Protein Structure Mapping:Unveiling the Mechanism of Diazirine Photo-reactions

Original link: https://communities.springernature.com/posts/protein-structure-mapping-unveiling-the-mechanism-of-diazirine-photo-reactions

Diazirine, a functional group known for its three-membered ring that opens upon irradiation, has been widely used in photo-chemistry. Despite its broad application, the photo-reaction mechanisms of alkyl diazirines, especially their intermediate pathways and preferences towards protein residues, remained poorly understood. Addressing this knowledge gap was crucial for leveraging photo-cross-linking (PXL)’s full potential in protein structure analysis.
In our recent paper, “Dissecting Diazirine Photo-reaction Mechanism for Protein Residue-Specific Cross-linking and Distance Mapping“, published in Nature Communication, we delved into the intricate mechanisms of diazirine photo-reactions. Our goal was to accurately identify the photo-cross-linked protein residue for protein strucure mapping, which has long been accomplished with chemical cross-linkers.

The Motivation: Overcoming Limitations of Existing Methods

PXL using alkyl diazirines offers shortest, and hence, stringent distance constraints, making it a powerful tool for studying protein structures. However, the ambiguity in identifying cross-linked residues has limited its effectiveness compared to chemical cross-linking (CXL). We have built an innovative system that systematically modulates light intensity and irradiation time, allowing for a quantitative evaluation of diazirine photolysis and photo-reaction mechanisms.

Key Findings: Diazo as the Main Intermediate

We proposed four models (Models I to IV) to describe the diazirine photolysis mechanism, focusing on the transformation pathways of diazirine (A) to diazo (B) and carbene (C) intermediates. By comparing the theoretical curves and experimental data, we identified Model II best accouting for the photolysis mechanism for sulfo-SDA, a common alkyl diazirine photo-cross-linker.
Our experiments revealed that the diazo intermediate, rather than carbene, plays a dominant role in alkyl diazirine photolysis. By monitoring the reactant and the production using in-line NMR spectroscopy and MS, we confirmed the sequential generation of diazo and carbene intermediates. This mechanistic insight allows us to fine-tune PXL conditions, enhancing selectivity towards polar residues and improving the accuracy of distance mapping against protein structures.

Application: Preferential Cross-Linking of Polar Residues

Our analysis revealed that an ideal combination of irradiation time and power density, specifically at 100 mW/cm² for 2 minutes, allows the cross-linker to preferetially react with polar residues, including aspartic acid (Asp), glutamic acid (Glu), and tyrosine (Tyr) in proteins. This optimization is crucial for ensuring reproducibility and efficiency in cross-linking experiments. By analyzing the cross-linked products, we observed that the distances between the cross-linked residues are consistent with the known protein structures. Additionally, most cross-linked residues are relatively buried, due to the competive reaction from water.

Conclusion: A Step Forward in Protein Research

Our study has laid the groundwork for more accurate and detailed protein structure analysis. We hope our approach will inspire further research and technological developments, contributing to the advancement of structural proteomics field.