Seminars2012 – 2016

The motions of particles in a viscous fluid confined within a spherical cell have been simulated using Brownian and Stokesian dynamics simulations. High volume fractions mimicking the crowded interior of biological cells were used. Importantly, although confinement yields an overall slowdown in motion, the qualitative effects of motion in the interior of the cell can be effectively modeled as if the system were an infinite periodic system. However, we observe layering of particles at the cell wall due to steric interactions in the confined space. Motions of nearby particles are also strongly correlated at the cell wall, and these correlations increase when hydrodynamic interactions are modeled. Further, particles near the cell wall have a tendency to remain near the cell wall. A consequence of these effects is that the mean contact time between particles is longer at the cell wall than in the interior of the cell. These findings identify a specific way that confinement affects the interactions between particles and points to a previously unidentified mechanism that may play a role in signal transduction and other processes near the membrane of biological cells.(Ref. Proc. Natl. Acad. Sci. USA, 112, 14846–14851 (2015).)

In-cell NMRとは細胞内蛋白質に対する選択的な高分解能異種核多次元NMR測定のことで、蛋白質の構造・動的挙動を原子レベルで解析することが可能である。我々は、HIV-1ウイルスのTat1蛋白質由来のCell Penetrating Peptide(CPP)を利用して、高効率に安定同位体標識された蛋白質を細胞質に導入することで、ヒト等高等哺乳動物体細胞におけるin-cell NMR測定に成功した。またその過程で、pyrenebutyrateによる細胞処理、細胞質におけるCPPの切断が、目的蛋白質の細胞質・核質への均一な導入に必須であることを見出した。更に、上記手法を用いて、細胞内における蛋白質の、細胞内在性の蛋白質との相互作用と、外部投与した低分子薬剤との相互作用を検出する試みを行い、一定の成果を得た。また、細胞内での蛋白質の構造安定性を重水素/軽水素交換実験によって解析したところ、in vitroに比べて不安定であることを示唆する結果を得た。上記に示すように、in-cell NMRという手法を用いることによって、高等哺乳動物体細胞内のような複雑な環境下において、特定の蛋白質の構造・動態を原子レベルで解析することが可能となった。本発表では、これまでの成果を概観するとともに、現在進行中のプロジェクト、特に細胞内蛋白質の構造安定性解析に関する進捗と今後の展望について述べる。

Solvation free energy is one of the most important physical quantities to elucidate the hydration effect on protein in solution phase. In this study, the solvation free energy of horse heart cytochrome c immersed in water was calculated using the molecular dynamics simulation coupled with the energy-representation method. The protein intramolecular energy and the solvation free energy are found to compensate each other in the course of equilibrium structural fluctuation, and the roles of the attractive and repulsive components in the protein-water interaction are examined for the solvation free energy.