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.
Highlights
Fluorescent protein biosensors have become widely utilized to monitor cellular activity in real-time via interactions of analytes with the protein [1,2,3]
Due to the presence of this conserved tyrosine residue at position 66 within the fluorophore, the fluorescence of green fluorescent protein (GFP) is modulated by the protonation state of the phenol moiety
unnatural amino acid (UAA) wasfor evolved and employed toUAAs introduce these novel aminoacyl-tRNA synthetase (aaRS) that degree of polyspecificity multiple fluorotyrosine was evolved residues into ribonuclease reductase to study the generation of tyrosyl radicals
Summary
Fluorescent protein biosensors have become widely utilized to monitor cellular activity in real-time via interactions of analytes with the protein [1,2,3]. UAAs wasfor evolved and employed toUAAs introduce these novel aaRS that degree of polyspecificity multiple fluorotyrosine was evolved residues into ribonuclease reductase to study the generation of tyrosyl radicals [10,23,24]. Utilizing this of and employed to introduce these residues into ribonuclease reductase to study the generation aaRS, it becomes feasible to incorporate mono-, di-, and trifluorotyrosines into. GFPs and investigate their photophysical relevant pHs. we envision exploiting this aaRS to express a series of and physiological sensing properties utilization as fluorescent protein biosensors. GFPs and investigate their for photophysical and physiological sensing properties for utilization as fluorescent protein biosensors
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