Selective dye-labeling of newly synthesized proteins in bacterial cells.
We describe fluorescence labeling of newly synthesized proteins in Escherichia coli cells by means of Cu(I)-catalyzed cycloaddition between alkynyl amino acid side chains and the fluorogenic dye 3-azido-7-hydroxycoumarin. The method involves co-translational labeling of proteins by the non-natural amino acids homopropargylglycine (Hpg) or ethynylphenylalanine (Eth) followed by treatment with the dye. As a demonstration, the model protein barstar was expressed and treated overnight with Cu(I) and 3-azido-7-hydroxycoumarin. Examination of treated cells by confocal microscopy revealed that strong fluorescence enhancement was observed only for alkynyl-barstar treated with Cu(I) and the reactive dye. The cellular fluorescence was punctate, and gel electrophoresis confirmed that labeled barstar was localized in inclusion bodies. Other proteins showed little fluorescence. Examination of treated cells by fluorimetry demonstrated that cultures supplemented with Eth or Hpg showed an 8- to 14-fold enhancement in fluorescence intensity after labeling. Addition of a protein synthesis inhibitor reduced the emission intensity to levels slightly above background, confirming selective labeling of newly synthesized proteins in the bacterial cell.
Beatty
Kimberly E
KE
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Xie
Fang
F
Wang
Qian
Q
Tirrell
David A
DA
D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry; D001386:Azides / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D003300:Copper / Q000737:chemistry; D003374:Coumarins / Q000737:chemistry*; D003500:Cyclization; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D007202:Indicators and Reagents; D015394:Molecular Structure
C507184:3-azido-7-hydroxycoumarin; D001386:Azides; D001426:Bacterial Proteins; D003374:Coumarins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; C408243:p-ethynylphenylalanine; C057438:barstar protein, Bacillus amyloliquefaciens; D003300:Copper; D000409:Alanine