Molecular Assembly of Protein with Liquid-Liquid Phase Separation : Structure, Mechanism, and Function of Elastic Fiber Protein Aggregates

弹性纤维蛋白生物合成的关键步骤是弹性蛋白原的自组装过程。弹性蛋白作为细胞外基质的多种功能来源于这些分子聚集过程中建立的分子结构。细胞外空间中的这些重要生物学过程相当于可溶性弹性蛋白相关肽的温度依赖性凝聚。本课题主要研究由α-弹性蛋白和人工合成的模型多肽组成的弹性蛋白凝聚体的结构、机制和功能。（1）弹性蛋白-水体系液-液相分离的研究采用湍流实验方法，初步研究了弹性蛋白肽的温度依赖性凝聚特性。静态和动态光散射实验也进行了进一步研究的分子组装过程的早期阶段。结果表明，弹性蛋白凝聚层的形成过程是由金属阳离子的具体影响。（2）弹性蛋白凝聚层是一种蛋白质液膜，弹性蛋白多肽的大凝聚层稳定性好，可以作为液体离子交换膜使用。观察到Ca^2+离子的选择性和特异性转运过程。研究表明，该体系可用于研究蛋白质与金属离子之间的相互作用。（3）弹性蛋白凝聚体的结构、作用机制和功能通过CD和NMR等多种方法观察到Ca^（2+）离子与Val-Pro-Gly-Val-Gly五肽骨架的选择性相互作用。这表明，负责产生弹性蛋白弹性功能的基本分子结构不可避免地受到Ca^<2+>的选择性相互作用。

The essential and key step for the biosynthesis of elastic fiber protein is a self-assembly process of tropoelastin, biogenic precursor protein of elastin. Multiple functions of elastin as an extracellular matrix are derived from the molecular structures established during these 'molecular aggregation process. These important biological processes in the extracellular space are equivalent to the temperature-dependent coacervation of soluble elastin-related peptides. In the present research project, structure, mechanism, and function of elastin coacervate composed of a-elastin or synthetic model polypeptide were investigated.(1)Examination of the liquid-liquid phase separation of elastin water system.Turbidity measurements were employed to survey primarily the characteristics of temperature-dependent coacervation of elastin peptides. Static and dynamic light scattering experiments were also carried out to further investigate the early stage of molecular assembly process. It was shown that the elastin coacervate formation process was affected specifically by metal cations.(2)Elastin coacervate as a protein liquid membrane.Macrocoacervate layer of elastin peptides was stable enough to utilize as a liquid ion-exchange membrane. Selective and specific transport processes for Ca^<2+> ions were observed. It was also demonstrated that the present system is useful to investigate the interactions between protein and metal cations.(3)Structure, mechanism, and function of elastin coacervate.Selective interactions of Ca^<2+> ions with pentapeptide, Val-Pro-Gly-Val-Gly, backbone were observed in a variety of measurements including CD and NMR sppectroscopy. It was suggested that the fundamental molecular structure responsible to generate elastomeric function of elastin is inevitably subjected to selective interaction of Ca^<2+>.