Liquid-liquid phase separation and crystallization in protein solutions induced by multivalent metal ions

蛋白质溶液中的液-液相分离（LLPS）（即分离成分别具有低蛋白质浓度和高蛋白质浓度的两个液相）是蛋白质结晶以及蛋白质缔合相关疾病（例如镰状细胞性贫血和白内障）的关键现象。然而，LLPS条件下的物理机制及其对后续蛋白质结晶的影响仍然知之甚少。在这个建议中，我们将进行一个全面的研究，蛋白质溶液中的多价金属离子的存在下，其中一个可重入的凝聚相行为和LLPS已经观察到最近在我们的小组。我们的目标是在分子水平上探索这种现象背后的基本机制以及关键参数，并最终控制蛋白质溶液的相行为。将解决以下问题：（a）当LLPS发生时，有效的蛋白质-蛋白质相互作用潜力是什么？(b)决定蛋白质溶液相行为的关键参数是什么（盐的性质和浓度、pH值、温度等）？(c)多价离子诱导的LLPS与聚合物（PEG）诱导的LLPS有何异同？一组互补的实验技术，包括小角散射（SAXS，SANS），静态/动态光散射（SLS/DLS），和光谱（FTIR，UV-vis和圆二色性）将用于解决这些复杂系统中的不同问题。

Liquid-liquid phase separation (LLPS) in protein solutions (i.e. the separation into two liquid phases with low and high protein concentration, respectively) is a crucial phenomenon for protein crystallization as well as protein-association related diseases, such as sickle cell anemia and cataracts. However, the physical mechanisms on the condition of LLPS and its effect on subsequent protein crystallization are still poorly understood. In this proposal, we will undertake a comprehensive study of protein solutions in the presence of multivalent metal ions, where a reentrant condensation phase behavior and LLPS have been observed recently in our group. Our aim is to explore the fundamental mechanisms as well as the key parameters behind of this phenomenon on a molecular level and finally to control the phase behavior of protein solutions. The following questions will be addressed: (a) What is the effective protein-protein interaction potential when LLPS occurs? (b) What are the key parameters determining the phase behavior of protein solutions (nature and concentration of salt, pH, T, …)? (c) What are the differences and similarities between LLPS induced by multivalent ions vs polymer (PEG)? A set of complementary experimental techniques including small-angle scattering (SAXS, SANS), static/dynamic light scattering (SLS/DLS), and spectroscopy (FTIR, UV-vis and circular dichroism) will be used to address the different issues in these complex systems.