Transient Crosslinking Mass Spectrometry: Taking Conformational Snapshots of Proteins

Abstract Protein structural analysis at the very moment of target binding or sensing incoming stimuli sheds light on how protein functions diversely with time or pathological conditions. To understand it, we need to intercept and see the intermediate conformation. Although conventional methods offer high resolution structural analysis, they do not address puzzling dynamic conformational changes. Herein, we developed a transient crosslinking mass spectrometry involving a novel photoreactive crosslinker that can capture intermediate conformers. The designed non-specific reactivity increased the crosslinking site diversity, thereby enhancing the resolution and broadening the scope of mass spectrometric-based structural analysis. A time-resolved crosslinking strategy was developed to take conformational snapshots for calmodulin, an important calcium sensor, and revealed the structural basis of its dynamic conformational response to calcium binding and target interaction. Therefore, the designed transient crosslinking makes short-lived conformers visible, which has the potential to tackle the question how variations in protein’s conformation change functions.