Prof. Tang's research group at Peking University has made a breakthrough in understanding the photoreaction mechanism of diazirines, commonly used photo-crosslinkers in protein structural analysis. Diazirines, characterized by their unique three-membered ring structure, generate highly reactive intermediates upon exposure to light. These intermediates have broad applications in photochemistry. However, their photoreaction mechanism has long been assumed to primarily involve carbene intermediates, which interact indiscriminately with protein residues. The details of alkyl diazirine reaction mechanism, particularly the existence of other potential intermediates and their preferences towards protein residues, have remained largely unknown. The work from Prof. Tang’s group, now published in Nature Communications (Nat. Commun. 2024, 15, 6060) titled “Dissecting Diazirine Photo-Reaction Mechanism for Protein Residue-Specific Cross-Linking and Distance Mapping”, reveals a nuanced reaction process that can be leveraged to achieve precise identification of protein conjugations sites. Their research demonstrates that the precision of diazirine application in protein structural photolabeling mass spectrometry can match that of traditional chemical crosslinking methods.
Fig. 1 Schematic diagram of diazirine photolysis intermediates and their preferences for protein residues.
Motivation: Overcoming Limitations of Existing Methods
Photo-crosslinking using alkyl diazirines (PXL) provides short-range constraints, making it a powerful tool for studying protein structure. However, ambiguity in identifying crosslinked residue sites has been a major limitation, leaving significant room for improvement. To address this, Prof. Tang and his team developed a novel illumination setup capable of systematically adjusting light intensity and exposure time, allowing for quantitative analysis of diazirine photolysis and photoreaction mechanisms.
Fig.2 Real-time photoreaction setup with online mass spectrometry (MS) monitoring, capable of simultaneously adjusting light power density and photoreaction time.
Discovery: Significance of Diazo Intermediates
In their study, the team proposed four models (I to IV) to describe the photolysis kinetics of diazirines, analyzing the conversion pathways from diazirine (A) to diazo (B) and carbene (C) intermediates. By comparing theoretical curves with experimental data, they identified model II as the best fit for explaining the photolysis mechanism of sulfo-SDA, a commonly used alkyl diazirine photo-crosslinker.
Their experiments revealed that diazo intermediates, rather than carbene intermediates, play a dominant role in alkyl diazirine photolysis. Kinetic curves of reactants and products confirmed the sequential formation of diazo and carbene intermediates. Kinetic analysis showed that by fine-tuning light exposure conditions, the selectivity towards polar residues could be enhanced, improving the precision of protein structural mass spectrometry analysis.
Fig.3 In the photolysis process described by Model II, diazo and carbene intermediates are generated sequentially.
Applications: Preferential Crosslinking of Polar Residues
Mechanistic analysis indicated that setting the exposure time and power density to an optimal combination of power and time allowed the crosslinker to preferentially react with polar protein residues, such as aspartic acid (Asp), glutamic acid (Glu), and tyrosine (Tyr). This optimization is crucial for ensuring the reproducibility and efficiency of crosslinking experiments. The crosslinking results showed that the theoretical distances between crosslinked sites matched known protein structures. Additionally, most crosslinked sites were located in relatively hydrophobic regions, suggesting competitive reactions with water.
Fig.4 Preferences of alkyl diazirine for protein residues vary at various combinations of irradiation time and power density.
Conclusion: A Small Step for Protein Structural Mass Spectrometry
This research lays the foundation for more precise and detailed protein structural mass spectrometry analysis. The method will contribute to the elucidation of protein time-resolved dynamics and also the advancement of protein-protein interactions on a proteomic scale.
Prof. Tang Chun, a Boya Distinguished Professor from the College of Chemistry and Molecular Engineering at Peking University, and also a senior investigator at the Peking-Tsinghua Center for Life Sciences, is the corresponding author of the work, while Jiang Yida, a doctoral student at the College of Chemistry and Molecular Engineering, is the first author. Staff scientists at the Analytical Instrumentation Center in Peking University provided significant assistance in MS and NMR measurement and analysis. This work was supported by the National Key Research and Development Program, the National Natural Science Foundation of China, and the Beijing National Laboratory for Molecular Sciences.
Link for the article: https://doi.org/10.1038/s41467-024-50315-y
Prof. Tang’s lab webpage: http://tanglab.pku.edu.cn
