Results and Discussion
Selection of an Aminoacyl-tRNA Synthetase with Activity Toward Dansylalanine. Dansylalanine (1, Fig. 1A) was initially chosen by virtue of its small size; relatively large Stokes shift; and the high degree of sensitivity of its emission wavelength and quantum yield to environmental effects such as ligand binding, complex formation, conformational changes, or protein unfolding (14, 17, 18). To encode this amino acid in yeast genetically, a previously reported Escherichia coli amber suppressor tRNA/leucyl-tRNA synthetase pair (tRNACUALeu5/LeuRS) was used. This pair is orthogonal in yeast, i.e., tRNACUALeu5 does not crossreact with any of the endogenous aminoacyl-tRNA synthetases, and LeuRS does not accept any endogenous tRNAs as substrates. This pair has been used to incorporate a number of structurally diverse amino acids into proteins with high fidelities and good efficiencies in yeast (19).
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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, -strands in yellow, and loops in green. |
To alter the amino acid specificity of LeuRS, a library of 107 mutants was generated by randomizing residues Met-40, Leu-41, Tyr-499, Tyr-527, and His-537 in the leucine-binding site. These amino acids form a hydrophobic pocket around one -methyl group of leucine in the x-ray crystal structure of the homologous Thermus thermophilus LeuRS (Fig. 1B) (20). Because this binding pocket largely consists of side-chain atoms, mutations might be expected to accommodate novel amino acid substrates without significant perturbation of the LeuRS polypeptide backbone. To evolve a LeuRS specific for 1, a selection scheme was used in which the codons for Thr-44 and Arg-110 of the gene for the transcriptional activator GAL4 were both converted to amber nonsense codons (TAG) (21). Suppression of these amber codons in the MaV203:pGADGAL4(2TAG) yeast strain leads to production of functional full-length GAL4, which drives expression of genomic HIS3 and URA3 reporter genes. These genes complement histidine and uracil auxotrophy, allowing clones harboring active synthetase mutants that aminoacylate endogenous amino acids or 1 to be positively selected on synthetic dropout (SD) medium (containing dextrose) supplemented with 1 mM 1. Negative selection of synthetases that accept endogenous amino acids is carried out by growth on SD medium lacking 1 but containing 0.1% 5-fluoroorotic acid, which is converted into a toxic product by the URA3 gene product (21, 22).
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