Biosynthetic substitution of tyrosine in green fluorescent protein with its surrogate fluorotyrosine in Escherichia coli

Abstract

Introduction of a fluorine moiety into green fluorescent protein offers an interesting novel spectral variant. The calculated binding energy of fluorotyrosine (F-Tyr) (-8.42 kcal/mol) for tyrosyl tRNA synthetase was moderately higher than that of tyrosine (Tyr) (-8.36 kcal/mol). This result directly correlated with the expression level of F-Tyr containing GFP (38 mg/l), which was comparably higher than that of the parent GFP expression level (34 mg/l). Finally, we generated a model structure for GFP to assess possible interaction in the chromophore of the protein structure, which plays an important role in determining the spectral and folding behaviors of the F-Tyr incorporated GFP variant.

Unnatural Amino Acid Mutagenesis of Fluorescent Proteins

“…To further explore the effects of steric/electronic perturbations to the fluorophore of a fluorescent protein, we previously substituted tyrosine 66 of the fluorophore in green fluorescent protein (GFP) with a series of unnatural amino acids 6–8. Furthermore, others have reported the use of unnatural amino acid mutagenesis to study the spectral properties and folding behavior of GFP,9 and to generate biosensors 10. 11 Herein we report the properties of four GFP (GFP UV , a GFP variant optimized for maximal fluorescence when excited by standard UV light; Clontech) mutants in which the hydroxy substituent of Tyr66 is replaced with boronate, azido, keto, and nitro substituents (Scheme ).…”

Mechanism of the AppABLUF Photocycle Probed by Site-Specific Incorporation of Fluorotyrosine Residues: Effect of the Y21 pKa on the Forward and Reverse Ground-State Reactions

“…The fluorinated derivatives alter both the pK a and the redox potential of the tyrosine but are expected to cause little or no perturbation to the structure. 21 This method has been used previously to study the mechanism of electron and proton transfer in numerous systems including photosystem II, 22 GFP, 23 and ribonucleotide reductase. 21,24-28 In addition, Mathes, Kennis and co-workers have used this approach to study the photoactivation and light state decay of PixD where they observed that replacement of Y8 (the Y21 homolog in PixD) with 2-fluorotyrosine reduced the rate of recovery ~4-fold whereas the 3-fluorotyrosine analog had only a small effect on the recovery rate.…”

Fluorinated Aromatic Amino Acids and its Therapeutic Applications

“…Introducing fluorinated tyrosine in the active of GST shifts the proton location and shows protonated Glutathione [15,33]. Incorporation of 3FTry into GFP increased its stability at higher pH compared to WT-GFP, indicates that 3FTry labeled GFP can be used as a pH sensor [34]. These studies clearly shows that the impact of fluorinated aromatic amino acids on enzymes particularly when they are present in the active site.…”

“…19F-NMR [45], Protein structure and function [15,32], Enzymatic studies [32], Fluorescence [31,34] Histidine 2 2-Fluoro-Histidine, 4-Fluoro-Histidine Protein structure and function [40][41][42], Enzymatic studies [39] * Fluorinated analogues was divided into two parts, Part I analogues has been used in various applications [15] and Some of Part II analogues are commercially available and no significant research studies have been reported. and tendency to form hydrogen bonds and salt bridges at local pH environment [35,36].…”