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

科学的前沿挑战之一是理解物质自我组装成定义和有序结构的方法。就利用和引导分子组装成具有所需分子性质的特定形式的能力而言，这些知识所产生的可能性是无限的。在生物学中，自组装的一些最显著的例子是通过相对简单的“积木”组合而成的，具有显著多样性、复杂性和美丽性的结构。很明显，大多数生物分子，无论是脂质、核酸还是蛋白质，实际上都是以组装的多聚体形式存在的，通过大量弱的非共价相互作用结合在一起。蛋白质在这种组装结构中代表了最大的多样性，形成从高度对称的病毒到不对称的多组分机器和延伸的丝状聚合物的结构。值得注意的是，似乎通常只需要在构建模块或环境条件中进行非常细微的变化就可以调整自组装途径，从而调整多聚体形式。随着材料科学和医学的应用，一些潜在的最有用的自组装生物结构是纳米尺度的笼子。它们为化学和粒子合成提供了相当大的可能性，但也许它们最令人兴奋的应用是作为生物治疗药物的运输工具。货物可以被封装在笼内，从而与周围介质隔离，因为笼本身直接靶向特定的细胞或组织。然而，目前，我们模仿自然并合理设计这种“纳米笼”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的方法，采样的建筑多样性跨越密切相关的自组装蛋白质。这将使我们能够开发一个“工具箱”的纳米笼，可以定制为特定的和不同的功能。我们将使用的蛋白质作为我们的研究重点是广泛的小热休克蛋白。尽管这些寡聚蛋白质的结构很难获得，但已经很明显的是，尽管有共同的模块化结构和高序列相似性的区域，这些蛋白质自组装成一系列具有引人注目的多面体结构的“纳米笼”。此外，自组装和拆卸的动态显示类似的多样性，并响应于微妙的变化，解决方案conditions.We建议进行广泛的调查，这些自然的纳米笼的建筑和动态多样性，以针指出的方式，自然调节其自组装的目的。这样的调查是通过一种新型实验管道实现的，该管道利用先进的质谱方法快速提供有关蛋白质组装体的寡聚化、结构和波动的信息。通过将这项技术以自动化的方式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的分子特性。在评估了自然赋予这些蛋白质组装体的可变性之后，这是如何在氨基酸水平上实现的，以及如何通过溶液条件进行调节，我们将通过重新组合从我们的初始筛选中选择的结构'盒'来设计新颖的纳米笼。通过这种方式，我们将能够构建一个广泛而多样的纳米笼库，在架构和自组装和拆卸特性方面都是可变的。这一点，再加上我们探索将这些笼子靶向特定细胞类型并用超快激光刺激其破坏的可能性，产生了将货物运送到体内特定位置的令人兴奋的潜在应用。

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.