Controlling the self-assembly of Small Heat-Shock Protein inspired nano-cages

科学中的前沿挑战之一是理解物质如何自我组装成定义和有序的结构。在利用和指导分子组装成具有理想分子特性的特定形式的能力方面，这种知识所产生的可能性是无限的。自组装的一些最显著的例子是在生物学中发现的，在生物学中，通过相对简单的‘积木’的组合产生了显著的多样性、复杂性和美观的结构。显然，大多数生物分子，无论是脂类、核酸还是蛋白质，实际上都是以组装的多聚体的形式存在的，通过大量弱的非共价相互作用将它们结合在一起。在这种组装结构中，蛋白质代表着最大的多样性，形成的结构范围从高度对称的病毒到不对称的多组分机器，以及延伸的丝状聚合物。值得注意的是，似乎通常只需要在构建块或环境条件中非常细微的变化来调整自组装途径，从而调整多聚体形式。由于在材料科学和医学中的应用，一些潜在的最有用的自组装生物结构是纳米级的笼子。它们作为用于化学和颗粒合成的微型反应容器提供了相当大的可能性，但它们最令人兴奋的应用可能是作为输送生物疗法的运输器。由于笼子本身直接针对特定的细胞或组织，因此可以将货物封装在笼子内，从而与周围的介质隔离。然而，目前我们模仿自然、合理设计这种“纳米笼子”的能力仍然有限。在这里，我们提出了一种新的策略，使用一种新的基于质谱学的方法来采样紧密相关的自组装蛋白质所跨越的结构多样性。这将使我们能够开发出一个可以为特定和不同功能量身定做的纳米笼子的“工具箱”。我们将作为研究重点的蛋白质是广泛存在的小分子热休克蛋白。尽管这些寡聚蛋白质的结构很难获得，但已经很明显的是，尽管这些蛋白质具有共同的模块结构和高度序列相似性的区域，但这些蛋白质自组装成一系列具有惊人多面体结构的“纳米笼子”。此外，自组装和分解的动力学表现出类似的多样性，并对溶液条件的细微变化做出反应。我们建议对这些天然纳米笼子的结构和动力学多样性进行广泛的调查，目的是指出自然调节它们自组装的方式。这种调查是由一种新型的实验管道实现的，该管道利用先进的质谱学方法快速提供关于蛋白质组装的寡聚、结构和波动的信息。通过以自动化的方式将这项技术与高通量蛋白质生产相结合，我们将能够以比传统方法快得多的速度确定这些纳米笼子的分子特性。在评估了大自然赋予这些蛋白质组件的可变性之后，这是如何在氨基酸水平上实现的，以及如何受到溶液条件的调节，我们将通过重新组合从初始筛选中选择的结构‘盒’来设计新型纳米笼子。通过这种方式，我们将能够构建一个广泛和多样化的纳米笼子库，在结构和自组装和拆卸特性上都是可变的。这一点，再加上我们对将这些笼子瞄准特定细胞类型并用超快激光刺激其破坏的可能性的探索，产生了令人兴奋的潜在应用，将货物运送到身体的特定位置。

One of the frontier challenges in science is to understand the means by which matter self-assembles into defined and ordered structures. The possibilities stemming from such knowledge, in terms of harnessing and directing the capability of molecules to assemble into specified forms with desirable molecular properties are boundless. Some of the most striking examples of self-assembly are found in biology, where structures of remarkable diversity, complexity and beauty arise through the combination of relatively simple 'building blocks'.It is apparent that the majority of biomolecules, be they lipids, nucleic acids, or proteins, actually exist in assembled multimeric forms, held together by a large number of weak non-covalent interactions. Proteins represent the greatest diversity in such assembled structures, forming structures ranging from highly symmetrical viruses, to asymmetric multi-component machines, and extended filamentous polymers. Remarkably, it appears that often only quite subtle changes in the building blocks, or environmental conditions, are required to adjust the self-assembly pathway, and consequently the multimeric form.With applications in both materials science and medicine, some of the potentially most useful self-assembled biological structures are nano-scale cages. They offer considerable possibilities as miniaturised reaction vessels for chemical and particle synthesis, but perhaps their most exciting application is as transporters for the delivery of biotherapeutics. Cargo could be encapsulated within the cages, and thereby sequestered from the surrounding medium, as the cages themselves are targeted directly at particular cells or tissue. Currently, however, our ability to mimic nature and rationally engineer such 'nano-cages' remains limited. Here we propose a novel strategy to sample the architectural diversity spanned by closely related self-assembling proteins using a novel mass spectrometry based approach. This will enable us to develop a 'tool-box' of nano-cages which can be tailored for particular and varied function.The proteins we will use as a focus for our studies are the widespread Small Heat-Shock Proteins. Even though structures of these oligomeric proteins has been hard to come by it is already apparent that, despite a common modular construction and regions of high sequence similarity, these proteins self-assemble into a range of 'nano-cages' with striking polyhedral architecture. Furthermore, the dynamics of self-assembly and disassembly display similar diversity, and are responsive to subtle changes in solution conditions.We propose to perform a wide survey of the architectural and dynamical diversity of these natural nano-cages, with the aim of pin-pointing the ways in which nature has regulated their self-assembly. Such a survey is enabled by a novel experimental pipeline which exploits the ability for advanced mass spectrometry approaches to rapidly provide information as to the oligomerization, structure, and fluctuations of protein assemblies. By coupling this technology in an automated fashion to high-throughput protein production we will be able to determine the molecular properties of these nano-cages at a rate dramatically faster than by means of traditional approaches.Having assessed the variability that nature has bestowed upon these protein assemblies, how this is achieved on the amino acid level and is regulated by solution conditions, we will engineer novel nano-cages by re-combining structural 'cassettes' selected from our initial screen. In this way we will be able to construct an extensive and diverse library of nano-cages, variable in both architecture and self-assembly and disassembly properties. This, together with our exploration of the possibilities in targeting these cages to specific cell types and to stimulate their disruption with ultra-fast lasers, yields the exciting potential application for delivery of cargo to defined locations in the body.