Determining protein structure by tyrosine bioconjugation

Exploration of protein structure by its solvent accessible surfaces has been widely exploited in structural biology. Amino acids most commonly targeted for covalent modification of the native folded protein are lysine and cysteine. Here we leveraged an ene-type chemistry targeting tyrosine residues to discriminate those solvent exposed from those buried. We find that 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD) can conjugate the phenolic group of tyrosine in a manner heavily influenced by the orientation of the residue with respect to the protein surface. We developed a strategy to investigate protein structure by analyzing PTAD conjugations with free tyrosine, peptides, and proteins. We found this conjugation-based approach robust, sensitive to shifts in protein structure, and adaptable to a wide range of analytic technologies, including fluorescence, chromatography, or mass spectrometry. These studies show how established tyrosine-specific bioconjugation chemistry can expand the toolkit for applications in structural biology.