Université Grenoble Alpes, Université Savoie Mont Blanc, CNRS, IRD, IFSTTAR, ISTerre, F-38000 Grenoble, France.
Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.
Nature. 2018 Apr 4;556(7699):89-94. doi: 10.1038/nature25971.
The formation of condensed (compacted) protein phases is associated with a wide range of human disorders, such as eye cataracts, amyotrophic lateral sclerosis, sickle cell anaemia and Alzheimer's disease. However, condensed protein phases have their uses: as crystals, they are harnessed by structural biologists to elucidate protein structures, or are used as delivery vehicles for pharmaceutical applications. The physiochemical properties of crystals can vary substantially between different forms or structures ('polymorphs') of the same macromolecule, and dictate their usability in a scientific or industrial context. To gain control over an emerging polymorph, one needs a molecular-level understanding of the pathways that lead to the various macroscopic states and of the mechanisms that govern pathway selection. However, it is still not clear how the embryonic seeds of a macromolecular phase are formed, or how these nuclei affect polymorph selection. Here we use time-resolved cryo-transmission electron microscopy to image the nucleation of crystals of the protein glucose isomerase, and to uncover at molecular resolution the nucleation pathways that lead to two crystalline states and one gelled state. We show that polymorph selection takes place at the earliest stages of structure formation and is based on specific building blocks for each space group. Moreover, we demonstrate control over the system by selectively forming desired polymorphs through site-directed mutagenesis, specifically tuning intermolecular bonding or gel seeding. Our results differ from the present picture of protein nucleation, in that we do not identify a metastable dense liquid as the precursor to the crystalline state. Rather, we observe nucleation events that are driven by oriented attachments between subcritical clusters that already exhibit a degree of crystallinity. These insights suggest ways of controlling macromolecular phase transitions, aiding the development of protein-based drug-delivery systems and macromolecular crystallography.
凝聚（致密）蛋白质相的形成与广泛的人类疾病有关，例如白内障、肌萎缩性侧索硬化症、镰状细胞性贫血和阿尔茨海默病。然而，凝聚的蛋白质相也有其用途：作为晶体，它们被结构生物学家用来阐明蛋白质结构，或者被用作药物应用的递送载体。同一大分子的不同形式或结构（“多晶型物”）的晶体的物理化学性质在很大程度上有所不同，并决定了它们在科学或工业环境中的可用性。为了控制新兴的多晶型物，需要对导致各种宏观状态的途径以及控制途径选择的机制有分子水平的理解。然而，目前尚不清楚大分子相的胚胎种子是如何形成的，或者这些核如何影响多晶型物的选择。在这里，我们使用时间分辨低温传输电子显微镜来观察蛋白质葡萄糖异构酶晶体的成核，并以分子分辨率揭示导致两种晶体状态和一种凝胶状态的成核途径。我们表明，多晶型物的选择发生在结构形成的最早阶段，并且基于每个空间群的特定构建块。此外，我们通过定点突变选择性地形成所需的多晶型物来展示对系统的控制，特别是通过特异性调节分子间键合或凝胶接种。我们的结果与目前的蛋白质成核图不同，因为我们没有将亚稳密集液体识别为晶体状态的前体。相反，我们观察到成核事件是由亚临界簇之间的取向附生驱动的，这些亚临界簇已经表现出一定程度的结晶度。这些见解为控制大分子相转变提供了思路，有助于开发基于蛋白质的药物递送系统和大分子晶体学。
