Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, PL-30239 Krakow, Poland.
Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, FI-00076 Aalto, Finland.
Biomacromolecules. 2021 Feb 8;22(2):690-700. doi: 10.1021/acs.biomac.0c01506. Epub 2021 Jan 6.
Liquid-liquid phase separation of biomacromolecules is crucial in various inter- and extracellular biological functions. This includes formation of condensates to control, e.g., biochemical reactions and structural assembly. The same phenomenon is also found to be critically important in protein-based high-performance biological materials. Here, we use a well-characterized model triblock protein system to demonstrate the molecular level formation mechanism and structure of its condensate. Large-scale molecular modeling supported by analytical ultracentrifuge characterization combined with our earlier high magnification precision cryo-SEM microscopy imaging leads to deducing that the condensate has a bicontinuous network structure. The bicontinuous network rises from the proteins having a combination of sites with stronger mutual attraction and multiple weakly attractive regions connected by flexible, multiconfigurational linker regions. These attractive sites and regions behave as stickers of varying adhesion strength. For the examined model triblock protein construct, the β-sheet-rich end units are the stronger stickers, while additional weaker stickers, contributing to the condensation affinity, rise from spring-like connections in the flexible middle region of the protein. The combination of stronger and weaker sticker-like connections and the flexible regions between the stickers result in a versatile, liquid-like, self-healing structure. This structure also explains the high flexibility, easy deformability, and diffusion of the proteins, decreasing only 10-100 times in the bicontinuous network formed in the condensate phase in comparison to dilute protein solution. The here demonstrated structure and condensation mechanism of a model triblock protein construct via a combination of the stronger binding regions and the weaker, flexible sacrificial-bond-like network as well as its generalizability via polymer sticker models provide means to not only understand intracellular organization, regulation, and cellular function but also to identify direct control factors for and to enable engineering improved protein and polymer constructs to enhance control of advanced fiber materials, smart liquid biointerfaces, or self-healing matrices for pharmaceutics or bioengineering materials.
生物大分子的液-液相分离在各种细胞内外的生物功能中至关重要。这包括形成凝聚物以控制，例如，生化反应和结构组装。同样的现象也被发现对基于蛋白质的高性能生物材料至关重要。在这里，我们使用一种经过充分表征的模型三嵌段蛋白系统来证明其凝聚物的分子水平形成机制和结构。通过分析超速离心特性支持的大规模分子建模以及我们之前的高倍精密冷冻电子显微镜成像，推断出凝聚物具有双连续网络结构。这种双连续网络源自于具有更强相互吸引力和多个弱吸引力区域的组合的蛋白质，这些区域通过柔性、多构象连接区连接。这些有吸引力的区域和连接区充当具有不同粘附强度的“贴纸”。对于所研究的模型三嵌段蛋白构建体，富含β-折叠的末端单元是较强的“贴纸”，而通过蛋白质柔性中间区域的弹簧状连接产生的额外较弱的“贴纸”则有助于凝聚亲和力。较强和较弱的贴纸状连接以及贴纸之间的柔性区域的组合产生了一种多功能、液态、自修复的结构。这种结构还解释了蛋白质的高灵活性、易变形性和扩散性，与在凝聚相中形成的双连续网络中的稀蛋白溶液相比，仅降低了 10-100 倍。通过更强结合区域和较弱的、柔性的牺牲键状网络的组合以及通过聚合物贴纸模型的通用性来演示模型三嵌段蛋白构建体的结构和凝聚机制，不仅提供了理解细胞内组织、调节和细胞功能的手段，而且还提供了直接控制因素的识别和工程改进蛋白质和聚合物构建体的方法，以增强对先进纤维材料、智能液体生物界面或用于药剂学或生物工程材料的自修复基质的控制。
