The E. Coli thioredoxin folding mechanism: The key role of the C-terminal helix
In this work, the unfolding mechanism of oxidized Escherichia Coli thioredoxin (EcTRX) was investigated experimentally and computationally. We characterized seven point mutants distributed along the C-terminal α-helix (CTH) and the preceding loop. The mutations destabilized the protein against globa...
Guardado en:
| Autores principales: | , , , , |
|---|---|
| Formato: | JOUR |
| Materias: | |
| Acceso en línea: | http://hdl.handle.net/20.500.12110/paper_15709639_v1854_n2_p127_Vazquez |
| Aporte de: |
| id |
todo:paper_15709639_v1854_n2_p127_Vazquez |
|---|---|
| record_format |
dspace |
| spelling |
todo:paper_15709639_v1854_n2_p127_Vazquez2023-10-03T16:26:54Z The E. Coli thioredoxin folding mechanism: The key role of the C-terminal helix Vazquez, D.S. Sánchez, I.E. Garrote, A. Sica, M.P. Santos, J. Folding kinetics Intermediate state Molecular dynamics Protein stability Transition state ensemble thioredoxin thioredoxin alpha helix Article beta sheet carboxy terminal sequence crystallization Escherichia coli genetic heterogeneity molecular dynamics nonhuman protein conformation protein folding protein stability protein unfolding thermodynamics chemistry Escherichia coli genetics kinetics point mutation protein conformation protein secondary structure protein unfolding Escherichia coli Escherichia coli Kinetics Molecular Dynamics Simulation Point Mutation Protein Conformation Protein Folding Protein Structure, Secondary Protein Unfolding Thermodynamics Thioredoxins In this work, the unfolding mechanism of oxidized Escherichia Coli thioredoxin (EcTRX) was investigated experimentally and computationally. We characterized seven point mutants distributed along the C-terminal α-helix (CTH) and the preceding loop. The mutations destabilized the protein against global unfolding while leaving the native structure unchanged. Global analysis of the unfolding kinetics of all variants revealed a linear unfolding route with a high-energy on-pathway intermediate state flanked by two transition state ensembles TSE1 and TSE2. The experiments show that CTH is mainly unfolded in TSE1 and the intermediate and becomes structured in TSE2. Structure-based molecular dynamics are in agreement with these experiments and provide protein-wide structural information on transient states. In our model, EcTRX folding starts with structure formation in the β-sheet, while the protein helices coalesce later. As a whole, our results indicate that the CTH is a critical module in the folding process, restraining a heterogeneous intermediate ensemble into a biologically active native state and providing the native protein with thermodynamic and kinetic stability. © 2014 Elsevier B.V. All rights reserved. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_15709639_v1854_n2_p127_Vazquez |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Folding kinetics Intermediate state Molecular dynamics Protein stability Transition state ensemble thioredoxin thioredoxin alpha helix Article beta sheet carboxy terminal sequence crystallization Escherichia coli genetic heterogeneity molecular dynamics nonhuman protein conformation protein folding protein stability protein unfolding thermodynamics chemistry Escherichia coli genetics kinetics point mutation protein conformation protein secondary structure protein unfolding Escherichia coli Escherichia coli Kinetics Molecular Dynamics Simulation Point Mutation Protein Conformation Protein Folding Protein Structure, Secondary Protein Unfolding Thermodynamics Thioredoxins |
| spellingShingle |
Folding kinetics Intermediate state Molecular dynamics Protein stability Transition state ensemble thioredoxin thioredoxin alpha helix Article beta sheet carboxy terminal sequence crystallization Escherichia coli genetic heterogeneity molecular dynamics nonhuman protein conformation protein folding protein stability protein unfolding thermodynamics chemistry Escherichia coli genetics kinetics point mutation protein conformation protein secondary structure protein unfolding Escherichia coli Escherichia coli Kinetics Molecular Dynamics Simulation Point Mutation Protein Conformation Protein Folding Protein Structure, Secondary Protein Unfolding Thermodynamics Thioredoxins Vazquez, D.S. Sánchez, I.E. Garrote, A. Sica, M.P. Santos, J. The E. Coli thioredoxin folding mechanism: The key role of the C-terminal helix |
| topic_facet |
Folding kinetics Intermediate state Molecular dynamics Protein stability Transition state ensemble thioredoxin thioredoxin alpha helix Article beta sheet carboxy terminal sequence crystallization Escherichia coli genetic heterogeneity molecular dynamics nonhuman protein conformation protein folding protein stability protein unfolding thermodynamics chemistry Escherichia coli genetics kinetics point mutation protein conformation protein secondary structure protein unfolding Escherichia coli Escherichia coli Kinetics Molecular Dynamics Simulation Point Mutation Protein Conformation Protein Folding Protein Structure, Secondary Protein Unfolding Thermodynamics Thioredoxins |
| description |
In this work, the unfolding mechanism of oxidized Escherichia Coli thioredoxin (EcTRX) was investigated experimentally and computationally. We characterized seven point mutants distributed along the C-terminal α-helix (CTH) and the preceding loop. The mutations destabilized the protein against global unfolding while leaving the native structure unchanged. Global analysis of the unfolding kinetics of all variants revealed a linear unfolding route with a high-energy on-pathway intermediate state flanked by two transition state ensembles TSE1 and TSE2. The experiments show that CTH is mainly unfolded in TSE1 and the intermediate and becomes structured in TSE2. Structure-based molecular dynamics are in agreement with these experiments and provide protein-wide structural information on transient states. In our model, EcTRX folding starts with structure formation in the β-sheet, while the protein helices coalesce later. As a whole, our results indicate that the CTH is a critical module in the folding process, restraining a heterogeneous intermediate ensemble into a biologically active native state and providing the native protein with thermodynamic and kinetic stability. © 2014 Elsevier B.V. All rights reserved. |
| format |
JOUR |
| author |
Vazquez, D.S. Sánchez, I.E. Garrote, A. Sica, M.P. Santos, J. |
| author_facet |
Vazquez, D.S. Sánchez, I.E. Garrote, A. Sica, M.P. Santos, J. |
| author_sort |
Vazquez, D.S. |
| title |
The E. Coli thioredoxin folding mechanism: The key role of the C-terminal helix |
| title_short |
The E. Coli thioredoxin folding mechanism: The key role of the C-terminal helix |
| title_full |
The E. Coli thioredoxin folding mechanism: The key role of the C-terminal helix |
| title_fullStr |
The E. Coli thioredoxin folding mechanism: The key role of the C-terminal helix |
| title_full_unstemmed |
The E. Coli thioredoxin folding mechanism: The key role of the C-terminal helix |
| title_sort |
e. coli thioredoxin folding mechanism: the key role of the c-terminal helix |
| url |
http://hdl.handle.net/20.500.12110/paper_15709639_v1854_n2_p127_Vazquez |
| work_keys_str_mv |
AT vazquezds theecolithioredoxinfoldingmechanismthekeyroleofthecterminalhelix AT sanchezie theecolithioredoxinfoldingmechanismthekeyroleofthecterminalhelix AT garrotea theecolithioredoxinfoldingmechanismthekeyroleofthecterminalhelix AT sicamp theecolithioredoxinfoldingmechanismthekeyroleofthecterminalhelix AT santosj theecolithioredoxinfoldingmechanismthekeyroleofthecterminalhelix AT vazquezds ecolithioredoxinfoldingmechanismthekeyroleofthecterminalhelix AT sanchezie ecolithioredoxinfoldingmechanismthekeyroleofthecterminalhelix AT garrotea ecolithioredoxinfoldingmechanismthekeyroleofthecterminalhelix AT sicamp ecolithioredoxinfoldingmechanismthekeyroleofthecterminalhelix AT santosj ecolithioredoxinfoldingmechanismthekeyroleofthecterminalhelix |
| _version_ |
1807314792835186688 |