記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/acsomega.1c06699

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.molliq.2020.114129

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-020-61966-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/acs.jcim.0c00063

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1063/1.5140499

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.molliq.2019.111374

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/acs.jcim.9b00226

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1063/1.5093110

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1063/1.5082217

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1039/c8cp04377a

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1063/1.5042111

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/acs.jpcb.8b00443

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1039/c8cp00155c

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.molliq.2018.02.089

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.molliq.2017.09.108

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1063/1.4999376

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1039/c7cp05160c

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1063/1.4975165

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1039/c6cp06000e

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1088/0953-8984/28/34/344003

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bpj.2016.05.006

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AIP Publishing  

It is of great interest from both scientific and practical viewpoints to theoretically predict the thermal-stability changes upon mutations of a protein. However, such a prediction is an intricate task. Up to now, significantly many approaches for the prediction have been reported in the literature. They always include parameters which are adjusted so that the prediction results can be best fitted to the experimental data for a sufficiently large set of proteins and mutations. The inclusion is necessitated to achieve satisfactorily high prediction performance. A problem is that the resulting values of the parameters are often physically meaningless, and the physicochemical factors governing the thermal-stability changes upon mutations are rather ambiguous. Here, we develop a new measure of the thermal stability. Protein folding is accompanied by a large gain of water entropy (the entropic excluded-volume (EV) effect), loss of protein conformational entropy, and increase in enthalpy. The enthalpy increase originates primarily from the following: The energy increase due to the break of protein-water hydrogen bonds (HBs) upon folding cannot completely be cancelled out by the energy decrease brought by the formation of protein intramolecular HBs. We develop the measure on the basis of only these three factors and apply it to the prediction of the thermal-stability changes upon mutations. As a consequence, an approach toward the prediction is obtained. It is distinguished from the previously reported approaches in the following respects: The parameters adjusted in the manner mentioned above are not employed at all, and the entropic EV effect, which is ascribed to the translational displacement of water molecules coexisting with the protein in the system, is fully taken into account using a molecular model for water. Our approach is compared with one of the most popular approaches, FOLD-X, in terms of the prediction performance not only for single mutations but also for double, triple, and higher-fold (up to sevenfold) mutations. It is shown that on the whole our approach and FOLD-X exhibit almost the same performance despite that the latter uses the adjusting parameters. For multiple mutations, however, our approach is far superior to FOLD-X. Five multiple mutations for staphylococcal nuclease lead to highly enhanced stabilities, but we find that this high enhancement arises from the entropic EV effect. The neglect of this effect in FOLD-X is a principal reason for its ill success. A conclusion is that the three factors mentioned above play essential roles in elucidating the thermal-stability changes upon mutations.

DOI： 10.1063/1.4931814

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/acs.jpcb.5b08513

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.cplett.2014.10.038

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/jp507930e

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AIP Publishing  

CLN025, a peptide with only 10 residues, folds into a specific β-hairpin structure (this is referred to as "native structure"). Here we investigate the stabilization mechanism for CLN025 using our free-energy function F. F comprises two components, the hydration entropy and the component related to the energetic dehydration effect. The former component is calculated using the hybrid of the angle-dependent integral equation theory (ADIET) and our recently developed morphometric approach. The ADIET is a statistical-mechanical theory applied to a molecular model for water. The latter component is calculated in a simple but judicious manner accounting for physically the most important factors: the break of polypeptide-water hydrogen bonds and formation of polypeptide intramolecular hydrogen bonds upon structural change to a more compact one. We consider the native structure, compact nonnative structures newly generated, and a set of random coils mimicking the unfolded state. F and its components are calculated for all the structures considered. The loss of the polypeptide conformational entropy upon structural transition from the unfolded state to a compact structure is also estimated using a simple but physically reasonable manner. We find that the key factor is the water-entropy gain upon folding originating primarily from an increase in the total volume available to the translational displacement of water molecules in the system, which is followed by the reduction of water crowding. The amino-acid sequence of CLN025 enables it not only to closely pack the backbone and side chains including those with large aromatic groups but also to assure the intramolecular hydrogen bonding upon burial of a donor and an acceptor when the backbone forms the native structure. The assurance leads to essentially no enthalpy increase upon folding. The close packing brings a water-entropy gain which is large enough to surpass the conformational-entropy loss. By contrast, it is not possible for the design template of CLN025, GPM12, to realize the same type of structure formation. There are significantly many compact structures which are equally stable in terms of F, and due to the conformational-entropy effect, the unfolded state is favorably stabilized.

DOI： 10.1063/1.4894753

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press  

It is a central issue to elucidate the new type of molecular recognition accompanied by a global structural change of a molecule upon binding to its targets. Here we investigate the driving force for the binding of R12 (a ribonucleic acid aptamer) and P16 (a partial peptide of a prion protein) during which P16 exhibits the global structural change. We calculate changes in thermodynamic quantities upon the R12-P16 binding using a statistical-mechanical approach combined with molecular models for water which is currently best suited to studies on hydration of biomolecules. The binding is driven by a water-entropy gain originating primarily from an increase in the total volume available to the translational displacement of water molecules in the system. The energy decrease due to the gain of R12-P16 attractive (van der Waals and electrostatic) interactions is almost canceled out by the energy increase related to the loss of R12-water and P16-water attractive interactions. We can explain the general experimental result that stacking of flat moieties, hydrogen bonding and molecular-shape and electrostatic complementarities are frequently observed in the complexes. It is argued that the water-entropy gain is largely influenced by the geometric characteristics (overall shapes, sizes and detailed polyatomic structures) of the biomolecules.

DOI： 10.1093/nar/gku382

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.cplett.2012.12.040

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.cplett.2009.11.074

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/jcc.20432

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