Abstract
Proteins can adopt different conformational states that are important for their biological function and, in some cases, can be responsible for their dysfunction. The essential roles that proteins play in biological systems make distinguishing the structural differences between these conformational states both fundamentally and practically important. Here, we demonstrate that collision-induced unfolding (CIU), in combination with ion mobility-mass spectrometry (IM-MS) measurements, distinguish subtly different conformational states for protein complexes. Using the open and closed states of the β-lactoglobulin (βLG) dimer as a model, we show that these two conformational isomers unfold during collisional activation to generate distinct states that are readily separated by IM-MS. Extensive molecular modeling of the CIU process reproduces the distinct unfolding intermediates and identifies the molecular details that explain why the two conformational states unfold in distinct ways. Strikingly, the open conformational state forms new electrostatic interactions upon collisional heating, while the closed state does not. These newly formed electrostatic interactions involve residues on the loop differentially positioned in the two βLG conformational isomers, highlighting that gas-phase unfolding pathways reflect aspects of solution structure. This combination of experiment and theory provides a path forward for distinguishing subtly different conformational isomers for protein complexes via gas-phase unfolding experiments. Our results also have implications for understanding how protein complexes dissociate in the gas phase, indicating that current models need to be refined to explain protein complex dissociation.