Biophysical dissection of protein nucleation using a combined experimental and computational approach

Proteins are responsible for the vast majority of functions in living organisms, where they make structural scaffolds, transport cargo from A to B, pass messages from cell to cell, recognise and repel invaders, and catalyse the reactions essential for life. The self-assembly of proteins into well-defined structures and into constructs of many molecules is also essential to our well-being. Occasionally, however, protein self-assembly takes place inappropriately, perhaps due to a mutation or a change in environment. When this happens in the body it typically causes disease, and diseases such as emphysema, Alzheimer's Disease, Parkinson's Disease, cataract and type II diabetes are all recognised to be the result of improper protein self-assembly. Protein self-assembly can also cause havoc in industrial processes including the production of biopharmaceuticals such as insulin. When this occurs, the pharmaceutical is often lost as an irretrievably tangled mass of gelled protein. All is not lost, however: the self-assembly of proteins also underpins the texture of foodstuffs including egg, meat and milk products. We are interested on one specific form of protein self-assembly that appears to be common to all proteins. It is possibly counter intuitive that a specific form of self-assembly seems to apply to a wide range of chemically very different species (proteins range from hundreds of atoms to hundreds of thousands of atoms), however this form of self-assembly is driven by groups in the backbone of the protein chain, and this backbone is a polymer common to all proteins. The outcome of self-assembly in this case is the formation of 'amyloid' fibrils, rope-like structures consisting of thousands of copies of the same protein. We are interested in the earliest stages that start the assembly of these fibrils. If all proteins can undergo this form of self-assembly, and if all proteins form the same final fibrillar structure, do they all also follow the same pathway? We propose to use a range of very different complementary techniques from the fields of chemistry, biophysics and physics, and a combination of state-of-the-art experimental and computational approaches to detect, identify and characterise the earliest species in self-assembly.

This proposal focuses on the self-assembly of polypeptides into the aggregates known as 'amyloid' or amyloid-like fibrils. Such fibrillar aggregates are implicated in the 'amyloidoses', a family of diseases including Alzheimer's disease. The actual role of fibrils in these diseases is still a matter of debate and remains the subject of intense study. The misfolding and self-assembly of proteins into amyloid-like fibrils is, however, of broader significance because it appears to represent a generic property of the polypeptide chain, a fundamental state of proteins alongside the folded, unfolded and molten globule states. This proposal addresses the earliest stage of amyloid fibril assembly, the formation of the fibril nucleus. Fibril assembly follows a sigmoidal relationship: nucleation occurs during a lag phase, following which fibrils grow at an exponential rate. If assembly into amyloid-like fibrils is truly a generic phenomenon, do all fibrils assemble in the same way? Do all fibril nuclei 'look' similar, and if not, how are they different? We know that in some cases fibril assembly can be cross-seeded (where nuclei formed by one protein species can catalyse the self-assembly of another protein) however this appears to be the exception rather than the rule. If nucleation occurs via a number of different pathways, what are the key factors that determine the final common fibrillar architecture? We have formed an interdisciplinary team with expertise in biophysical chemistry, computational physics and biological physics to address this problem. We propose to use a wide range of very different but complementary techniques including ion mobility mass spectrometry, atomic force microscopy, transmission electron microscopy, dynamic light scattering and neutron scattering alongside computational simulations. Our unique strength is our combined experimental and computational approach to detect, identify and characterise the earliest species in self-assembly.

The immediate beneficiaries of this research will be those involved in the study of protein aggregation and self-assembly, whether in the life, chemical or physical sciences. Inappropriate protein aggregation can cause problems with the scale-up and production of biopharmaceuticals such as polypeptide drugs. Our research will possibly suggest ways to prevent aggregation while maintaining function. Similarly, protein self-assembly is implicated in the protein misfolding disorders (including most famously Alzheimer's Disease) and a common pathway to nucleation of aggregation or, conversely, evidence for multiple different pathways to self-assembly, will benefit those looking for new therapeutic interventions. Protein aggregation and self-assembly are also of interest to the food industry, where they underlie phenomena such as food texturing, 'mouth feel', and/ or longevity, and an understanding of the physical basis for self-assembly may be of benefit to the development of new foodstuffs (either by promoting or preventing self-assembly, as appropriate). The research will also be of benefit to those unaware of the strength of mass spectrometry as an analytical tool for the life sciences. The development of new mass spectrometry methods and their wider application will benefit those responsible for the development of instrumentation. This is a interdisciplinary and collaborative project, in an environment when 'multidisciplinarity' is being promoted. Projects such as this one serve as case studies for those in government and the research councils looking for evidence that interdisciplinary collaboration brings 'added value' to research projects. The staff involved in the project will benefit from the development of advanced experimental skills, and the experience of working on a complex project requiring good organisation and time-management skills. They will also acquire experience of working in a multidisciplinary and collaborative environment that emphasises team work and cooperation. To ensure the research outcomes reach the widest range of beneficiaries we will publish in high-impact journals and present the scientific outcomes of research at national and international meetings. In the past we have presented projects similar to this one to the Philosophy of Science community as 'Case Studies' of interdisciplinary research, and we will continue to engage with this community. Our industrial collaborators include instrumentation developers, the biopharmaceutical industry and the food industry, and we will continue to disseminate our findings to them. Track Record The three investigators named in this proposal have a long-standing commitment to reaching out to a wider audience. We publish in high-impact and (crucially) multidisciplinary journals. We present at conferences ranging from small focused meetings to large general science conferences. All have experience of working at the interface between disciplines and therefore have direct understanding of the requirement to translate research into a different disciplinary 'language' and the importance of disseminating information to as wide a scientific audience as possible. All are involved in student recruitment. They sit on committees of the learned societies (the Institute of Physics, the Royal Society of Chemistry and the Biochemical Society). One of the named researchers publishes a pseudonymous blog about the nature of scientific research. With regard to economic impact, MacPhee has founded one start-up company and is currently seeking seed funding to spin out a second. She also acts on the Scientific Advisory Board of two SMEs. Barran has substantial experience of translating modifications to laboratory instrumentation into commercial equipment in collaboration with Mass Spectrometry companies. Crain has led large collaborative research programs at the physical/life science interface in industrial sectors and holds 5 patents.