be handled without difficulty; and since it yields not only the HFE but also its many physically insightful energetic and entropic components, it is best suited to the elucidation of mechanisms of diverse phenomena such as the receptor-ligand binding, different types of molecular recognition, and protein folding, denaturation, and association.

DOI： 10.1063/1.5093110

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AIP Publishing  

DOI： 10.1063/1.5082217

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/c8cp04377a

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1063/1.5042111

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.jpcb.8b00443

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/c8cp00155c

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Language：English   Publishing type：Research paper (scientific journal)  

We calculate potential of mean force (PMF) between the spherical particles in an ionic liquid by using an integral equation theory, a statistical mechanical theory for liquids. The PMFs between like-charged particles, unlike-charged particles, charged-uncharged particles, and uncharged particles are calculated. Effect of the particle diameter on the PMF is also studied. It is found that the pairs of the like charged particles, unlike charged particles, and uncharged particles are stabilized when they are closely coupled, whereas the charged-uncharged particles are destabilized by the contact. To investigate roles of the energy and entropy in the PMF, it is decomposed into the energetic and entropic components by a temperature differentiation. It is found that the energetic component has relatively high dependency on the surface charges of the immersed particles. On the other hand, the entropic component does not change much upon change in the surface charges of the immersed particles. Generally, an interaction between two particles in an ionic liquid is often explained by the electrostatic and van der Waals interactions, which are classified as the energetic component. However, we found that the entropic component also plays an important role in the interaction.

DOI： 10.1016/j.molliq.2018.02.089

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.molliq.2017.09.108

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1063/1.4999376

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/c7cp05160c

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1063/1.4975165

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/c6cp06000e

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1088/0953-8984/28/34/344003

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bpj.2016.05.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AIP Publishing  

DOI： 10.1063/1.4931814

CiNii Article

CiNii Books

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.jpcb.5b08513

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.cplett.2014.10.038

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/jp507930e

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AIP Publishing  

DOI： 10.1063/1.4894753

CiNii Article

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press  

DOI： 10.1093/nar/gku382

CiNii Article

CiNii Books

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.cplett.2012.12.040

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Biophysical Society of Japan  

Herein, the absorption maximum of bacteriorhodopsin (bR) is calculated using our recently developed method in which the whole protein can be treated quantum mechanically at the level of INDO/S-CIS//ONIOM (B3LYP/6-31G(d,p): AMBER). The full quantum mechanical calculation is shown to reproduce the so-called opsin shift of bR with an error of less than 0.04 eV. We also apply the same calculation for 226 different bR mutants, each of which was constructed by replacing any one of the amino acid residues of the wild-type bR with Gly. This substitution makes it possible to elucidate the extent to which each amino acid contributes to the opsin shift and to estimate the inter-residue synergistic effect. It was found that one of the most important contributions to the opsin shift is the electron transfer from Tyr185 to the chromophore upon excitation. We also indicate that some aromatic (Trp86, Trp182) and polar (Ser141, Thr142) residues, located in the vicinity of the retinal polyene chain and the <symbol>b</symbol>-ionone ring, respectively, play an important role in compensating for the large blue-shift induced by both the counterion residues (Asp85, Asp212) and an internal water molecule (W402) located near the Schiff base linkage. In particular, the effect of Trp86 is comparable to that of Tyr185. In addition, Ser141 and Thr142 were found to contribute to an increase in the dipole moment of bR in the excited state. Finally, we provide a complete energy diagram for the opsin shift together with the contribution of the chromophore-protein steric interaction.<br>

DOI： 10.2142/biophysics.8.115

CiNii Article

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.cplett.2009.11.074

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Language：English   Publishing type：Research paper (scientific journal)  

Here we improved our hybrid QM/MM methodology (Houjou et al. J Phys Chem B 2001, 105, 867) for evaluating the absorption maxima of photoreceptor proteins. The renewed method was applied to evaluation of the absorption maxima of several retinal proteins and photoactive yellow protein. The calculated absorption maxima were in good agreement with the corresponding experimental data with a computational error of 10 nm. In addition, our calculations reproduced the experimental gas-phase absorption maxima of model chromophores (protonated all-trans retinal Schiff base and deprotonated thiophenyl-p-coumarate) with the same accuracy. It is expected that our methodology allows for definitive interpretation of the spectral tuning mechanism of retinal proteins. © 2006 Wiley Periodicals, Inc.

DOI： 10.1002/jcc.20432

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