A selection for mutants that interfere with folding of Escherichia coli thioredoxin-1 in vivo

Research output: Contribution to journal › Article › peer-review

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A selection for mutants that interfere with folding of Escherichia coli thioredoxin-1 in vivo. / Huber, Damon; Cha, Myoung-Il; Debarbieux, Laurent; Planson, Anne-Gaëlle; Cruz, Nelly; López, Gary; Tasayco, María Luisa; Chaffotte, Alain; Beckwith, Jon.

In: National Academy of Sciences. Proceedings, Vol. 102, No. 52, 27.12.2005, p. 18872-7.

Research output: Contribution to journal › Article › peer-review

Harvard

Huber, D, Cha, M-I, Debarbieux, L, Planson, A-G, Cruz, N, López, G, Tasayco, ML, Chaffotte, A & Beckwith, J 2005, 'A selection for mutants that interfere with folding of Escherichia coli thioredoxin-1 in vivo', National Academy of Sciences. Proceedings, vol. 102, no. 52, pp. 18872-7. https://doi.org/10.1073/pnas.0509583102

APA

Huber, D., Cha, M-I., Debarbieux, L., Planson, A-G., Cruz, N., López, G., Tasayco, M. L., Chaffotte, A., & Beckwith, J. (2005). A selection for mutants that interfere with folding of Escherichia coli thioredoxin-1 in vivo. National Academy of Sciences. Proceedings, 102(52), 18872-7. https://doi.org/10.1073/pnas.0509583102

Vancouver

Huber D, Cha M-I, Debarbieux L, Planson A-G, Cruz N, López G et al. A selection for mutants that interfere with folding of Escherichia coli thioredoxin-1 in vivo. National Academy of Sciences. Proceedings. 2005 Dec 27;102(52):18872-7. https://doi.org/10.1073/pnas.0509583102

Author

Huber, Damon ; Cha, Myoung-Il ; Debarbieux, Laurent ; Planson, Anne-Gaëlle ; Cruz, Nelly ; López, Gary ; Tasayco, María Luisa ; Chaffotte, Alain ; Beckwith, Jon. / A selection for mutants that interfere with folding of Escherichia coli thioredoxin-1 in vivo. In: National Academy of Sciences. Proceedings. 2005 ; Vol. 102, No. 52. pp. 18872-7.

Bibtex

@article{f57e71402c12485b8a33b2b138eea98d,
title = "A selection for mutants that interfere with folding of Escherichia coli thioredoxin-1 in vivo",
abstract = "Escherichia coli thioredoxin is normally a cytoplasmic protein involved in the reduction of disulfide bonds. However, thioredoxin can be translocated to the periplasm when it is attached to a cotranslational signal sequence. When exported to the periplasm, it can partially replace the activity of DsbA in promoting the formation of disulfide bonds. In contrast, when thioredoxin is fused to a posttranslational signal sequence, very little of it appears in the periplasm. We propose that this absence of posttranslational export is due to the rapid folding of thioredoxin in the cytoplasm. We sought mutants of thioredoxin that retarded its folding in the cytoplasm, which we accomplished by fusing thioredoxin to a posttranslational signal sequence and selecting for mutants in which thioredoxin was exported to the periplasm, where it could replace DsbA. The collection of mutants obtained represents a limited number of amino acid changes in the protein. In vitro studies on purified mutant proteins show that all but one are defective in the kinetics and thermodynamics of protein folding. We propose that the slower folding of the thioredoxin mutant proteins in the cytoplasm allows their export by a posttranslational pathway. We discuss some implications of this class of mutants for aspects of the folding pathway of thioredoxin and for its mechanism of export. In particular, the finding that a folding mutant that allows protein translocation alters an amino acid at the C terminus of the protein suggests that the degree to which thioredoxin folds during its translation must be severely restricted.",
keywords = "Disulfides, Thioredoxins, Magnetic Resonance Spectroscopy, Mutagenesis, Mutagenesis, Site-Directed, Bacterial Proteins, Databases, Protein, Cytoplasm, Escherichia coli, Time Factors, Protein Biosynthesis, Protein Disulfide-Isomerases, Models, Molecular, Protein Processing, Post-Translational, Subcellular Fractions, Circular Dichroism, Protein Sorting Signals, Plasmids, Protein Binding, Oxygen, Blotting, Western, Genetic Techniques, Kinetics, Escherichia coli Proteins, Protein Folding, Protein Structure, Tertiary, Calorimetry, Differential Scanning, Mutation",
author = "Damon Huber and Myoung-Il Cha and Laurent Debarbieux and Anne-Ga{\"e}lle Planson and Nelly Cruz and Gary L{\'o}pez and Tasayco, {Mar{\'i}a Luisa} and Alain Chaffotte and Jon Beckwith",
year = "2005",
month = dec,
day = "27",
doi = "10.1073/pnas.0509583102",
language = "English",
volume = "102",
pages = "18872--7",
journal = "Proceedings of the National Academy of Sciences",
issn = "1091-6490",
publisher = "National Academy of Sciences",
number = "52",

}

RIS

TY - JOUR

T1 - A selection for mutants that interfere with folding of Escherichia coli thioredoxin-1 in vivo

AU - Huber, Damon

AU - Cha, Myoung-Il

AU - Debarbieux, Laurent

AU - Planson, Anne-Gaëlle

AU - Cruz, Nelly

AU - López, Gary

AU - Tasayco, María Luisa

AU - Chaffotte, Alain

AU - Beckwith, Jon

PY - 2005/12/27

Y1 - 2005/12/27

N2 - Escherichia coli thioredoxin is normally a cytoplasmic protein involved in the reduction of disulfide bonds. However, thioredoxin can be translocated to the periplasm when it is attached to a cotranslational signal sequence. When exported to the periplasm, it can partially replace the activity of DsbA in promoting the formation of disulfide bonds. In contrast, when thioredoxin is fused to a posttranslational signal sequence, very little of it appears in the periplasm. We propose that this absence of posttranslational export is due to the rapid folding of thioredoxin in the cytoplasm. We sought mutants of thioredoxin that retarded its folding in the cytoplasm, which we accomplished by fusing thioredoxin to a posttranslational signal sequence and selecting for mutants in which thioredoxin was exported to the periplasm, where it could replace DsbA. The collection of mutants obtained represents a limited number of amino acid changes in the protein. In vitro studies on purified mutant proteins show that all but one are defective in the kinetics and thermodynamics of protein folding. We propose that the slower folding of the thioredoxin mutant proteins in the cytoplasm allows their export by a posttranslational pathway. We discuss some implications of this class of mutants for aspects of the folding pathway of thioredoxin and for its mechanism of export. In particular, the finding that a folding mutant that allows protein translocation alters an amino acid at the C terminus of the protein suggests that the degree to which thioredoxin folds during its translation must be severely restricted.

AB - Escherichia coli thioredoxin is normally a cytoplasmic protein involved in the reduction of disulfide bonds. However, thioredoxin can be translocated to the periplasm when it is attached to a cotranslational signal sequence. When exported to the periplasm, it can partially replace the activity of DsbA in promoting the formation of disulfide bonds. In contrast, when thioredoxin is fused to a posttranslational signal sequence, very little of it appears in the periplasm. We propose that this absence of posttranslational export is due to the rapid folding of thioredoxin in the cytoplasm. We sought mutants of thioredoxin that retarded its folding in the cytoplasm, which we accomplished by fusing thioredoxin to a posttranslational signal sequence and selecting for mutants in which thioredoxin was exported to the periplasm, where it could replace DsbA. The collection of mutants obtained represents a limited number of amino acid changes in the protein. In vitro studies on purified mutant proteins show that all but one are defective in the kinetics and thermodynamics of protein folding. We propose that the slower folding of the thioredoxin mutant proteins in the cytoplasm allows their export by a posttranslational pathway. We discuss some implications of this class of mutants for aspects of the folding pathway of thioredoxin and for its mechanism of export. In particular, the finding that a folding mutant that allows protein translocation alters an amino acid at the C terminus of the protein suggests that the degree to which thioredoxin folds during its translation must be severely restricted.

KW - Disulfides

KW - Thioredoxins

KW - Magnetic Resonance Spectroscopy

KW - Mutagenesis

KW - Mutagenesis, Site-Directed

KW - Bacterial Proteins

KW - Databases, Protein

KW - Cytoplasm

KW - Escherichia coli

KW - Time Factors

KW - Protein Biosynthesis

KW - Protein Disulfide-Isomerases

KW - Models, Molecular

KW - Protein Processing, Post-Translational

KW - Subcellular Fractions

KW - Circular Dichroism

KW - Protein Sorting Signals

KW - Plasmids

KW - Protein Binding

KW - Oxygen

KW - Blotting, Western

KW - Genetic Techniques

KW - Kinetics

KW - Escherichia coli Proteins

KW - Protein Folding

KW - Protein Structure, Tertiary

KW - Calorimetry, Differential Scanning

KW - Mutation

U2 - 10.1073/pnas.0509583102

DO - 10.1073/pnas.0509583102

M3 - Article

C2 - 16357193

VL - 102

SP - 18872

EP - 18877

JO - Proceedings of the National Academy of Sciences

JF - Proceedings of the National Academy of Sciences

SN - 1091-6490

IS - 52

ER -