Localization of GroEL determined by in vivo incorporation of a fluorescent amino acid
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Localization of GroEL determined by in vivo incorporation of a fluorescent amino acid. / Charbon, Godefroid; Wang, Jiangyun; Brustad, Eric; Schultz, Peter G; Horwich, Arthur L; Jacobs-Wagner, Christine; Chapman, Eli.
In: Bioorganic & Medicinal Chemistry Letters, Vol. 21, No. 20, 2011, p. 6067-6070.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Localization of GroEL determined by in vivo incorporation of a fluorescent amino acid
AU - Charbon, Godefroid
AU - Wang, Jiangyun
AU - Brustad, Eric
AU - Schultz, Peter G
AU - Horwich, Arthur L
AU - Jacobs-Wagner, Christine
AU - Chapman, Eli
N1 - Copyright © 2011 Elsevier Ltd. All rights reserved.
PY - 2011
Y1 - 2011
N2 - 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.
AB - 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.
KW - Amino Acids/analysis
KW - Chaperonin 60/analysis
KW - Escherichia coli/cytology
KW - Fluorescent Dyes/analysis
KW - Microscopy, Fluorescence
U2 - 10.1016/j.bmcl.2011.08.057
DO - 10.1016/j.bmcl.2011.08.057
M3 - Journal article
C2 - 21890355
VL - 21
SP - 6067
EP - 6070
JO - Bioorganic & Medicinal Chemistry Letters
JF - Bioorganic & Medicinal Chemistry Letters
SN - 0960-894X
IS - 20
ER -
ID: 201155966