Direct observation of biomolecular mechanisms by single molecule mass measurement

We will develop a novel experimental platform aimed at directly visualising the biomolecular mechanisms driving cellular processes. In contrast to the state-of-the-art, our approach aims at identifying and quantifying biomolecular interactions in terms of both energetics and kinetics by following the activity of individual macromolecular machines. To achieve this, we will combine mass photometry – single molecule mass measurement by light scattering – with surface functionalisation and lithography. We will drive, motivate and optimise these technological efforts by simultaneous application to a long-standing puzzle: the molecular details of ubiquitin chain priming and extension, and the tug-of-war with deubiquitination enzymes. Despite the fundamental importance of these processes to cellular function and regulation, and the emergence of next generation therapeutics using targeted protein degradation, our mechanistic understanding remains poor, largely as a consequence of the technological challenges associated with ensemble-based experimental approaches that dominate (de)ubiquitination studies. Our innovative approach of dedicated trapping and activation strategies at the single molecule level will facilitate detailed characterisation of priming and extension rates from a variety of biologically significant ubiquitin ligase complexes. Our advancements create a comprehensive, quantifiable approach for characterizing other ubiquitin ligases, and a general platform transforming our approach to study biomolecular function and regulation.