Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 103, Issue 26, Pages 9785-9789Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0603965103
Keywords
molecular evolution; fluorescent probes; genetic code expansion; protein design; unnatural amino acids
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Funding
- Medical Research Council [MC_U105181009] Funding Source: researchfish
- MRC [MC_U105181009] Funding Source: UKRI
- Medical Research Council [MC_U105181009] Funding Source: Medline
- NIGMS NIH HHS [GM 62159, R01 GM062159] Funding Source: Medline
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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-amino3-(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.
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