KCS

Characterization of the protein-protein interactions is crucial for the study of a wide variety of biological systems. Numerous research has been reported that various methods are developed to support the identification, characterization, and analysis of protein complexes. Since the protein-protein interactions are easily influenced by post-translational modification (PTM), metal ions, and ion concentration, the development of methods to probe protein-protein interaction induced by environmental factors is needed. Here, we have investigated the ATP effect on the quaternary structure of insulin using multiple biophysical analyses. Firstly, we have conducted the solution small-angle X-ray scattering (SAXS) experiments to understand insulin overall size information, protein association state from their scattering profiles. Then, to identify, characterize, and quantify intermolecular protein-protein interactions in the presence and absence of ATP, we performed crosslinking-mass spectrometry (XL-MS) using two crosslinkers. Finally, we executed molecular docking simulation using our obtained information from the XL-MS regarding residue-residue interaction, and simulated structures were compared to the experimental SAXS profiles to suggest possible protein structures. The experimental methodology using diverse analytical techniques would be highly helpful in understanding the protein-protein interactions.