Structural and spectrophotometric investigation of two unnatural amino‐acid altered chromophores in the superfolder green fluorescent protein

The spectrophotometric properties of the green fluorescent protein (GFP) result from the post‐translationally cyclized chromophore composed of three amino acids including a tyrosine at the center of the β‐barrel protein. Altering the amino acids in the chromophore or the nearby region has resulted in numerous GFP variants with differing photophysical properties. To further examine the effect of small atomic changes in the chromophore on the structure and photophysical properties of GFP, the hydroxyl group of the chromophore tyrosine was replaced with a nitro or a cyano group. The structures and spectrophotometric properties of these superfolder GFP (sfGFP) variants with the unnatural amino acids (UAAs) 4‐nitro‐l‐phenylalanine or 4‐cyano‐l‐phenylalanine were explored. Notably, the characteristic 487 nm absorbance band of wild‐type (wt) sfGFP is absent in both unnatural amino‐acid‐containing protein constructs (Tyr66pNO2Phe‐sfGFP and Tyr66pCNPhe‐sfGFP). Consequently, neither Tyr66pNO2Phe‐sfGFP nor Tyr66pCNPhe‐sfGFP exhibited the characteristic emission of wt sfGFP centered at 511 nm when excited at 487 nm. Tyr66pNO2Phe‐sfGFP appeared orange due to an absorbance band centered at 406 nm that was not present in wt sfGFP, while Tyr66pCNPhe‐sfGFP appeared colorless with an absorbance band centered at 365 nm. Mass spectrometry and X‐ray crystallography confirmed the presence of a fully formed chromophore and no significant structural changes in either of these UAA‐containing protein constructs, signaling that the change in the observed photophysical properties of the proteins is the result of the presence of the UAA in the chromophore. This work explores the structural and spectrophotometric implications of replacing a tyrosine residue in the chromophore of a fluorescent protein with two different unnatural amino acids.