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, 103 (26), 9785-9

A Genetically Encoded Fluorescent Amino Acid

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A Genetically Encoded Fluorescent Amino Acid

Daniel Summerer et al. Proc Natl Acad Sci U S A.

Abstract

The ability to introduce fluorophores selectively into proteins provides a powerful tool to study protein structure, dynamics, localization, and biomolecular interactions both in vitro and in vivo. Here, we report a strategy for the selective and efficient biosynthetic incorporation of a low-molecular-weight fluorophore into proteins at defined sites. The fluorescent amino acid 2-amino-3-(5-(dimethylamino)naphthalene-1-sulfonamide)propanoic acid (dansylalanine) was genetically encoded in Saccharomyces cerevisiae by using an amber nonsense codon and corresponding orthogonal tRNA/aminoacyl-tRNA synthetase pair. This environmentally sensitive fluorophore was selectively introduced into human superoxide dismutase and used to monitor unfolding of the protein in the presence of guanidinium chloride. The strategy described here should be applicable to a number of different fluorophores in both prokaryotic and eukaryotic organisms, and it should facilitate both biochemical and cellular studies of protein structure and function.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Structures of fluorescent amino acid and complex of aminoacyl-tRNA synthetase and natural amino acid used for library design. (A) Structure of dansylalanine (1). (B) Structure of Thermus thermophilus leucyl-tRNA synthetase (LeuRS) active site with the bound inhibitor leucyl-adenylate sulfamoyl analogue (Protein Data Bank ID code 1H3N). Side chains corresponding to the randomized region of E. coli LeuRS are shown as sticks. Protein helices are colored in red, β-strands in yellow, and loops in green.
Fig. 2.
Fig. 2.
Analysis of incorporation efficiency and fidelity of 1 assayed with hSOD-33TAG-His6. (A) Expression of hSOD-33TAG-His6 in the presence (+) and absence (−) of 5 mM 1 with the orthogonal tRNACUALeu5 and LeuRSB8T252A mutant. Protein was detected after Ni-NTA affinity purification and SDS/PAGE by using GelCode Blue staining. Left two lanes, expression with tRNACUALeu5/LeuRSB8T252A from the vector pLeuRSB8T252A. Right two lanes, expression with tRNACUALeu5/LeuRSB8T252A from the modified vector pLeuRSB8T252A′ harboring three copies of tRNACUALeu5 with flanking regions of yeast suppressor tRNA SUP4 under control of the PGK1 promoter. (B) MALDI-TOF MS analysis of Ni-NTA-purified hSOD-33TAG-His6 expressed in the presence of 1 and the orthogonal tRNACUALeu5 and LeuRSB8T252A. m/z = mass to charge ratio; (M+H)+ = protonated molecular ion. The peaks with m/z = 16,575 and m/z = 16,718 do not correspond to hSOD-33TAG-His6 with an endogenous amino acid incorporated at position 33.
Fig. 3.
Fig. 3.
Structure of hSOD (Protein Data Bank ID code 1PU0). Protein is colored in green, and Greek-key 1 is shown in red. Amino acids Gln-16 and Trp-33 of β-strands 2 and 3 and the disulfide bridge between Cys-57 and Cys-146 are highlighted. Copper and zinc ions at the active site are shown as spheres and are colored in orange and gray, respectively.
Fig. 4.
Fig. 4.
Fluorescence spectra of different unfolding states of hSOD containing 1 at position 16 or 33. (A) Fluorescence spectra of hSOD with 1 at position 16 in the presence of different concentrations of GdmCl (■, 0 M; □, 0.5 M; ▴, 1.5 M; ▵, 2.0 M; ●, 3.5 M; ○, 5.0 M). Samples were measured in 50 mM sodium phosphate (pH 7.2) and 100 mM NaCl containing 3 μM hSOD and 2 eq of ZnCl2 and CuSO4, respectively. Spectra were recorded in 1-nm steps at 22°C by using excitation at 340 nm and a bandpass of 5 nm for both excitation and emission. (B) Fluorescence spectra of hSOD with 1 at position 33 conducted under conditions identical to those in A. (C) Fluorescence intensity of 1 at positions 16 and 33 of hSOD at unfolding states of the protein present at different GdmCl concentrations. The fluorescence intensity of spectra shown in A (♦) and B (■) at the wavelength where maximal fluorescence is observed (λmax) is plotted against the GdmCl concentration. (D) Wavelength of maximal fluorescence intensity (λmax) of 1 at positions 16 and 33 of hSOD at unfolding states of the protein present at different GdmCl concentrations. λmax of spectra shown in A (♦) and B (■) is plotted against GdmCl concentration. AU, arbitrary units.

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