Localization of GroEL determined by in vivo incorporation of a fluorescent amino acid

BIOORGANIC & MEDICINAL CHEMISTRY LETTERS

卷:21

Article

Charbon, Godefroid²  Wang, Jiangyun³  Brustad, Eric³  Schultz, Peter G.³  Horwich, Arthur L.⁴  Jacobs-Wagner, Christine^2,5  Chapman, Eli¹ 

[1] Scripps Res Inst, Dept Mol Biol, La Jolla, CA 92037 USA

[2] Yale Univ, Dept Mol Cellular & Dev Biol, New Haven, CT 06520 USA

[3] Scripps Res Inst, Dept Chem, La Jolla, CA 92037 USA

[4] Yale Univ, Sch Med, Dept Genet, Howard Hughes Med Inst, New Haven, CT 06510 USA

[5] Yale Univ, Sch Med, Microbial Pathogenesis Sect, Howard Hughes Med Inst, New Haven, CT 06510 USA

关键词: GroEL;    Chaperonin;    Chaperone;    Protein localization;    Unnatural amino acid;    tRNA;   

DOI  :  10.1016/j.bmcl.2011.08.057

来源: Elsevier

【 摘 要 】

The molecular chaperone GroEL is required for bacterial growth under all conditions, mediating folding assistance, via its central cavity, to a diverse set of cytosolic proteins; yet the subcellular localization of GroEL remains unresolved. An earlier study, using antibody probing of fixed Escherichia coli cells, indicated colocalization with the cell division protein FtsZ at the cleavage furrow, while a second E. coli study of fixed cells indicated more even distribution throughout the cytoplasm. Here, for the first time, we have examined the spatial distribution of GroEL in living cells using incorporation of a fluorescent unnatural amino acid into the chaperone. Fluorescence microscopy indicated that GroEL is diffusely distributed, both under normal and stress conditions. Importantly, the present procedure uses a small, fluorescent unnatural amino acid to visualize GroEL in vivo, avoiding the steric demands of a fluorescent protein fusion, which compromises proper GroEL assembly. Further, this unnatural amino acid incorporation avoids artifacts that can occur with fixation and antibody staining. (C) 2011 Elsevier Ltd. All rights reserved.

【 授权许可】

Free