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

Abstract

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.

Directed Expression of Tracheal Antimicrobial Peptide as a Treatment for Bovine-Associated Staphylococcus Aureus-Induced Mastitis in Mice

“…Thus, we can visualize it as a paint bucket with pigment. The outer wall of the bucket effectively prevents its influence on the spatial structure of the target protein ( 54 – 56 ). The GFP does not require any reaction substrate and cofactor, has no species restriction, and can be expressed in a variety of biological cells to emit stable fluorescence.…”

Cellular and Biophysical Applications of Genetic Code Expansion

“…Recent research focuses on engineering fluorescent proteins capable of labeling multiple biological targets simultaneously, aiming to achieve enhanced brightness and fluorescence. A critical approach in this research involves the incorporation of ncAAs using GCE technology. ,− An exemplary case is the GFP, which features a chromophore formed by the condensation of Tyr, Gly and Ser . This condensation reaction establishes a new bond among these three amino acids, leading to the formation of a conjugated double bond system integrated with the aromatic ring of Tyr.…”

“…Researchers have developed a variety of fluorescent proteins with distinct chromophores by incorporating ncAAs such as 81 , 94 , 102 , 109 , 119 , and 120 (Table ). ,− ,, The Schultz group demonstrated the effectiveness of GCE technology by replacing GFP’s chromophore Tyr with ncAAs 17 , 96 , and 108 , achieving fluorescent proteins with diverse emission spectra. Specifically, the ncAA 17 mutant exhibited emission at 442 nm, the ncAA 96 mutant at 498 nm, and the ncAA 108 mutant at 510 nm .…”