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 L iving cells by extracellular administration of 4',5'-bis(1,3,2-dithioa rsolan-2-yl)fluor This designed small Ligand is membrane-permeant and nonfluorescent until it binds with high affinity and specificity to th e tetracysteine domain, Such in situ Labeling adds much Less mass than does green fluorescent protein and offers greater versatility in atta chment sites as well as potential spectroscopic and chemical propertie s. This system provides a recipe for slightly modifying a target prote in so that it can be singled out from the many other proteins inside L ive cells and fluorescently stained by small nonfluorescent dye molecu les added from outside the cells.