FACS-based selection of tandem tetracysteine peptides with improved ReAsH brightness in live cells.

Non-perturbing and site-specific protein labeling methods are highly desired as they allow researchers to probe complex cellular functions. The biarsenical/tetracysteine labeling system allows in situ fluorescent labeling of intracellular proteins which have been appended with small (12 amino acids) genetically encoded peptide tags. In this work we present the selection of semi-randomized tandem tetracysteine peptides with improved biarsenical (ReAsH) fluorescent brightness (2-fold) relative to a single tetracysteine motif or rationally designed 3-fold tetracysteine repeat. We found that Fluorescence Activated Cell Sorting by direct ReAsH excitation as opposed to FRET-mediated ReAsH excitation was optimal for selecting 3×Tetracysteine peptides with enhanced brightness. The selected multimer-tetracysteine peptides display enhanced properties due to higher order ReAsH/3×Tetracysteine dye stoichiometries as opposed to enhancement of the individual core tetracysteine photophysical properties. In summary, we have isolated new 3×Tetracysteine motifs with improved ReAsH brightness in live cells. These modular tags should provide enhanced contrast for live cell imaging applications where small tag size (4.8 KDa) is a requisite for protein labeling.