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Cell is a minimum unit of life phenomena, and its physical entity is a mixture of biopolymers (e.g., protein, DNA and RNA) and low-weight molecules (e.g., water, ion, lipid etc). Such a mixture of molecules generate a series of chemical reactions which can be seen as life phenomena. However, the mechanism is still elusive at the molecular level, and thus its understanding remains to be one of the central issues of the modern science. In our laboratory, we are addressing full understanding of life phenomena from the physicochemical aspect employing "bottom-up" approach, where chemical processes in the cell, attributed to biomolecules, are examined by molecular simulations. Our goal is to give an answer "what is life?" as basic field of science, and also to contribute to develop de-novo drugs for specific disease and de-novo diagnostic methods based on molecular properties as applied filed of science and engineering.

Statistical analysis of multiple oxygen entry pathways in hemoglobin subunits

Human hemoglobin (HbA) is an oxygen transport protein composed of two α and two β subunits. Each HbA subunit stores an oxygen molecule (O₂) in a cavity called "heme pocket" to transport O₂. However, in the crystal structures, there is no static channel from solvent to the heme pocket. Thus there is an ongoing debate over the O₂ entry and escape pathways. Experimental studies with mutated HbA revealed that O₂ directly escapes from the heme pocket to solvent through the channel called "histidine gate" (His-gate) which is located near the distal histidine (J. Biol. Chem. 2011, 286, 10515). Meanwhile, computational studies with molecular dynamics (MD) simulations revealed that O₂ escapes from the heme pocket through multiple pathways ( J. Am. Chem. Soc. 2012, 134, 11177).

In order to determine O₂ entry pathway, we executed 128 independent MD simulations in O₂-rich aqueous solvent with Hemoglobin. As a result, we obtained hundreds of O₂ entry events in the α and two β subunits. We visualized the entry pathway distributions using the newly developed IPIC (intrinsic pathway identification by clustering) method with the help of clustering (Fig.1). In both subunits, O₂ entry mainly occurs through multiple pathways and rarely through the His-gate. In addition, there are big entry pathways in α subunit which do not exist in β subunit. This result at the molecular level is in accordance with the experimental finding that O₂ entry occurs twice as fast in β subunit.

Non-site-specific allosteric effect of O₂ on human hemoglobin

Protein allostery is essential for vital activities. Allosteric regulation of human hemoglobin (HbA) with two quaternary states T and R has been a paradigm of allosteric structural regulation of proteins (learn in Biology Ⅱ in high school). It is widely accepted for a long time that O₂ act as a "site-specific" homotropic effector, or the successive O₂ binding to the heme brings about the quaternary regulation. However, we succeeded to demonstrate that the effect of "site-specific" allostery is not the only mechanism for O₂ allostery.

In fact, our ensemble MD simulations of T-state HbA in O₂-rich aqueous solution revealed that the solution environment of high O₂ partial pressure enhances the quaternary change from T to R without binding to the heme, suggesting an additional "non-site-specific" allosteric effect of O₂. The non-site-specific effect should play a complementary role in the quaternary change by affecting the intersubunit contacts, i.e., hydrogen bonds and salt bridges. This kind of analyses must become a milestone in comprehensive understanding of the allosteric regulation of HbA from the molecular point of view.

Theoretical study on relaxation process of myoglobin in aqueous solvent

Myoglobin contains heme groups, which binds a ligand molecule (i.e. O₂, CO, NO …) with the iron atom. Visible light absorption produces photodissociation of CO bound myoglobin (MbCO), whose excessive energy is released into solvent as heat flow.

By utilizing MD simulations, we analyzed isotropic deformation of myoglobin 3D structure and relaxation processes of excessive energy release. Since the ligand dissociation triggers expression of allosteric effect of myglobin, understating the mechanism is important to clarify the relationship between the protein structure and function.

Theoretical study on the effect of compatible solutes on dynamics and reactivity of biomacromolecules

Water molecules play important roles in the stability and the dynamics of protein conformation. Although there are a number of factors to alter the property of water molecules in a cellular environment, it has been recently elucidated that compatible solutes make the protein conformation stabilized and maintain the biological activity. Compatible solutes are such compounds with low molecular weights that are produced by the cells of organisms and are accumulated in them in response to such high environmental stresses as the high salt concentration or the high temperature. Although they do not interact directly with macromolecules themselves, they play an indirect role to modify the stability of proteins by altering the solvent properties. We have focused on an ectoine molecule, which is the most common compatible solutes found in the cytosol of aerobic heterotrophic bacteria, and examined the effect of the compatible solutes on the dynamic property of water molecules.

As a model, we executed MD simulations of chymotrypsin inhibitor 2 (CI2) in ectoine-water mixture and pure water solvent. From the results obtained, some characteristic differences in coordination of water molecules and their dynamic behavior around the protein were found. Although ectoin molecules do not interact directly with macromolecules themselves, they significantly slow down the diffusion of water molecules near the protein surface in addition to that in the bulk solution. This should play an important role on the stabilization of protein conformation at the molecular level. We are now addressing the elucidation of the effect of cosolvents on the dynamics and reactivity in aqueous solution using relatively small and flexible peptide hormone such as enkephalin compared with CI2.

Study of the stability of partial chains from apo-myoglobin

mRNA is translated into the corresponding protein upon ribosome. In the step of the extension of polypeptide chain, a part of the chain is located out of ribosome and partially folds (cotranslational folding). The earlier experimental studies indicated that HbA α subunit captures heme group while binding to ribosome. It was also revealed that the contents of helix formation increase with the elongation of partial chain of myoglobin. However, the information we can access about such phenomena is still insufficient and it remains elusive how cotranslational folding of myoglobin proceeds.

As a first step to elucidate the mechanism of cotranslational folding of partial myoglobin chain, we performed MD simulations for the helix domain obtained from X-ray crystallography structure, and assessed the stability. Then the analyses indicated that short partial chain cannot form alpha helix stably, but long partial chain can.

キーワード ： protein ・ compatible solute ・ ectoine ・ chymotrypsin inhibitor 2(CI2) ・ enkephalin ・ vibrational excitation ・ ribosome ・ folding ・ myoglobin ・ αhelix ・ βsheet ・ residue ・ side chain ・ allosteric interaction