PRE2023-Ultradynamic protein assemblies and network channeling (PID2022-141460NB-I00)

A classical view sustains that sequential biochemical reactions can be efficiently executed by proteins in a crowded intracellular environment, but some evidence suggests that these enzymes would be somehow organized in protein assemblies. As the existence of stable complexes holding a large number of different enzymes by multiple keylock interactions is rather exceptional, we propose that functional channeling in protein networks is driven by extremely dynamic protein-protein interactions. Based on fluorescence fluctuation analysis, we found that protein pairs performing enzymatic steps of glycolysis display strong spatiotemporal coincidence compared to unrelated proteins acting in other pathways. Notably, spatiotemporal coincidence was inversely correlated with biochemical distance within the glycolytic pathway. Here we will analyze a large number of proteins belonging to different paradigmatic pathways and networks by proximity labeling. Next, we will apply fluorescence-lifetime data to raster-image correlation spectroscopy to determine the spatiotemporal coincidence as an orthogonal approach. We also propose to dissect the molecular determinants underlying protein flock formation, with special attention to quinary structural properties. Finally, we will use the armamentarium offered by the latest developments in molecular and cell biology to study the relevance of protein flock identity in the efficiency of paradigmatic pathways and networks.