Dissecting In Vivo Cross-Linker Performance: Insights into Structural Dynamics and Molecular Rigidity

Understanding protein structure and dynamics is crucial to elucidating their biological functions. Recent advancements in experimental techniques and AI-driven predictions, such as AlphaFold, have significantly enhanced our ability to determine protein structures. However, most methods are based on in vitro studies, which often fail to capture the complexities of protein behavior in vivo. This study focuses on in vivo chemical cross-linking mass spectrometry (XL-MS) as a promising approach to investigate protein structures within their native cellular environments. We systematically evaluate the performance of four commonly used cross-linkers, analyzing their structural characteristics, like length and dihedral angles, as well as the resulting thermodynamic and kinetic behaviors. By combining extensive in vivo XL-MS data with all-atom molecular dynamics simulations, we elucidate the relationship between cross-linker structure and their effectiveness in probing protein dynamics. The results reveal the relationship between the flexibility of cross-linkers and their structural characteristics and further elucidate that more rigid cross-linkers are able to capture more protein dynamic information, especially the dynamics of disordered regions. This study provides critical insights into cross-linker selection and design, offering a framework for future in vivo XL-MS applications aimed at advancing our understanding of protein dynamics and interactions in living cells.