New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications.

来自 ACS

喜欢 0

阅读量：

164

作者：

SR Adams，RE Campbell，LA Gross，BR Martin，GK Walkup，Y Yao，J Llopis，RY Tsien

展开

摘要：

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.

展开

关键词：

Humans Hela Cells Fluoresceins Organometallic Compounds Cysteine Recombinant Proteins Fluorescent Dyes Electrophoresis, Polyacrylamide Gel Fluorometry Spectrometry, Fluorescence