Adaptability of the GroEL/GroES chaperonin to proteostatic challenge revealed by directed evolution
Abstract
The folding of newly synthesized proteins is assisted by an essential network of proteins known as molecular chaperones. In bacteria, the chaperones Trigger Factor (TF), DnaK/DnaJ (Hsp70 system), and GroEL/GroES (chaperonin system) are key players in proteostasis maintenance. Here, we further explore the cooperation and substrate preference within the bacterial chaperone network by performing directed evolution of GroEL/GroES in Escherichia coli lacking both TF and DnaK chaperone pathways. We found that single amino acid substitutions in GroEL were sufficient to significantly improve its chaperone activity in vivo at high temperature in the absence of TF and DnaKJ, with a combination of selected mutations further enhancing functionality. In vitro analysis showed that high-performing GroEL variants have a higher rate of ATP hydrolysis and an increased folding rate for certain substrates at low ATP concentration, thereby compensating for the significant drop in ATP level observed in vivo in cells lacking TF and DnaKJ. Analysis of substrates bound to GroEL in vivo showed that experimentally evolved chaperones likely adapted toward obligate GroEL substrates with low solubility, thus bypassing cooperation with upstream chaperones like TF and DnaKJ.
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BibTeX
@article{castaniecornet2026adaptability,
title = {{Adaptability of the GroEL/GroES chaperonin to proteostatic challenge revealed by directed evolution}},
author = {Marie-Pierre Castanié-Cornet and Tatsuya Niwa and Émile Dupuy and Anne-Marie Cirinesi and Manajit Hayer-Hartl and Hideki Taguchi and F. Ulrich Hartl and Pierre Genevaux},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
year = {2026},
month = sep,
doi = {10.1073/pnas.2608599123},
url = {https://hal.science/hal-05772584v1},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Adaptability of the GroEL/GroES chaperonin to proteostatic challenge revealed by directed evolution
AU - Marie-Pierre Castanié-Cornet
AU - Tatsuya Niwa
AU - Émile Dupuy
AU - Anne-Marie Cirinesi
AU - Manajit Hayer-Hartl
AU - Hideki Taguchi
AU - F. Ulrich Hartl
AU - Pierre Genevaux
PY - 2026
DA - 2026/09/23
JO - Proceedings of the National Academy of Sciences of the United States of America
DO - 10.1073/pnas.2608599123
UR - https://hal.science/hal-05772584v1
AB - The folding of newly synthesized proteins is assisted by an essential network of proteins known as molecular chaperones. In bacteria, the chaperones Trigger Factor (TF), DnaK/DnaJ (Hsp70 system), and GroEL/GroES (chaperonin system) are key players in proteostasis maintenance. Here, we further explore the cooperation and substrate preference within the bacterial chaperone network by performing directed evolution of GroEL/GroES in Escherichia coli lacking both TF and DnaK chaperone pathways. We found that single amino acid substitutions in GroEL were sufficient to significantly improve its chaperone activity in vivo at high temperature in the absence of TF and DnaKJ, with a combination of selected mutations further enhancing functionality. In vitro analysis showed that high-performing GroEL variants have a higher rate of ATP hydrolysis and an increased folding rate for certain substrates at low ATP concentration, thereby compensating for the significant drop in ATP level observed in vivo in cells lacking TF and DnaKJ. Analysis of substrates bound to GroEL in vivo showed that experimentally evolved chaperones likely adapted toward obligate GroEL substrates with low solubility, thus bypassing cooperation with upstream chaperones like TF and DnaKJ.
SN - 0027-8424
SN - 1091-6490
ER -