A Multidimensional Approach to Explore the Use of a Small Heterobifunctional Crosslinker based on a Metabolite of the Kynurenine Pathway

This study describes the use of a new small heterobifunctional crosslinker for crosslinking of proteins (e.g. lysozyme). This crosslinker is based on 3-hydroxy anthranilic acid (3HAA) that is part of the kynurenine pathway of the degradation of Tryptophan. 3HAA is found in enhanced amounts in disease states in the human body. Small crosslinkers capture interacting protein interfaces better, while the larger ones are more useful for identifying interacting partners. The new crosslinker described here, functions presumably via a ‘long lived’ transient, leading to enhanced rate of intermolecular crosslinking, which is otherwise difficult to achieve. It contains a photo labile azido group and an amine reactive N-hydroxysuccinimide (NHS) group. Successful crosslinking in two steps (incubation followed by photolysis (366 nm, 6W UV lamp) , has been confirmed using SDS-PAGE, ESI-MS/MS, and bioinformatics analysis via StavroX 3.6.0.1 Docking followed by molecular dynamics simulation studies, have provided detailed structural insights into the ‘dimer’ formation of lysozyme. Identical conclusions have been obtained, using two different software, and providing a more refined 3D view of the interfaces during protein-protein interactions.