2022, Volume 8, Issue 5-6
Transient and weak protein–protein interactions are essential to many biochemical reactions, yet are technically challenging to study. Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) provides a powerful tool in the analysis of such interactions. Central to this technology are chemical cross-linkers. Here using two transient heterodimeric complexes EIN/HPr and EIIAGlc/EIIBGlc as the model systems, the authors evaluated the effects of two amine-specific homo-bifunctional cross-linkers with different reactivities. They showed previously that DOPA2 (di-ortho-phthalaldehyde with a di-ethylene glycol spacer arm) cross-links proteins 60–120 times faster than DSS (disuccinimidyl suberate). They found that though most of the intermolecular cross-links of either cross-linker are consistent with the encounter complexes (ECs), an ensemble of short-lived binding intermediates, more DOPA2 intermolecular cross-links could be assigned to the stereospecific complex (SC), the final lowest-energy conformational state for the two interacting proteins. The finding suggests that faster cross-linking captures the SC more effectively and cross-linkers of different reactivities potentially probe protein–protein interaction dynamics across multiple timescales.