Figure 1. Schematic illustration of the approach for the generation of N-terminal functionalized green fluorescent protein (GFP).

Step 1: generation of internal methionine (Met) free GFP by semi-rational mutagenesis using similar physicochemical amino acids. Step 2: introduction of folding enhancement mutations to rescue the fluorescence activity of internal Met-free native GFP. Step 3: N-terminal functionalization of active GFP by Met residue specific incorporation methodology.