title: New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications.
abstract: We recently introduced a method (Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269-272 and Griffin, B. A.; Adams, S. R.; Jones, J.; Tsien, R. Y. Methods Enzymol. 2000, 327, 565-578) for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is an noncysteine amino acid, is genetically fused to or inserted within the protein, where it can be specifically recognized by a membrane-permeant fluorescein derivative with two As(III) substituents, FlAsH, which fluoresces only after the arsenics bind to the cysteine thiols. We now report kinetics and dissociation constants ( approximately 10(-11) M) for FlAsH binding to model tetracysteine peptides. Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa = Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed alpha-helix. Many analogues of FlAsH have been synthesized, including ReAsH, a resorufin derivative excitable at 590 nm and fluorescing in the red. Analogous biarsenicals enable affinity chromatography, fluorescence anisotropy measurements, and electron-microscopic localization of tetracysteine-tagged proteins.
authors.affiliation: Department of Pharmacology, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, and Biomedical Sciences Program, University of California, San Diego, La Jolla, California 92093-0647, USA.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Adams
authors.forename: Robert E
authors.initials: RE
authors.lastname: Campbell
authors.forename: Larry A
authors.initials: LA
authors.lastname: Gross
authors.forename: Brent R
authors.initials: BR
authors.lastname: Martin
authors.forename: Grant K
authors.initials: GK
authors.lastname: Walkup
authors.forename: Yong
authors.initials: Y
authors.lastname: Yao
authors.forename: Juan
authors.initials: J
authors.lastname: Llopis
authors.forename: Roger Y
authors.initials: RY
authors.lastname: Tsien
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D016880:Anisotropy; D003545:Cysteine / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005452:Fluoresceins / Q000737:chemistry*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry*; D005470:Fluorometry; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009942:Organometallic Compounds / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
chemical_list: C113276:4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D011994:Recombinant Proteins; D003545:Cysteine