Periodic Reporting for period 2 - PHOTOMASS (Imaging Biomolecular Self-Assembly with a Molecular Photonic Scale) Reporting period: 2020-12-01 to 2022-05-31 Summary of the context and overall objectives of the project Biomolecular self-assembly is central to a wide range of physiological processes. Protein function often requires the assembly of cellular machines spanning length scales from small complexes consisting of several molecules1, to tens of nm-scale objects such as viral capsids, all the way to the mesoscale for biological filaments. By contrast, mis-assembly often leads to debilitating conditions, such as sickle-cell anaemia, cataract formation, Alzheimer’s and Parkinson’s disease. In addition, rational self-assembly is becoming a powerful approach for de novo drug design, which has numerous applications, such as in the development of novel therapeutics.Our goal is to revolutionise the way we can monitor and quantify biomolecular interactions by imaging single molecules in solution in a mass-sensitive fashion using light scattering detection and quantification of single biomolecules in solution. This will address a pressing current gap between existing approaches capable of characterising the atomic structure of individual molecules or simple complexes at one extreme and tools for studying ensemble dynamics at the other. This advance is what will allow us to reveal the energetic landscapes that govern the thermodynamics and kinetics of self-assembly in nature.The overarching aim of this project is to uncover the physicochemical basis of biological self-assembly at the single molecule level across different species, architectures, length scales and associated functions. To achieve this, we have defined five objectives:1. Determine the physical origins of polydispersity in proteins2. Visualise how viral capsids assemble3. Understand how proteins form filaments4. Capture the mechanism underlying protein aggregation associated with disease5. Implement the technological advances required for objective 1-4 Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far To date, we have made considerable progress on objective 1,3 and 5, with 2 and 4 being largely held back by lack of appropriate samples from collaborators strongly impacted by the Covid19 epidemic.For objective 1, we performed a series of experiments starting from the basics of detecting and counting single biomolecules using mass photometry. We began by understanding and quantifying the basics of the technology: does it count and identify molecules correctly, and can one use the results to determine the interaction strengths and dynamics between molecules. We used commonly employed antibodies, given their therapeutic importance and method of action based on (un)binding to specific targets. We then continued by expanding our approach to more complex systems beyond 1:1 interactions, and showed that the range of species formed for some systems depends not only on environmental factors, but in fact controls protein function and varies substantially between different organisms.Objective 3 benefitted considerably from the advances made in objective 1: we could now reveal how strongly proteins interact with each other at the single molecule level, as well as obtaining quantitative information on distributions ranging from single proteins to complexes containing a few hundred molecules. Equipped with this capability, we could directly observe how individual proteins dissolved in solution can turn into long filaments, a process that is central to a number of physiological and pathological processes. Interestingly, the mechanism we observed did not match that developed from ensemble observations made over the past 50 years.Objective 5 has made considerable progress, most importantly through the development of a completely new approach to mass photometry. Until now, mass photometry was almost exclusively performed on glass surfaces, meaning that essentially everything present in solution produced a signal. This meant that we could only study systems consisting of highly purified proteins. Inspired by nature's use of lipid bilayer membranes, we used a related approach to passivate and at the same time selectively activate glass surfaces. In this way, we could remove the need for purification, making mass photometry applicable to a much broader range of systems. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Our achievements within objective one shed new light on the importance of how proteins interact with themselves to form active constructs, and the (dis)assembly of these structures can be used both for regulation of function, and differentiation between different organisms. Similarly, our results on how biomolecular filaments are formed have the potential to transform our understanding of biomolecular self-assembly given that they are clearly distinct from the status quo that has been accepted since the early 1960s. Our focus for the remainder of the project will be on achieving objectives two and four, and thereby producing a more holistic view of how protein-protein interactions are involved in both physiological and pathological process. Together with the completion of the remaining sub-objective of objective five, we believe that we can achieve the original goal of the proposal: understanding the physicochemical basis of biological self-assembly at the single molecule level across different species, architectures, length scales and associated functions.