Deciphering Diazirine Reaction Mechanism: through Irradiation Modulation for Residue-Specific Distance Restraints in Protein Photo-Crosslinking

The diazirine photoreactive group has been widely used for photo-labeling and photo-cross-linking (PXL) of proteins. Yet, due to the mechanistic complexity of diazirine photo-reaction, site-specific and quantitative analysis of protein PXL data remains challenging. Herein, we have built an in-line photo-reaction monitoring system, capable of systematically varying optical power density and irradiation time. Using this system, we have determined the kinetic parameters of diazirine photo-reactions, and shown that an alkyl diazirine undergoes rearrangement upon irradiation to yield a diazo intermediate. A carbene intermediate is produced from the diazo intermediate upon further irradiation, whereas at a relatively small proportion, it is produced directly from the diazirine. Both diazo and carbene intermediates react with protein side-chains. Significantly, the diazo intermediate preferentially reacts with polar side-chains, exhibiting much higher specificity than the carbene intermediate. As the diazo and carbene intermediates are generated sequentially, modulating irradiation time and optical power density can achieve site- or space- selective PXL results through different mechanisms. Together, we have established a new approach for the kinetic mechanism of photo-reactions, and paved the way for quantitative analysis of PXLs to visualize protein structure and dynamics.