A genetically encoded fluorescent amino acid.
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.
Summerer
Daniel
D
Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, SR202, La Jolla, CA 92037, USA.
Chen
Shuo
S
Wu
Ning
N
Deiters
Alexander
A
Chin
Jason W
JW
Schultz
Peter G
PG
D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism; D018389:Codon, Nonsense / Q000378:metabolism; D003619:Dansyl Compounds / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D005815:Genetic Code; D006801:Humans; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013482:Superoxide Dismutase / Q000737:chemistry / Q000378:metabolism
C512587:2-amino-3-(5-(dimethylamino)naphthalene-1-sulfonamide)propanoic acid; D000596:Amino Acids; D018389:Codon, Nonsense; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D012343:RNA, Transfer; D013482:Superoxide Dismutase; D000604:Amino Acyl-tRNA Synthetases; D000409:Alanine