Position‐specific incorporation of dansylated non‐natural amino acids into streptavidin by using a four‐base codon

Abstract

Novel non-natural amino acids carrying a dansyl £uorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG fourbase codon in an Escherichia coli in vitro translation system. 2,6-Dansyl-aminophenylalanine (2,6-dnsAF) was found to be incorporated into the protein more e⁄ciently than 1,5-dansyllysine, 2,6-dansyl-lysine, and 1,5-dansyl-aminophenylalanine. Fluorescence measurements indicate that the position-speci¢c incorporation of the 2,6-dnsAF is a useful technique to probe protein structures. These results also indicate that well-designed non-natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

A Genetically Encoded Fluorescent Amino Acid

“…To demonstrate the utility of dansylamino acid 1 in studies of protein structure and function, it was used as an environmentally sensitive reporter of protein unfolding (13,17,18). hSOD has a Greek-key ␤-barrel fold with an external loop and short helical regions at one barrel face near the active site, which contains the copper and zinc ions (27).…”

“…However, all of these approaches rely on the introduction of specific dye-acceptor motifs, peptides or proteins that restrict the sites of modification and can introduce undesired structural perturbations into the protein to be analyzed. Finally, in vitro mutagenesis with suppressor tRNAs chemically modified with fluorescent amino acids can be used to label proteins with fluorescent probes site-specifically, but this method is largely limited to in vitro systems affording small quantities of proteins (13)(14)(15)(16).…”

Genetically engineered avidins and streptavidins

“…There would seem to exist significant differences in the biotin-binding capacity of resultant mutants when non-natural amino acids are incorporated into streptavidin. For example, substitution in positions 85 and 87 has always yielded inactive streptavidin in respect of biotin binding [116][117][118].…”

Development of a Novel PPARγ Ligand Screening System Using Pinpoint Fluorescence-Probed Protein

“…In this study, we developed a novel ligand assay using the four-base codon fluorescence-probing method, in which a fluorescence-probed amino acid is inserted into a chosen position of a protein without denaturation, [8][9][10][11] in screening PPAR ligands. In the assay developed, sterol receptor co-activator-1 (SRC-1), which plays a critical role in insulin sensitivity and diabetes by modulating for PPAR activity, 12) was used as a fluorescence-probed co-activator.…”