New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications.
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.
Adams
Stephen R
SR
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.
Campbell
Robert E
RE
Gross
Larry A
LA
Martin
Brent R
BR
Walkup
Grant K
GK
Yao
Yong
Y
Llopis
Juan
J
Tsien
Roger Y
RY
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
C113276:4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D011994:Recombinant Proteins; D003545:Cysteine