Center for Biomimetic Sensor Science, School of Materials Science and Engineering, Nanyang Technological University (NTU), 50 Nanyang Drive, Singapore, 637553, Singapore.
Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, Kemistintie 1, 02150, Espoo, Finland.
Nat Commun. 2019 Nov 29;10(1):5465. doi: 10.1038/s41467-019-13469-8.
Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) is involved in both intracellular membraneless organelles and extracellular tissues. Despite growing understanding of LLPS, molecular-level mechanisms behind this process are still not fully established. Here, we use histidine-rich squid beak proteins (HBPs) as model IDPs to shed light on molecular interactions governing LLPS. We show that LLPS of HBPs is mediated though specific modular repeats. The morphology of separated phases (liquid-like versus hydrogels) correlates with the repeats' hydrophobicity. Solution-state NMR indicates that LLPS is a multistep process initiated by deprotonation of histidine residues, followed by transient hydrogen bonding with tyrosine, and eventually by hydrophobic interactions. The microdroplets are stabilized by aromatic clustering of tyrosine residues exhibiting restricted molecular mobility in the nano-to-microsecond timescale according to solid-state NMR experiments. Our findings provide guidelines to rationally design pH-responsive peptides with LLPS ability for various applications, including bioinspired protocells and smart drug-delivery systems.
液液相分离（LLPS）涉及到细胞内无膜细胞器和细胞外组织中固有无序蛋白质（IDPs）。尽管对 LLPS 的理解不断加深，但该过程的分子水平机制仍未完全建立。在这里，我们使用富含组氨酸的鱿鱼喙蛋白（HBPs）作为模型 IDPs，以阐明控制 LLPS 的分子相互作用。我们表明，HBPs 的 LLPS 是通过特定的模块重复介导的。分离相（液态与水凝胶）的形态与重复序列的疏水性相关。溶液状态 NMR 表明，LLPS 是一个多步过程，由组氨酸残基的去质子化引发，随后与酪氨酸发生瞬时氢键相互作用，最终是疏水相互作用。微滴通过酪氨酸残基的芳族聚集稳定，根据固态 NMR 实验，酪氨酸残基在纳秒至微秒时间尺度上表现出受限的分子迁移性。我们的发现为合理设计具有 LLPS 能力的 pH 响应肽提供了指导，这些肽可用于各种应用，包括仿生原细胞和智能药物输送系统。
