Adenosine triphosphate (ATP) functions not only as the universal energy currency of the cell but also as a key allosteric modulator of protein structure and activity. Although ATP-binding sites are well characterized in many enzymes, the proteome-wide structural consequences of ATP variation in plants remain largely unexplored. Here, we employed limited proteolysis mass spectrometry (LiP‑MS) to systematically examine ATP-dependent conformational changes in the root proteome of Medicago truncatula.
Root tissue extracts were treated with three defined concentrations of ATP to mimic physiologically relevant energetic states. Samples were subjected to controlled proteolysis using proteinase K under native conditions followed by tryptic digestion and liquid chromatography ion mobility tandem mass spectrometry (LC-IM-MS/MS) analysis using data independent (DIA) acquisition. Differential peptide protease accessibility was used as a readout to detect conformational or interaction changes induced by ATP.
Across the ATP gradient, LiP-MS identified a set of proteins exhibiting concentration-dependent changes in proteolytic susceptibility, indicating ATP-mediated conformational changes. Functional enrichment analysis revealed an overrepresentation of proteins involved in primary metabolism, mitochondrial energy production, and redox balance. Several kinases, helicases, and chaperones also displayed altered accessibility profiles, suggesting ATP influences both catalytic activity and protein–protein interaction networks.
Notably, in addition to known ATP-binding enzymes, multiple uncharacterised proteins demonstrated consistent ATP-responsive structural signatures. Mapping of significant LiP sites to predicted tertiary structures highlighted localized rearrangements within nucleotide-binding, catalytic, or regulatory domains. These findings expand the known repertoire of ATP-sensitive proteins in plants and reveal potential new layers of metabolic and signalling regulation.
This study demonstrates the value of LiP-MS as a powerful approach for capturing energy-dependent proteome remodelling in complex plant systems. By integrating structural and semiquantitative proteomics, our results provide new insight into how ATP availability dynamically shapes protein conformation, interaction networks, and ultimately, cellular physiology in roots. These data establish a foundation for future research linking energy signalling to growth and stress adaptation in plants.