Photo-Activated Crosslinking Mass Spectrometry for Studying Biomolecular Interactions

Abstract

The structural and mechanistic analysis of biomolecular interaction is important for understanding the molecular basis of a wide range of biological phenomenon. Relatively weak interactions between functional molecules may play crucial roles in regulating highly networked and dynamically controlled biological systems. This type of interaction, however, is more challenging to study because of its transient nature. As a general solution to the problem, we propose the use of chemical crosslinking in combination with mass spectrometry and apply it to protein-protein interactions. Ultraviolet-activatable crosslinker molecules that are incorporated to the protein of interest can be utilized to capture transient interactions under a physiological condition by forming covalent bonds between interacting molecules in close proximity via carbene chemistry. The crosslinked sample is then enzymatically digested and analyzed by liquid chromatography-mass spectrometry. High mass resolution analysis ensures identification of crosslinked peptide species and enables spatial mapping onto available structural models obtained from crystallography or NMR spectroscopy. The proposed methodology is demonstrated with a model system of cytochrome c and its oxidase, where we find multiple binding modes and explore their possible role in controlling enzymatic activity.