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

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.

The proposed work is directed at the current EPSRC signpost in the Physical Sciences for 'Control of Self-Assembly', and will ultimately have practical importance within academia, industry, and have potential impact on human health. The downstream research outcomes will be directly applicable to EPSRC's general themes of 'manufacturing the future' and 'healthcare technologies', and core scientific areas of Physical Sciences and Engineering.
In general, the ability to harness and direct the process of self-assembly to create structures with the desired molecular properties is one of the frontier challenges in physical science and engineering. The scope of novel materials which could result from such control of self-assembly is essentially limitless. The research proposed here is focussed on investigating the self-assembly of protein-based cage-like structures. Specifically we will uncover the interplay between the large number of weak interactions which govern the structures and dynamics of their multimeric forms, providing us with a means for engineering novel nano-cages.

Healthcare technology
There is a strong desire in biomedicine to not only synthesise novel drugs, but also to target their administration and thereby avoid undesirable systemic effects. Encapsulation of therapeutics within nano-sized cages, functionalised to modulate their biodistribution, represents an exciting means of achieving this feat. The nano-cages we are proposing to develop have potential utility in enabling this by transporting payloads of drugs or biotherapeutics. Our cages are based on Small Heat-Shock Proteins, which have been show to be able to activate dendritic cells. Coupled with their ability to bind non-native proteins, this renders them an intriguing prospect for enhancing antigen presentation and stimulating the immune response. The opportunities that such cages may ultimately present are therefore considerable. We will investigate these downstream applications through our collaboration with Prof Ben Davis (Oxford), a world leader in such drug-delivery technologies, and our relationships with biopharmaceutical companies, and the intellectual property company of the University.
Aside from the nano-cages themselves, the technology we are proposing to develop for their rapid analysis will also have an impact on the UK's health and economic competitiveness. The mass spectrometry based pipeline we have envisaged will effectively become a rapid means for assessing protein architecture and dynamics. This approach is widely applicable, and will be of considerable use for structural biology, which ultimately contributes to medicine. The UK is world-leading in mass spectrometry design, and we will ensure that our advances are exploited through our ongoing collaborations with both Waters UK (mass spectrometers), and Advion Biosciences (robotic sample infusion).

Manufacturing the future
The nano-cages we will study and engineer have intriguing potential for nano-particle synthesis. Hollow virus particles have previously been shown to allow the generation of nano-particles, by nucleating growth on the inside of their cages. Here we are proposing to generate a suite of nano-cages, of not only variable cavity size, but also of variable morphology. As such they could potentially be used to template the synthesis of monodisperse nano-particles, of accurately defined size and shape. Alternatively, mirroring their role in nature, cavities can also be used to catalyse chemical reactions, or to sequester reactive molecules. We will take advantage of the diverse experience in inorganic and organic chemistry in our department to ascertain the potential of our cages in this regard.