Nagaoka Laboratory Graduate School of Information Science, Nagoya University

From Molecules to Life Phenomena

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).

Figure1. Visualized O2 entry pathways from solvent to the heme pocket in the HbA α and two β subunits. Green spheres represent portals into the cavities.

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.

[Reference] M. Takayanagi, I. Kurisaki, M. Nagaoka, J. Phys. Chem. B, 117, 6082 (2013).

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.

[Reference] M. Takayanagi, I. Kurisaki, M. Nagaoka, Scientific Reports, 4, 4601 (2014).

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.

Figure 2. Schematic view of relaxation pathway for excessive energy flow from the heme.

I. Okazaki, Y. Hara, M. Nagaoka, Chem. Phys. Lett., 337, 151 (2001).

M. Takayanagi, C. Iwahashi, M. Nagaoka, J. Phys. Chem. B, 111, 864 (2007).

M. Takayanagi, H. Okumura, M. Nagaoka, J. Phys. Chem. B, 114, 12340 (2010).

Figure 3. 3D structure of myoglobin.

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

Figure 4. Molecular structure of ectoine.

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.

Figure 5. Increase of residence time of water molecules in the vicinity of protein surface under the presence ectoine 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.

[references] I.Yu and M.Nagaoka, Chem. Phys. Lett., 388, 316 (2004).

I.Yu, M.Takayanagi, M.Nagaoka, J. Phys. Chem. B, 113, 3543 (2009).

I.Yu, T.Tasaki, K.Nakada, M.Nagaoka, J. Phys. Chem. B, 114, 12392 (2010).

Study of the stability of partial chains from apo-myoglobin

Figure 6. Schematic view of cotranslational folding

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.

[references] M.Takayanagi, I.Yu, M.Nagaoka, Chem. Phys. Lett., 421, 300 (2006).

(Yu, Tahakayangi, Okumura, Iwahashi, Kurisaki)



compatible solute



エクトイン(ectoine (2-methyl-4-carboxyl-1,4,5,6-tetrahydropyrimidine))は塩水湖や塩分を含んだ砂漠などの過酷な環境に生息する微生物などがもつ、代表的な補償溶質である。水溶液中では、双性イオン状態(単一分子内でプラスとマイナスの電荷が分離したイオン状態)となり、水分子を強く引き付ける。分子動力学シミュレーションを用いた当グループの研究によって、タンパク質表面の水分子の拡散が、エクトイン添加によって大幅に遅くなる事が明らかになった。

chymotrypsin inhibitor 2(CI2)




vibrational excitation









アミノ酸残基がタンパク質中で右巻きの螺旋(らせん)構造をとっている箇所をαヘリックスという。αヘリックスは、n 番目のアミノ酸残基のカルボニル酸素原子Oと n+4 番目の残基のアミド基 NH が水素結合を形成しているため安定化している。この螺旋は3.6残基で1回転し、側鎖は外側に向かって飛び出している。


タンパク質を構成する複数のアミノ酸によって形成される特徴的な立体構造のひとつ。並行に隣り合ったペプチド鎖の間で、一方の鎖の N-H(アミド基)の部分が、隣接する鎖のC=O (カルボニル基)の部分と水素結合を形成し、全体として平面構造を形成している。多数の水素結合によって構造が保持されるため、非常に安定している。



side chain