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

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 - 2011/10/15

Y1 - 2011/10/15

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

SN - 0960-894X

VL - 21

SP - 6067

EP - 6070

JO - Bioorganic & Medicinal Chemistry Letters

JF - Bioorganic & Medicinal Chemistry Letters

IS - 20

ER -