Renner-Rao Max, Jehle Franziska, Priemel Tobias, Duthoo Emilie, Fratzl Peter, Bertinetti Luca, Harrington Matthew J
Dept. of Chemistry, McGill University, Montreal, Quebec H4A 0B8, Canada.
Dept. of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam 14476, Germany.
ACS Nano. 2022 Dec 27;16(12):20877-20890. doi: 10.1021/acsnano.2c08410. Epub 2022 Nov 22.
Mussels () adhere to hard surfaces in intertidal marine habitats with a porous underwater glue called the byssus plaque. The plaque is an established role model for bioinspired underwater glues and comprises at least six proteins, most of which are highly cationic and enriched in the post-translationally modified amino acid 3,4-dihydroxyphenylalanine (DOPA). While much is known about the chemistry of plaque adhesion, less is understood about the natural plaque formation process. Here, we investigated plaque structure and formation using 3D electron microscopic imaging, revealing that micro- and nanopores form spontaneously during secretion of protein-filled secretory vesicles. To better understand this process, we developed a method to purify intact secretory vesicles for assembly studies. We discovered that each vesicle contains a sulfate-associated fluid condensate consisting of ∼9 histidine- and/or DOPA-rich proteins, which are presumably the required ingredients for building a plaque. Rupturing vesicles under specific buffering conditions relevant for natural assembly led to controlled multiphase liquid-liquid phase separation (LLPS) of different proteins, resulting in formation of a continuous phase with coexisting droplets. Rapid coarsening of the droplet phase was arrested through pH-dependent cross-linking of the continuous phase, producing native-like solid porous "microplaques" with droplet proteins remaining as fluid condensates within the pores. Results indicate that histidine deprotonation and sulfates figure prominently in condensate cross-linking. Distilled concepts suggest that combining phase separation with tunable cross-linking kinetics could be effective for microfabricating hierarchically porous materials via self-assembly.
贻贝通过一种叫做足丝斑的多孔水下胶水附着在潮间带海洋栖息地的坚硬表面上。足丝斑是一种公认的仿生水下胶水的典范，它至少由六种蛋白质组成，其中大多数是高度阳离子化的，并且富含翻译后修饰的氨基酸3,4 -二羟基苯丙氨酸（DOPA）。虽然人们对足丝斑粘附的化学原理了解很多，但对其自然形成过程却知之甚少。在这里，我们使用三维电子显微镜成像技术研究了足丝斑的结构和形成过程，发现微孔隙和纳米孔隙在充满蛋白质的分泌囊泡分泌过程中自发形成。为了更好地理解这一过程，我们开发了一种纯化完整分泌囊泡用于组装研究的方法。我们发现每个囊泡都含有一种与硫酸盐相关的流体凝聚物，它由大约9种富含组氨酸和/或DOPA的蛋白质组成，这些蛋白质可能是构建足丝斑所需的成分。在与自然组装相关的特定缓冲条件下使囊泡破裂，会导致不同蛋白质发生可控的多相液 - 液相分离（LLPS），从而形成一个含有共存液滴的连续相。通过连续相的pH依赖性交联阻止了液滴相的快速粗化，产生了类似天然的固体多孔“微足丝斑”，液滴蛋白质作为流体凝聚物保留在孔隙中。结果表明，组氨酸去质子化和硫酸盐在凝聚物交联中起重要作用。由此提炼出的概念表明，将相分离与可调交联动力学相结合可能对通过自组装微制造分级多孔材料有效。
