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.
The biarsenical-tetracysteine tagging system was the first of (and inspiration for) the now numerous methods for site-specifically labeling proteins in living cells with small molecules such as fluorophores. This historical recollection describes its conception and the trial-and-error chemical development required to become a versatile technique.
Interactions between proteins or protein domains can be imaged by fusing them to cyan (CFP) and yellow (YFP) mutants of Green Fluorescent Protein and observing fluorescence resonance energy transfer (FRET). For example, fusions of CFP, calmodulin, a calmodulin-binding peptide, and YFP are transfectable emission-ratioing Ca2+ indicators with many uses. They are highly suitable for twophoton excitation at 770-810 nm, even at video rates. Applications not possible with previous indicators include detection of submicroscopic domains of Ca2+ by fusion of the indicators to key proteins, and dynamic imaging of Ca2+ in transgenic animals. YFPs have been improved as FRET acceptors by reducing their sensitivity to pH changes. Many other applications of GFP mutants to detect fluctuating protein-protein interactions are underway.A synthetic alternative to GFPs for protein tagging arises from the ability of membrane-permeant biarsenical dyes to seek out and light up alpha-helical Cys-Cys-X-X-Cys-Cys motifs placed in recombinant proteins in live cells. The new system is much smaller than GFP (6 residues vs. 238), can label internal domains not just N- and C-terminii, and offers novel readouts (e.g. red emission peaking > 600 nm) and better temporal control of the labeling.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.