基于肽自组装纳米金属酶的设计、结构构筑与性能研究

Control of self-assembly processes and tuning of biological functions represent the formidable challenge of current and future self-assembling design and experimentation. In this respect, metalloproteins provide a natural inspiration, in which metal ions can either act as a structural co-factor by triggering folding of polypeptides and stabilizing their tertiary structures or serve as an active co-factor by catalyzing biochemical reactions and biologically signalling, or both. In spite of decades of global endeavors, our ability to mimic these naturally evolved structure-function relations remain primitive, let alone our ambition to surpass the nature to fabricate new designer enzymes that could display functions and stability far superior to the natural ones. In this proposal, we propose to embark on this challenge by designing and experimenting designer peptide enzymes. The central line of the project will rely on combining the structural self-assembly of many short peptides from our accumulated knowledge and experience with the de novo design of artificial enzymes, taking advantage of metal ion-triggered peptide self-assembly processes. Specifically, we will focus our efforts on: (1) the design of novel self-assembling units, which consider both functional amino acids with catalytic, coordinational and binding roles and their cooperation and impact on peptide self-assembly processes, (2) the self-assembly of designer peptides and characterizations of their self-assembled nanostructures, the former being triggered through addition of metal ions and the latter including the assessments of self-assembled morphologies, nanostructures, and coordination chemistry, (3) the properties of self-assembled nanostructures, including catalytic activity and structural stability. The completion of the proposed studies shall enable us to understand how to de novo design well-defined nanostructures with good catalytic efficiency and high structural stability through metal ion-triggered self-assembly processes. This project will also enhance our understanding of folding and misfolding of proteins and polypeptides and even the origin of life.

组装过程调控和生物学功能导向的组装体系设计是自组装研究的重点，也是其今后发展的突破点。在天然蛋白质中，金属离子既可以作为结构辅因子，又可以作为功能辅因子。本项目拟利用金属离子与氨基酸残基侧链的配位作用调控小分子肽的折叠和自组装，构筑具有较高催化效率的纳米金属酶，从而达到从头设计功能蛋白质的目的。研究主要包括：（1）构建新颖的组装基元分子（肽），序列中含有不同的功能氨基酸残基（配位和催化）；（2）利用金属离子的配位作用以相对精确的方式调控自组装，系统表征组装体的结构特征，获得尺度可控、结构有序的组装体系；（3）系统考察纳米组装体的催化性能和稳定性，最终实现生物学功能组装体系的构建，获得稳定性好、催化效率高的纳米金属酶。项目的特色之处在于将肽的分子设计、可控组装与功能蛋白质（酶）的从头合成相结合。此外，项目的开展对于理解蛋白质和长链多肽的折叠或错误折叠、甚至生命的起源也具有重要的指导意义。

多肽自组装近年来备受关注，人们期望通过肽自组装过程研究来揭示蛋白质的折叠机制，在体外构筑具有生物学功能的纳米组装体或器件。本项目设计了三个系列的肽组装基元，这些基元分子可以形成特定的二级结构，且分子中含有特定功能残基。一方面，通过金属离子配位，启动α-helix和β-hairpin肽的分子内折叠，进而通过分子自组装形成结构有序的功能体，并研究了其生物催化性能。另一方面，通过特定残基侧链之间的氢键作用，首次发现了极性氨基酸拉链结构也可以在β-sheet之间形成，从而促进β-sheet的侧向堆积，形成较宽的纳米带状结构。主要的研究内容和结果如下：.在β-hairpin肽中引入功能性的组氨酸和酪氨酸，这些残基侧链可以与Cu2+配位，诱导肽链由无规则卷曲形成具有规则疏/亲水面的β-hairpin结构，进而通过分子间氢键和疏水作用形成β-sheet纳米纤维。该Cu2+–肽复合纳米纤维具有类似水解酶和氧化酶的催化活性，当金属离子和配位氨基酸侧链官能团（活性中心）位于β-hairpin结构亲水面上并远离转角序列时，催化效率最高。.α-helix肽自组装展示出典型的分级组装过程，项目通过分子设计和改变溶液条件，对其自组装过程、组装体结构甚至功能进行了有效调控。功能氨基酸组氨酸的引入使得α-helix肽在Cu2+配位诱导下折叠、自组装，最终形成不同纳米结构形貌的Cu2+–肽组装体，可用于人工酶的模拟。.在两亲性短肽I3K的疏水肽段和亲水残基之间引入不带电荷的极性氨基酸残基，所设计的I3QGK、I3NGK和I3SGK可自组装形成较宽的纳米带状结构，进一步的分析表明β-sheet之间形成了极性氨基酸拉链结构，这种结构源于极性侧链之间的氢键相互作用。.这些研究不仅有助于深入理解肽自组装的机制和规律，且可在此基础上有针对性的设计肽组装基元、调控肽自组装过程，构筑出具有生物学功能的结构体，所构筑的金属离子-肽纳米结构体可以用来模拟一些金属蛋白酶。基于该项目的研究，已经发表SCI期刊论文17篇，其中包括1篇Nat. Commun.和2篇Small研究论文、1篇Coordin. Chem. Rev.综述论文，研究成果被国家自然科学基金委、英国科学技术基础设施理事会ISIS等国内外多个学术网站作为研究亮点报道。此外，基于该项目研究已培养3名博士和6名硕士生。