Protein-protein interactions play crucial roles in the regulation of many biological processes, but the identification of dynamic, transient and weak protein-protein interactions in living cells still remains challenging with traditional methodology.

Using genetically encoding photo-crosslinkers to form covalent linkage with any proximal residue is an attractive strategy to address the challenge of capturing protein interactions. However, such non-selective crosslinking complicates mass spectrometric analysis (MS), and artificially leads to false positive interacting partners for protein-protein interactions.  

Recently, scientists from Shanghai Institute of Materia Medica (SIMM) of the Chinese Academy of Sciences (CAS) and Chengdu Institute of Biology of CAS developed a conceptually new approach that genetically encoded, residue-selective photo-crosslinker to capture protein-protein interactions in temporal controlled manner. This study was published in the journal of Chem. 

The novel photo-crosslinker is efficient to capture protein-protein interactions in vitro and living cells, upon UV light activation, providing lysine selective crosslinking sites and predictable crosslinked peptides. The MS identification of the crosslinked peptides also severs as direct evidence for protein-protein interactions, facilitating mass spectrometric analysis and mapping accurate interaction interfaces.  

More importantly, this photo-crosslinker enables the capture of elusive enzyme-substrate interaction with directly interacting lysine and validation of acetylation site of the substrate, which could exclude false positive substrates for enzymes and directly provide the post-translation modification site of substrates, thus, enabling the specificity, accuracy and reliability for enzyme-substrate interactions.  

Combining the advantages of temporal control and the unique ability of residue selectivity, this genetically encoded photo-crosslinker will be a powerful tool for the identification of the dynamic, weak, and transient protein interactions in living cells with specificity, accuracy, and reliability, thus, leads to a better understanding of biological processes and pathophysiology.  

The concept that photo-crosslinking with residue-selectivity may lead to new findings of photo-crosslinkers to expand the repertoire of current protein crosslinking technology and interactive proteomics.  

The current approach should be of general interest to researchers who apply crosslinking strategy to protein studies, such as proteomics, drug target identification and validation, structural biology research and signaling pathway. It will have an effect on a wide range of fields involving protein-protein and ligand-receptor interactions such as basic biological studies, interactive proteomics, protein structural studies and chemical biology.  

Schematic illustration of the genetically encoded residue-selective photo-crosslinker to capture and identify protein interactions in living cells (Image by HU Wei)