Fluorescence Modulation of Green Fluorescent Protein Using Fluorinated Unnatural Amino Acids

Abstract

The ability to modulate protein function through minimal perturbations to amino acid structure represents an ideal mechanism to engineer optimized proteins. Due to the novel spectroscopic properties of green fluorescent protein, it has found widespread application as a reporter protein throughout the fields of biology and chemistry. Using site-specific amino acid mutagenesis, we have incorporated various fluorotyrosine residues directly into the fluorophore of the protein, altering the fluorescence and shifting the pKa of the phenolic proton associated with the fluorophore. Relative to wild type GFP, the fluorescence spectrum of the protein is altered with each additional fluorine atom, and the mutant GFPs have the potential to be employed as pH sensors due to the altered electronic properties of the fluorine atoms.

Playing with the Molecules of Life

“…193, 194 In addition ncAAs have been incorporated at residue 66 in GFP to alter the fluorescence properties of the protein. 37, 190, 195–198 , and into the coelenterazine binding site of aequorin to dramatically red-shift the bioluminescence of the interaction to afford in vivo bioluminescence reporters in mice. 197 …”

“…In addition to ions, this approach has been employed for the detection of H 2 O 2 with a boronate ncAA, H 2 S with a azidophenylalanine ncAA, and sirtuins using an actylated lysine which can be cleaved by these enzymes . Finally, the p- boronophenylalanine has also been exploited to detect peroxynitrite in mammalian cells in order to monitor the production of this cell-signaling molecule at physiologically relevant concentrations. , In addition ncAAs have been incorporated at residue 66 in GFP to alter the fluorescence properties of the protein, ,,− and into the coelenterazine binding site of aequorin to dramatically red-shift the bioluminescence of the interaction to afford in vivo bioluminescence reporters in mice …”

Photoinduced Proton Transfer of GFP-Inspired Fluorescent Superphotoacids: Principles and Design

“…It is notable that other studies on the modified GFP-core chromophore in a protein or solution environment have shed more light on the influence of structural flexibility and EWGs on photoacidity. For instance, GFP with a fluorinated chromophore has a lower p K a and p K a * than the nonfluorinated GFP, and the photoacidity increase as well as spectral changes exhibit the site dependence of fluorination. , Such engineered GFP derivatives have found important applications in live-cell imaging of RNAs and protein-binding fluorogenic dyes for live-cell imaging . When outside of the protein matrix, the difluorinated p -HBDI is more photoacidic than p -HBDI but less than the conformationally locked difluorinated p -HBDI. , …”

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

“…The utility of unnatural amino acids to explore and/or alter the chemical underpinnings of various proteins has resulted in a wide array of chemically diverse UAAs. For instance, numerous UAAs have been incorporated site-specifically into the chromophore of GFP, replacing the tyrosine at site 66 (Reddington et al, 2013;Young, Jockush et al, 2011;Wang et al, 2003Wang et al, , 2012Chang et al, 2019;Villa et al, 2017). This work has resulted in a range of protein constructs with altered photophysical properties: most notably different fluorescent properties.…”