Specific covalent labeling of recombinant protein molecules inside live cells.
Recombinant proteins containing four cysteines at the i, i + 1, i + 4, and i + 5 positions of an alpha helix were fluorescently labeled in living cells by extracellular administration of 4',5'-bis(1,3, 2-dithioarsolan-2-yl)fluorescein. This designed small ligand is membrane-permeant and nonfluorescent until it binds with high affinity and specificity to the tetracysteine domain. Such in situ labeling adds much less mass than does green fluorescent protein and offers greater versatility in attachment sites as well as potential spectroscopic and chemical properties. This system provides a recipe for slightly modifying a target protein so that it can be singled out from the many other proteins inside live cells and fluorescently stained by small nonfluorescent dye molecules added from outside the cells.
Griffin
B A
BA
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093-0647, USA.
Adams
S R
SR
Tsien
R Y
RY
D000595:Amino Acid Sequence; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002463:Cell Membrane Permeability; D002470:Cell Survival; D003545:Cysteine / Q000737:chemistry*; D004735:Energy Transfer; D019855:Ethylene Glycol; D005452:Fluoresceins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008024:Ligands; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014162:Transfection
C113276:4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; D002147:Calmodulin; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008024:Ligands; D008164:Luminescent Proteins; D009942:Organometallic Compounds; D010455:Peptides; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D019855:Ethylene Glycol; D003545:Cysteine