Mammalian cell–based optimization of the biarsenical- binding.pdfVIP

L E T T E R S Mammalian cell–based optimization of the biarsenical- binding tetracysteine motif for improved fluorescence y and affinity g o l o n h Brent R Martin1,2, Ben N G Giepmans1,3,4, Stephen R Adams1 Roger Y Tsien1,5,6 c e t o i b e r Membrane-permeant biarsenical dyes such as FlAsH and The earliest designs of tetracysteine sequences were intended to u t a ReAsH fluoresce upon binding to genetically encoded tetra- encourage a-helicity under the assumption that the biarsenical dye n / cysteine motifs expressed in living cells1,2, yet spontaneous would ideally fit into the i, i+1, i+4, and i+5 positions of an a-helix1. m o c nonspecific background staining can prevent detection of With these sequences, nonspecific biarsenical background staining was . e 2,3 r weakly expressed or dilute proteins . If the affinity of the estimated to equal the fluorescence of labeled protein of several u t 2,3 a tetracysteine peptide could be increased, more stringent micromolar . Partial reduction of the background fluorescence was n . dithiol washes should increase the contrast between specific achieved by increasing the concentration of the dithiols 1,2-ethane- w w w and nonspecific staining. Residues surrounding the tetra- dithiol (EDT) or 2,3-dimercaptopropanol (BAL) in washes to remove / / : cysteine motif were randomized and fused to GFP, retrovirally thiol-dependent background or by including nonfl