Exploring the Potential of Site-Selective Labeling on a Green Fluorescent Protein Through Lys–His Linchpin-Directed Modification

Protein-based biosensors require controlled and site-selective functionalization strategies to enable stable and oriented immobilization without compromising protein structure and signal transduction efficiency. We evaluated a chemoselective linchpin-directed modification (LDM) approach targeting Lys–His pairs as a tool for site-specific labeling of the model fluorescent biosensor green fluorescent protein (GFP). LDM molecules with variable spacer lengths were prepared, and a structure-guided computational workflow was implemented to map Lys–His distances on the protein and identify candidate modification sites. Experimental validation by UV-Vis spectroscopy and mass spectrometry demonstrated efficient conjugation and a final degree of labeling close to unity, consistent with single-site modification, with all LDM molecules selectively targeting the same histidine residue (His181), independently of spacer length. Structural analysis revealed that this residue is located within an accessible internal cavity that favors productive interactions with the reactive group. Importantly, the modification preserves GFP fluorescence and pH response, confirming retention of sensing functionality. These results demonstrate that LDM enables selective modification not only of surface residues, but also of structurally guided, non-surface residues. This approach provides the proof of concept of a new, promising strategy for the controlled functionalization and immobilization of protein-based biosensors.