Residue choice defines efficiency and influence of bioorthogonal protein modification via genetically encoded strain promoted Click chemistry.

GFP and a FRET compatible dye were used to assess the influence of genetically encoded aryl azide positioning on Click chemistry-based protein conjugation. While modification efficiency of the sampled mutants using a strain promoted reaction varied by as much as ∼10 fold, there was no simple correlation with accessibility of the aryl azide on GFP's surface. One labeled GFP mutant (Gln204AzPhe) exhibited high efficiency FRET (∼90%) and an unprecedented pseudo-Stokes shift of 126 nm.