X-ray footprinting mass spectrometry (XFMS) is a cutting-edge structural biology technique that utilizes broadband X-rays for in situ hydroxyl radical labeling, enabling the detailed mapping of protein interactions and conformation in solution. While XFMS offers crucial structural insights into biomolecules, the complex landscape of protein conformational dynamics demands more comprehensive approaches at high spatial and temporal resolution. At the advanced light source’s XFMS facility, we developed a hybrid data collection approach that integrates fluorescent spectroscopy with XFMS to simultaneously understand the time evolution of both local protein interactions and global conformational changes. Here, we report the design of the system featuring rapid mixing inline to the optofluidic environments and an automated data collection platform to produce robust time-resolved spectroscopy-XFMS hybrid data. We monitored the interaction of the SpyCatcher-SpyTag split protein system during the formation of the covalent isopeptide bond. Our results demonstrate the capability of our new system to resolve the time frame of the global SpyTag-SpyCatcher binding interaction via FRET, while providing detailed structural dynamics at the residue level through side-chain hydroxyl reactivities. We developed and demonstrated a real-time orthogonal toolset that enhances our understanding of protein interactions and dynamics, offering a precise workflow for engineering these interactions.
Gupta et al. (Sun,) studied this question.