Dynamic 3D proteomes reveal functional pathway alterations in situ

Many biological processes are regulated via molecular events, such as intermolecular interactions and chemical modifications, which do not affect protein levels and escape detection in classical proteomic screens. Reasoning that these events affect protein structure, we propose here that a global protein structural readout could detect many functional alterations simultaneously and in situ. We test this idea using limited proteolysis-mass spectrometry (LiP-MS), which monitors structural changes in thousands of proteins within a native-like environment. In bacteria undergoing nutrient adaptation and in yeast responding to acute stress, this global structural readout captures enzyme activity changes, allosteric regulation, phosphorylation, protein aggregation and protein complex formation, with sufficiently high resolution to identify alterations of single functional sites such as active site occupancy and binding interfaces. Comparison with prior knowledge, including flux, phosphoproteomics and metabolomics data, shows that LiP-MS captures most known enzyme functional alterations and suggests novel metabolite-protein binding events, enabling identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics screen coverage and generates mechanistic hypotheses, thus better linking holistic and reductionist approaches and paving the way for a new in situ structural systems biology.