New Biarsenical Ligands and Tetracysteine Motifs for Protein Labeling in Vitro and in Vivo: Synthesis and Biological Applications

@article{citeulike:1046361, abstract = {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 (\~{}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 -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.}, address = {Contribution from the 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}, author = {Adams, S. R. and Campbell, R. E. and Gross, L. A. and Martin, B. R. and Walkup, G. K. and Yao, Y. and Llopis, J. and Tsien, R. Y.}, citeulike-article-id = {1046361}, doi = {10.1021/ja017687n}, journal = {J. Am. Chem. Soc.}, keywords = {flash, reash, tetracysteine}, month = {May}, number = {21}, pages = {6063--6076}, posted-at = {2008-09-23 17:10:18}, priority = {2}, title = {New Biarsenical Ligands and Tetracysteine Motifs for Protein Labeling in Vitro and in Vivo: Synthesis and Biological Applications}, url = {http://dx.doi.org/10.1021/ja017687n}, volume = {124}, year = {2002} }

TY - JOUR ID - citeulike:1046361 L3 - citeulike-article-id:1046361 N2 - 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 (~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 -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. IS - 21 JF - J. Am. Chem. Soc. AD - Contribution from the 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 EP - 6076 TI - New Biarsenical Ligands and Tetracysteine Motifs for Protein Labeling in Vitro and in Vivo: Synthesis and Biological Applications VL - 124 SP - 6063 KW - flash KW - reash KW - tetracysteine AU - Adams, SR AU - Campbell, RE AU - Gross, LA AU - Martin, BR AU - Walkup, GK AU - Yao, Y AU - Llopis, J AU - Tsien, RY PY - 2002/05/29/ UR - http://dx.doi.org/10.1021/ja017687n ER -