The Japanese archipelago is comprised of four main islands—Hokkaido, Honshu, Shikoku, and Kyushu—which contain high mountainous areas that likely allowed for lineage differentiation and population genetic structuring during the climatic changes of the late Pleistocene. Here, we assess the historical background of the evolutionary dynamics of herbivorous red-backed voles (Myodes) in Japan, examining the evolutionary trends of mitochondrial cytochrome b gene (Cytb) sequence variation. Four apparent signals from rapid expansion events were detected in three species, M. rufocanus and M. rutilus from Hokkaido and M. smithii from central Honshu. Taken together with results from previous studies on Japanese wood mice (Apodemus spp.), three of the expansion events were considered to be associated with predicted bottleneck events at the marine isotope stage (MIS) 4 period, in which glaciers are thought to have expanded extensively, especially at higher elevations. In the late Pleistocene, the possible candidates are transitions MIS 6/5, MIS 4/3, and MIS 2/1, which can be characterized by the cold periods of the penultimate glacial maximum, MIS 4, and the last glacial maximum, respectively. Our data further reveal the genetic footprints of repeated range expansion and contraction in the northern and southern lineages of the vole species currently found in central Honshu, namely M. andersoni and M. smithii, in response to climatic oscillation during the late Pleistocene. The time-dependent evolutionary rates of the mitochondrial Cytb presented here would provide a possible way for assessing population dynamics of cricetid rodents responding to the late Pleistocene environmental fluctuation.

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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.

No abstract

The thermal stability of a protein is lowered by the addition of a monohydric alcohol, and this effect becomes larger as the size of hydrophobic group in an alcohol molecule increases. By contrast, it is enhanced by the addition of a polyol possessing two or more hydroxyl groups per molecule, and this effect becomes larger as the number of hydroxyl groups increases. Here, we show that all of these experimental observations can be reproduced even in a quantitative sense by rigid-body models focused on the entropic effect originating from the translational displacement of solvent molecules. The solvent is either pure water or water-cosolvent solution. Three monohydric alcohols and five polyols are considered as cosolvents. In the rigid-body models, a protein is a fused hard spheres accounting for the polyatomic structure in the atomic detail, and the solvent is formed by hard spheres or a binary mixture of hard spheres with different diameters. The effective diameter of cosolvent molecules and the packing fractions of water and cosolvent, which are crucially important parameters, are carefully estimated using the experimental data of properties such as the density of solid crystal of cosolvent, parameters in the pertinent cosolvent-cosolvent interaction potential, and density of water-cosolvent solution. We employ the morphometric approach combined with the integral equation theory, which is best suited to the physical interpretation of the calculation result. It is argued that the degree of solvent crowding in the bulk is the key factor. When it is made more serious by the cosolvent addition, the solvent-entropy gain upon protein folding is magnified, leading to the enhanced thermal stability. When it is made less serious, the opposite is true. The mechanism of the effects of monohydric alcohols and polyols is physically the same as that of sugars. However, when the rigid-body models are employed for the effect of urea, its addition is predicted to enhance the thermal stability, which conflicts with the experimental fact. We then propose, as two essential factors, not only the solvent-entropy gain but also the loss of protein-solvent interaction energy upon protein folding. The competition of changes in these two factors induced by the cosolvent addition determines the thermal-stability change.