生物大分子组装的介晶机制研究

Macromolecular crystallization has many implications in biological and materials science. Like crystallization of other molecules, macromolecular crystallization conventionally considers a critical nucleus followed by crystallographic packing of macromolecules to drive further crystal growth. In this project, the applicant proposes a distinctive macromolecular crystallization pathway by developing a concept of macromolecular mesocrystal. The applicant underlines this nonclassical polymer crystallization by mesoscale self-assembly of biomacromolecular nanocrystals. This new understanding on macromolecular crystallization is fundamental, and relevant to many fields of material science, chemistry, biomimetics, nanoscience and structural biology. . The concept of mesocrystal represents a kind of suprastructure self-assembled by nanocrystalline intermediates in a crystallographic ordered way and this mechanism exhibits its powerful capability to fabricate hierarchical architecture and present close relevance to the formation of biogenic crystals in nature. The unprecedented features of mesocrystals thereby impose marvelous opportunities towards advanced crystalline and material engineering. Up to now, the acquisition of mesocrystals with a variety of constitution, morphology and structure is highly restrained to inorganic crystallization with few mesocrystal examples on small molecular amino acids and organic dyes. In contrast, so far (bio)organic macromolecular mesocrystal is never achieved. This project aims to underline the first non-classical biopolymer mesocrystal pathway by mesoscale self-assembly and cooperative transformation of macromolecular nanocrystalline intermediates across multiple length scales. Using an unfolded lysozyme as a model biopolymer with uniform molecular sequence and chain length, macromolecular nanocrystal architecture is designed as crystalline core with amorphous shell. It is expected that the formation of such core-shell nanocrystal could lead to unprecedented control over biopolymer crystallization, as a wide spectrum of flexible crystalline macromolecular materials with complex form and hierarchy including single-crystal platelets, polycrystalline multilayered hydrogel film and hybrid biopolymer-metal polycrystalline heterostructures could be facilely acquired through the assembly and crystallographic fusion of the core-shell macromolecular crystalline architectures. This project has significant implications for higher-order macromolecular assembly and template-directed formation of crystalline polymeric materials in biological as well as synthetic systems.

自然界中大部分矿物晶体的结晶形式多是按照非经典的结晶路线进行：分子经过两步成核（先聚集后成核）形成纳米晶，纳米晶进一步组装形成具有超结构的类单晶体—介晶。过去十几年的介晶研究始终无法证明大分子介晶的存在。本项目创新性地提出基于蛋白质相转变的新型类淀粉样聚集来实现大分子介晶结构的可控合成。通过以溶菌酶为模型蛋白质进行水相温和的类淀粉样组装，研究在热力学准平衡条件下β-sheet短程有序聚集体的可控制备，继而通过调控这些纳米聚集体的进一步介观组装行为，发展大分子介晶路径。通过分析蛋白质中对介晶组装起核心调控作用的氨基酸序列结构及空间立构变化规律，实现对溶菌酶介晶组装的精准调控。探索纳米聚集体与最终晶态材料的微观/宏观形貌、性能之间的构效关系，并利用后化学和物理修饰技术对溶菌酶介晶结构进行改性和功能化。尝试拓展到其他淀粉样蛋白质体系，从而全面建立和发展生物大分子介晶组装化学和材料制备新体系。

蛋白质的淀粉样聚集行为往往会引起神经退行性疾病，例如帕金森综合征、阿尔兹海默症等，类淀粉样蛋白结晶对于理解和揭示蛋白质淀粉样聚集行为中分子机制以及发展相关治疗手段至关重要，而在另一方面，蛋白质淀粉样聚集所提供的高度有序结构为新材料的合成与设计提供了崭新的平台和模板。然而在热力学上难以区分蛋白质淀粉样纤维化和结晶，淀粉样晶体的获得通常被限定在淀粉样相关的多肽，对于淀粉样相关的蛋白质，由于熵的限制更易于形成淀粉样纤维而难以形成晶体，获得淀粉样蛋白质晶体仍然是一项巨大的挑战。为此，本项目通过蛋白质解折叠，发展了动力学驱动的类淀粉样蛋白质非经典结晶途径，主要包括类淀粉样纳米晶的两步形核和基于介观组装的生长，实现了类淀粉样蛋白质晶体的结构和功能调控。.研究工作总结如下:.（1）首先我们研究类淀粉样蛋白质纳米晶的两步形核过程。通过三（2-羧乙基）膦（TCEP）还原溶菌酶中二硫键使其解折叠，从而激发类淀粉样转变。解折叠链之间形成的短程有序β-sheets聚集体，动力学控制的准平衡态组装能够抑制蛋白质之间过强作用，促使短程有序的β-sheets聚集体能够进行有序堆积，形成类淀粉样纳米晶体，该晶体具有典型的“核-壳”结构，即纳米晶体作为“核”嵌入在由解折叠链构成的“壳”中。.（2）以（1）为基础，我们发现了第一例（生物）大分子介晶，在此之前介晶的获得被限制在无机物结晶以及一些有机小分子结晶中，如氨基酸和有机染料。由解折叠链形成无定型壳提供动力学能垒稳定蛋白质纳米晶，在一定条件下克服动力学的能垒，类淀粉样纳米晶体结构能够通过介观晶体学有序组装形成蛋白质介晶，蛋白质介晶进一步融合形成具有分级结构的柔性大面积片状单晶。.（3）以（1）和（2）为基础，我们提出基于动力学调控蛋白纳米晶有序组装的介晶化途径来实现淀粉样蛋白质晶体结构和形貌调控。这种核壳结构的蛋白质纳米晶具有“外柔内刚”的特点，具有多层分级结构的“刚性”结晶核能够抵抗β-sheets结构本身的柔性扰动，同时解折叠链构成的柔性“壳”能够释放组装过程中的弹性形变，这两者能够使得纳米晶即使在较高温度下仍不受β-sheets本身结构扭曲的影响，而实现晶态自组装。