Dynamic 3D proteomes reveal protein functional alterations at high resolution in situ

Area of Science:

• Proteomics
• Structural Biology
• Systems Biology
• Biochemistry

Background:

• Many biological processes rely on protein modifications and interactions, not just protein levels, which are often missed by standard proteomics.
• Understanding these dynamic functional alterations is crucial for deciphering cellular regulation and disease mechanisms.

Purpose of the Study:

• To introduce and validate a global protein structural readout technique, limited proteolysis-mass spectrometry (LiP-MS), for detecting functional protein changes.
• To demonstrate the application of LiP-MS in complex biological systems, including bacteria and yeast, under various conditions.

Main Methods:

• Limited proteolysis-mass spectrometry (LiP-MS) was employed to generate a global protein structural readout.
• The technique was applied to bacterial and yeast models undergoing nutrient adaptation and stress responses, respectively.
• Structural changes were visualized as 'structural barcodes' and analyzed for functional insights.

Main Results:

• LiP-MS successfully detected functional alterations including enzyme activity changes, phosphorylation, protein aggregation, and complex formation.
• The method provided high resolution, identifying changes at specific functional sites like binding and active sites.
• LiP-MS identified a novel fructose-1,6-bisphosphate regulatory mechanism for glucose uptake in E. coli and corroborated known pathway alterations.

Conclusions:

• LiP-MS significantly expands the coverage of classical proteomics by detecting functional protein dynamics.
• The technique generates mechanistic hypotheses and facilitates the identification of novel regulatory mechanisms.
• LiP-MS enables in situ structural systems biology, paving the way for a deeper understanding of cellular functions.