Researchers from Tokyo Medical and Dental University have pioneered a novel technique that sheds light on the intricate world of protein interactions. Published in Protein Science, their study introduces a method combining genetic code expansion (GCE) and site-specific cleavage, enabling the identification and characterization of binding interfaces on interacting proteins.
Proteins, the fundamental building blocks of the human body, often collaborate to drive essential biological processes. However, deciphering the molecular dynamics of these partnerships poses a formidable challenge. The TMDU team's innovative approach involves introducing a non-natural amino acid, pBpa, into a protein using GCE. Subsequent light irradiation induces cross-linking between interacting proteins, facilitating their study. To pinpoint the interaction site on the second protein, a cleavage site named AllocLys-OH is strategically introduced using GCE.
The researchers validated their method by investigating the homophilic interaction of LAMP2A molecules, crucial components of lysosomal membranes involved in chaperone-mediated autophagy. Through a series of crosslinking and cleavage experiments, they unveiled the geometry of the LAMP2A homophilic interaction, suggesting the formation of a trimeric or higher oligomeric structure.
This breakthrough technique holds promise for researchers studying protein-protein interactions, offering a means to characterize binding interfaces and unravel complex protein geometries. Furthermore, its applicability to all mammalian cells opens doors for potential therapeutic applications.