MDシミュレーション（Coarse-Grained Model）によるLLPS様の FUS Proteinの凝集体の解析
Tags: llps, gromacs, mdanalysis, protein
Published: 2021-04-28 13:12:19

# Abstract

## 導入

* 変性タンパク質と核酸が凝集してLLPSを起こす

* MD simulationsにより物理化学的な特性やLLPSの組成や大きさを決めるfactorを決定するユニークな手法

Disordered proteins and nucleic acids can condense into droplets that resemble the membraneless organelles observed in living cells. MD simulations offer a unique tool to characterize the molecular interactions governing the formation of these biomolecular condensates, their physicochemical properties, and the factors controlling their composition and size. 

## 従来の問題点

* 生体高分子の凝集はエネルギーまたはエントロピーの寄与の影響のバランスにセンシティブに依存する

However, biopolymer condensation depends sensitively on the balance between different energetic and entropic contributions. 

## 新規にわかったこと

* 生体高分子の相分離の動力学シミュレーションのポテンシャルエネルギー関数をfine-tuneする一般的な手法を開発した

* 溶媒およびエントロピー的な寄与に対するタンパク質-タンパク質の相互作用のバランスを、「希薄溶液と縮合物の間でタンパク質が移動する際の過剰な自由エネルギーに合わせて」見直した。

Here, we develop a general strategy to fine-tune the potential energy function for molecular dynamics simulations of biopolymer phase separation. We rebalance protein–protein interactions against solvation and entropic contributions to match the excess free energy of transferring proteins between dilute solution and condensate. 

## 実験の内容

* FUSのlow-complexity domain (LCD) のドロップレット形成を、rebalanced MARTINI model（?）でシミュレーションすることでこの関数を実証した。

* 粗視化したMARTINI potential energy functionのなかで、非結合相互作用の強さをスケーリング（?）することで、「タンパク質濃度」と「相互作用の強さ」の状態図（phase diagram）を描いた。（map out）

We illustrate this formalism by simulating liquid droplet formation of the FUS low-complexity domain (LCD) with a rebalanced MARTINI model. By scaling the strength of the nonbonded interactions in the coarse-grained MARTINI potential energy function, we map out a phase diagram in the plane of protein concentration and interaction strength. 

* critical scaling factor　αc が 0.6以上の時、FUS-LCD の凝集が観察された。ここで、α = 1 と α = 0はMARTINI modelにおける完全な相互作用と反発相互作用に対応する。

* scaling factor α が 0.65 の時、実験的に希薄相と密集相の密度を測定した。

* それにより、タンパク質がドロップレットに入るときの過剰な自由エネルギーとFUS-LCDが凝集する時の飽和濃度が得られた。

Above a critical scaling factor of αc ≈ 0.6, FUS-LCD condensation is observed, where α = 1 and 0 correspond to full and repulsive interactions in the MARTINI model. For a scaling factor α = 0.65, we recover experimental densities of the dilute and dense phases, and thus the excess protein transfer free energy into the droplet and the saturation concentration where FUS-LCD condenses. 

* 相分離の領域では、希薄相とFUS-LCD密集板の間でFUS-LCDのドロップレットを数十ミリ秒間、４つの異なるサイズでシミュレーションした。

* 1ms以上、凝集した状態のシミュレーションを行なった。

* 表面張力を、ドロップレットの形の変動や、２つの相の間のcapillary-wave-like broadeningから0.01–0.4 mN/m の範囲で決定した。

In the region of phase separation, we simulate FUS-LCD droplets of four different sizes in stable equilibrium with the dilute phase and slabs of condensed FUS-LCD for tens of microseconds, and over one millisecond in aggregate. We determine surface tensions in the range of 0.01–0.4 mN/m from the fluctuations of the droplet shape and from the capillary-wave-like broadening of the interface between the two phases. 

* タンパク質の端から端までの距離から、scaling factors α を 0.625–0.75（相分離が観察できた範囲）に設定して0.001 から 0.02 Pa sまでの範囲でFUS-LCD のドロップレットの粘度を測定した。

* 液滴の内部が著しく水和することでタンパク質と液滴の流動性が保たれる。

From the dynamics of the protein end-to-end distance, we estimate shear viscosities from 0.001 to 0.02 Pa s for the FUS-LCD droplets with scaling factors α in the range of 0.625–0.75, where we observe liquid droplets. Significant hydration of the interior of the droplets keeps the proteins mobile and the droplets fluid.

# Introduction

* LLPSの例

* stress granules, processing bodies, nucleoli（核小体）, Cajal bodies（mRNAのプロセッシング）

Intracellular compartmentalization into organellar structures is crucial for the organization of cellular biochemistry in time and space. Cell nuclei and mitochondria are examples of subcellular compartments bounded by a lipid membrane, whereas stress granules, processing bodies, nucleoli, or Cajal bodies are membraneless.(1−6) Disordered proteins and nucleic acids can cluster to form biomolecular condensates with liquid-like properties.(1,2,7,8) Such condensates formed by liquid–liquid phase separation (LLPS) in vitro mimic the membraneless organelles in cells.(1,2)

* 

Individually weak but multivalent interactions between intrinsically disordered proteins (IDPs), in some cases amplified by condensing factors, are major drivers of LLPS.(4,8) The biomolecular condensates produced by LLPS behave as liquid droplets immersed in dilute solution.(7) Their liquid-like interior facilitates the rapid diffusion of reactants within the condensates and their exchange with the outside.(9) Their dynamic nature also makes biomolecular condensates a promising template for novel biomimetic materials.(10,11) The rational design of new materials will benefit from predictive models that relate the static and dynamic materials properties of biomolecular condensates to the protein and nucleic acid sequences(10) and the molecular interactions they encode.