Developing an efficient Tyrosine Reactive Crosslinker (TRCL) for protein crosslinking

Chemical crosslinking is a well-established strategy to probe the structures of proteins and decipher protein-protein interactions. The construction of crosslinkers with distance constraints adapted into the design gives a three-dimensional structure of the protein obtained from the crosslinked peptides. Currently, most crosslinkers are designed with reactive groups that target only a few amino acid residues, such as lysine, arginine, cysteine, and glutamic/aspartic acid. This narrow set of amino acid targets restricted the potential of chemical crosslinking strategy capturing protein-protein interactions and protein structure analysis. The demand for new crosslinkers targeting other residues is immense, but unfortunately, the chemistry associated with these residues is not very suitable to conjugate with reactive functional groups. Targeting the phenolic side chain of a tyrosine residue in aqueous media is challenging. This work demonstrates a new 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) based tyrosine reactive crosslinker, TRCL and its efficiency in proteins and cell lysate. In this report, we introduced a PTAD-based tyrosine reactive crosslinker which is stable in native biological settings. Proof of concept studies was demonstrated in several standard proteins, such as Myoglobin, bovine albumin (BSA), and viral spike protein from SARS-CoV-2. The large-scale studies were demonstrated in HeLa cell lysate. Expanding the scope of chemical crosslinkers to target aromatic amino acid residues like tyrosine would help uncover new protein-protein interactions that have biological significance and provide a valuable structural biology perspective utilizing mass spectrometry.