Conclusion
In summary, we have shown that a fluorescent amino acid can be incorporated into proteins in yeast at genetically specified sites in good yield and with high fidelity in response to the nonsense codon TAG. This amino acid should be useful for a large range of in vitro studies of protein structure, function, and bimolecular interactions because of its relatively small size and sensitivity to local environment. For example, it should be possible to use 1 to report directly ligand–antibody-binding interactions, protein conformational changes involved in catalysis or regulation, and as a reporter of protein posttranslational modifications. In addition, it may be possible to extend this approach to amino acids with increased fluorescent quantum yields or longer emission wavelengths, as well as apply this methodology to imaging directly in mammalian cells.
Methods
Synthesis of Dansylalanine (1). Dansyl chloride (2.4 g, 8.9 mmol; Sigma–Aldrich) and Et3N (2.6 ml; Fluka) were dissolved in CH2Cl2 (50 ml; Fisher). The solution was cooled to 0°C, Boc-Dap-OH (2.0 g, 9.8 mmol; Bachem) was added in one portion to the stirred solution, and the mixture was allowed to warm to room temperature. After stirring overnight, the volatiles were removed, and the residue was dissolved in CH2Cl2 (30 ml). Trifluoroacetic acid (15 ml; Aldrich) was added, and the reaction mixture was stirred at room temperature for 4 h. The solvent was removed in vacuo, and the residue was dissolved in H2O (50 ml). The solution was made basic with solid NaOH and then acidified with concentrated HCl until precipitation occurred. The precipitate was collected, washed with H2O and EtOH, and dried in vacuo, yielding dansylalanine (1.75 g, 58% yield) as a slightly yellowish solid. 1H NMR (400 MHz, DMSO):
Evolution of Aminoacyl-tRNA Synthetases for Incorporation of Dansylalanine. Selection was carried out in the presence of 1 mM 1 as described in ref. 19. Three different clones whose growth rate depended on the presence of 1 emerged after three rounds of selection (B8: M40A, L41N, Y499I, Y527G, and H537T; C6: M40G, L41N, Y499I, Y527G, and H537G; and D12: M40G, L41L, Y499V, Y527G, and H537E. B8 additionally had an S497C mutation, presumably introduced by PCR during library design. The T252A mutation was introduced by using the QuikChange method (Stratagene) with primers LRST252AFwd (5'-ACA CTA CCC GCC CGG ACG CGT TTA TGG GTT GTA CCT A-3') and LRST252ARev (5'-TAG GTA CAA CCC ATA AAC GCG TCC GGG CGG GTA GTG T-3').
Vector Construction. Vector pLeuRSB8T252A' was constructed as follows. E. coli tRNACUALeu5 and its flanking sequences were assembled by overlap PCR with the following primers (1Fwd, 5'-ATC CCG ACC GGT AAG CTG CTA GCC TCT TTT TC-3'; 1Rev, 5'-GAA GAA AGA GTA TAC TAC ATA ACA CAT ATA CAA TTG AAA AAG AGG CTA GC-3'; 2Fwd, 5'-GTA TAC TCT TTC TTC AAC AAT TAA ATA GCC CGG ATG GTG GAA TCG GTA GA-3'; 2Rev, 5'-CAG CGC GAA CGC CGA GGG ATT TAG AAT CCC TTG TGT CTA CCG ATT CCA CC-3'; 3Fwd, 5'-TCG GCG TTC GCG CTG TGC GGG TTC AAG TCC CGC TCC GGG TAT TTT TTT GT-3'; 3Rev, 5'-CGG CTC TAG ACA TAA AAA ACA AAA AAA TAC CCG-3'). The 5' and 3' sequences are identical to the flanking sequences of yeast suppressor tRNA gene SUP4, which is known to contain a pol III promoter (25, 26). This combined tRNACUALeu5-SUP4 sequence was digested with AgeI and XbaI and repetitively inserted into the AgeI/NheI sites of vector pLeuRSB8T252A to yield three copies of this gene in one direction in the same vector. Subsequently, a PCR DNA fragment including the 650-bp upstream sequence of yeast PGK1 ORF was inserted into the AgeI/NheI site to introduce an additional pol II promoter for transcription of the tRNA region. Vectors pC1SOD-33TAG-His6 and pC1SOD-16TAG-His6 were constructed as follows. S. cerevisiae strain 208708 was obtained from American Type Culture Collection. This strain produces Cu/Zn SOD from a 2-µm leu2-d plasmid. This plasmid was extracted, and a BamHI restriction fragment of
