New Approaches for Microscopic Analysis of Macromolecular Assemblies

The correct assembly of protein molecules into functional units allows them to carry out their roles in living cells, but incorrect assembly frequently leads to malfunction and disease. Much is known about the structures of functional states and how they are reached from newly synthesised proteins, but incorrect assembly has proven to be challenging to understand and control. Such questions lie at the heart of the nature of polypeptides and proteins as the molecules of life, but are also of crucial importance for problems of great practical value. Indeed, disorders resulting from aberrant assembly of proteins include Alzheimer's and Parkinson's disease, conditions which are increasingly prevalent and problematic in an ageing society. One of the key unknowns in protein aggregation phenomena concerns the nature of the first events that trigger proteins to undergo aberrant assembly from their normal soluble states. These processes are difficult to probe as the initial stages of aggregation phenomena involve only a few misfolded proteins amongst millions of soluble proteins, and such nucleation events are therefore very challenging to locate within the relatively large volumes that are used in traditional biochemical experiments. The proposed work addresses this challenge by devising strategies for performing measurements directly in volumes small enough to contain a single nucleation event. Through this approach, we will be able to probe directly the manner in which the conversion of proteins into insoluble structures propagates from the initial nucleation stage and eventually leads to significant loss of solubility of proteins. The practical strategy for enabling measurements in such small volumes will be through the creation of a large number of miniature water droplets within an inert oil. Using this approach, it is possible to study simultaneously under a microscope thousands of individual aggregation reactions in different microdroplets. The ability to perform large numbers of such experiments is particularly valuable for the systematic search for different compounds which have the potential to stop of reverse the pathological aggregation of proteins of the type associated with protein deposition diseases. The most prevalent class of such disorders includes Alzheimer's disease, and together with related forms of dementia, is rapidly becoming one of the greatest strains on the healthcare system. Indeed in the UK alone, the costs associated with these conditions reach tens of billions of pounds per year and are expected to rise sharply in the next few years. The basic technology required for large scale studies of protein solubility is therefore of increasing practical importance, and the microdroplet-based approach developed in this project has the potential to increase the throughput of such studies by many orders of magnitude compared to the current state of the art.

This project seeks to develop high-throughput assays for protein aggregation that allow access to aggregation phenomena in picolitre volumes characterised by confinement comparable to cellular or subcellular compartments. The small system size offers crucial advantages for studying the very early stages of protein aggregation, including the isolation of single nucleation sites for detailed study, which is not possible in bulk experiments. In order to achieve this objective, this project brings together droplet microfluidics and protein science. Microfluidic devices will be developed which allow a massively parallel measurements of individual aggregation reactions over time in aqueous micro-droplets generated and stored in an inert oil phase on single devices.

This project is part of a long-term vision to understand and control protein assembly phenomena. Aberrant protein assembly underlies a range of disorders, including Alzheimer's disease, which are becoming an increasingly severe and central problem in an ageing society. The technological innovation that the present application delivers, namely new high-throughput and ultra-small protein aggregation assays through the use of droplet microfluidics, has direct implications for enabling a more effective search for mechanisms and modulators that control aberrant protein aggregation. The microdroplet based approach has the potential to increase the throughput of such experiments by several orders of magnitude compared to currently available methods, and in order to ensure that these technologies are of as wide use as possible to the biological community an important element in the project is making available the results and the device designs. Increased throughput has also an impact on the practical process of finding inhibitors against aberrant aggregation, and in order to explore this possibility to the full, we will seek contacts with industrial partners.