Grant: Alliance Grants

Extracellular proteins interact to form a vast number of functional complexes and aggregates with important microscopic and large-scale characteristics. The elementary kinetics of protein-protein interactions regulate many vital processes of biomolecular recognition and cellular signaling. The aggregation of proteins can also lead to deadly diseases arising from the misfolding, aggregation, and extracellular deposition of soluble proteins, causing cell death and organ failure. Progress in understanding the factors that govern all pathways of protein assembly and aggregation requires insight into the elementary dynamics of primary protein-protein interactions under biological conditions in solution. Established spectroscopic approaches cannot provide mass-resolved information on aggregation, or capture initial oligomers, which play a critical role in the initiation of disease or resolve the cytotoxic interactions of specific agonists on cell membranes. Progress will require a comprehensive method that can resolve protein-protein rate processes in real-time, under label-free conditions, with sub-monomer mass resolution. Here, we propose a novel approach that employs single-molecule mass photometry in a closed microfluidic system with electrophoretic detection (EPD-iSCAT) to investigate the thermochemical kinetics of individual protein-protein reactions leading to oligomerization and aggregation. Similar time-resolved images of landings on anchored cells will isolate specific protein-membrane interactions that result in cell damage and death. Work in collaboration with a University of Milan structural biologist, Stefano Ricagno, will apply this probe to monitor the elementary kinetics of protein-protein and protein-membrane interactions associated with AL amyloidosis. This work linking protein structure to single-molecule dynamics will engage industrial partners, ScopeSys, a Canadian maker of nanoconfinement instrumentation, and Cardiomix, a leading Milan systems bioanalytical and cardiological diagnosis company. These Alliance partners will form a parallel collaboration that draws value from the instrumental breakthroughs that this work achieves to strengthen their market positions.