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.

虽然使用烷基二氮丙啶的光交联 (PXL) 可以提供严格的距离限制并提供对蛋白质结构的深入了解，但交联残基的明确识别阻碍了数据解释达到与化学交联 (CXL) 所实现的相同水平。我们通过开发一种能够系统调节光强度和照射时间的在线系统来应对这一挑战，该系统可以对二氮丙啶光解和光反应机制进行定量评估。我们的结果揭示了一个两步途径，主要是连续生成重氮和卡宾中间体。重氮中间体优先靶向埋藏的极性残基，其中许多残基由于其有限的反应性而无法用已知的 CXL 探针接近。此外，我们证明，调整光强度和持续时间可以通过使重氮介导的交联反应偏向于卡宾交联反应来增强对极性残基的选择性。这种机械解剖释放了 PXL 的全部潜力，为针对蛋白质结构进行精确距离映射并最终揭示蛋白质动态行为铺平了道路。