ABSTRACT
While photo-cross-linking (PXL) with alkyl diazirines can provide stringent distance restraints and offer insights into protein structures, unambiguous identification of cross-linked residues hinders data interpretation to the same level that has been achieved with chemical cross-linking (CXL). We address this challenge by developing an in-line system with systematic modulation of light intensity and irradiation time, which allows for a quantitative evaluation of diazirine photolysis and photo-reaction mechanism. Our results reveal a two-step pathway with mainly sequential generation of diazo and carbene intermediates. Diazo intermediate preferentially targets buried polar residues, many of which are inaccessible with known CXL probes for their limited reactivity. Moreover, we demonstrate that tuning light intensity and duration enhances selectivity towards polar residues by biasing diazo-mediated cross-linking reactions over carbene ones. This mechanistic dissection unlocks the full potential of PXL, paving the way for accurate distance mapping against protein structures and ultimately, unveiling protein dynamic behaviors.