Department of Physical Chemistry II, Ruhr University Bochum, Bochum, Germany.
Department of Biochemistry of Neurodegenerative Diseases, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany.
Phys Chem Chem Phys. 2023 Oct 25;25(41):28063-28069. doi: 10.1039/d3cp03299j.
Understanding how protein rich condensates formed upon liquid-liquid phase separation (LLPS) evolve into solid aggregates is of fundamental importance for several medical applications, since these are suspected to be hot-spots for many neurotoxic diseases. This requires developing experimental approaches to observe in real-time both LLPS and liquid-solid phase separation (LSPS), and to unravel the delicate balance of protein and water interactions dictating the free energy differences between the two. We present a vibrational THz spectroscopy approach that allows doing so from the point of view of hydration water. We focus on a cellular prion protein of high medical relevance, which we can drive to undergo either LLPS or LSPS with few mutations. We find that it is a subtle balance of hydrophobic and hydrophilic solvation contributions that allows tuning between LLPS and LSPS. Hydrophobic hydration provides an entropic driving force to phase separation, through the release of hydration water into the bulk. Water hydrating hydrophilic groups provides an enthalpic driving force to keep the condensates in a liquid state. As a result, when we modify the protein by a few mutations to be less hydrophilic, we shift from LLPS to LSPS. This molecular understanding paves the way for a rational design of proteins.
理解富含蛋白质的凝聚物如何在液-液相分离（LLPS）后演变成固体聚集体，对于许多医学应用都具有重要的基础意义，因为这些聚集体被怀疑是许多神经毒性疾病的热点。这需要开发实验方法来实时观察 LLPS 和液-固相分离（LSPS），并揭示决定两种状态之间自由能差异的蛋白质和水相互作用的微妙平衡。我们提出了一种振动太赫兹光谱方法，从水合的角度可以做到这一点。我们专注于一种具有高度医学相关性的细胞朊病毒蛋白，我们可以通过少量突变来驱动它发生 LLPS 或 LSPS。我们发现，正是疏水性和亲水性溶剂化贡献之间的微妙平衡，使得 LLPS 和 LSPS 之间的转换得以实现。疏水性水合作用通过将水合水释放到体相中来提供相分离的熵驱动力。水合亲水性基团提供了一个焓驱动力，以保持凝聚物处于液态。因此，当我们通过几个突变来修饰蛋白质，使其亲水性降低时，我们就会从 LLPS 转变为 LSPS。这种分子理解为蛋白质的合理设计铺平了道路。
