Disordered protein conformation upon crowding and phase separation

PGL-3 (693 a.a., ~75 kDa) is a C. elegans germ granule scaffold protein that phase separates into membrane-less condensates in vivo and in vitro. Liquid-liquid phase separation depends on temperature and salt concentration, and results in dense and dilute co-existing phases with over 100-fold difference in protein concentration. This vast concentration disparity between the dense and dilute regimes poses the question: what are the protein's conformational states as the concentration increases from dilute towards dense? PGL-3 has two globular domains followed by an intrinsically disordered tail. While one of the folded domains participates in dimerization, the role of the other one and the disordered tail is unclear. We aim to obtain structural information in dilute, concentrated below phase separation, and phase separated conditions to establish the effect of salt, protein concentration and temperature on structure.

PGL-3（693个氨基酸残基，约75 kDa）是秀丽隐杆线虫（C. elegans）的生殖颗粒支架蛋白，可在体内与体外发生液-液相分离（Liquid-liquid phase separation），形成无膜凝聚体（membrane-less condensates）。液-液相分离过程受温度与盐浓度调控，会形成蛋白质浓度差异超100倍的致密相与稀相共存的体系。致密相与稀相之间的巨大浓度差异引出了核心科学问题：当蛋白质浓度从稀相向致密相逐步升高时，该蛋白质的构象状态究竟如何？PGL-3包含两个球状结构域，其后接续一段内在无序尾巴（intrinsically disordered tail）。其中一个折叠结构域参与蛋白质二聚化过程，而另一个折叠结构域以及这段内在无序尾巴的功能目前仍不明确。本研究旨在获取稀相、相分离临界浓度以下以及相分离状态下的结构信息，以明确盐浓度、蛋白质浓度与温度对该蛋白质结构的调控作用。