Calorimetric force spectroscopy experiments in DNA and protein folding
[eng] In the present thesis, single-molecule experiments have been carried out using optical tweezers with a temperature controller. Employing equilibrium and non-equilibrium experiments, the free energy, enthalpy, and entropy during the molecular folding of the protein barnase and different DNA hai...
| Autor: | |
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2022 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/185906 |
| Acceso en línea: | https://hdl.handle.net/2445/185906 http://hdl.handle.net/10803/674311 |
| Access Level: | acceso abierto |
| Palabra clave: | Termodinàmica Biomolècules Mecànica estadística Thermodynamics Biomolecules Statistical mechanics |
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| dc.title.none.fl_str_mv |
Calorimetric force spectroscopy experiments in DNA and protein folding |
| title |
Calorimetric force spectroscopy experiments in DNA and protein folding |
| spellingShingle |
Calorimetric force spectroscopy experiments in DNA and protein folding Rico Pastó, Marc Termodinàmica Biomolècules Mecànica estadística Thermodynamics Biomolecules Statistical mechanics |
| title_short |
Calorimetric force spectroscopy experiments in DNA and protein folding |
| title_full |
Calorimetric force spectroscopy experiments in DNA and protein folding |
| title_fullStr |
Calorimetric force spectroscopy experiments in DNA and protein folding |
| title_full_unstemmed |
Calorimetric force spectroscopy experiments in DNA and protein folding |
| title_sort |
Calorimetric force spectroscopy experiments in DNA and protein folding |
| dc.creator.none.fl_str_mv |
Rico Pastó, Marc |
| author |
Rico Pastó, Marc |
| author_facet |
Rico Pastó, Marc |
| author_role |
author |
| dc.contributor.none.fl_str_mv |
Ritort Farran, Fèlix Universitat de Barcelona. Facultat de Física |
| dc.subject.none.fl_str_mv |
Termodinàmica Biomolècules Mecànica estadística Thermodynamics Biomolecules Statistical mechanics |
| topic |
Termodinàmica Biomolècules Mecànica estadística Thermodynamics Biomolecules Statistical mechanics |
| description |
[eng] In the present thesis, single-molecule experiments have been carried out using optical tweezers with a temperature controller. Employing equilibrium and non-equilibrium experiments, the free energy, enthalpy, and entropy during the molecular folding of the protein barnase and different DNA hairpins have been determined as a function of temperature. From these measurements, we derived the heat capacity change per base pair during the DNA hybridization reaction. In addition, with a new analysis developed in this thesis, the data obtained have allowed us to characterize the thermodynamic properties of the transition state in the free energy landscape governing molecular folding. These results have revealed the nature of the transition state. In particular, the results have shown that the transition state exhibits the thermodynamic characteristics predicted by the molten globule model developed to explain molecular folding in the 1980s-90s. Characterizing this state is highly difficult in bulk experiments, where many molecules are studied simultaneously. However, the single-molecule experiments presented in this Ph.D. thesis have allowed characterizing all the thermodynamic potentials with unprecedented accuracy. In parallel, a fluctuation theorem (FT) has been derived to investigate non-equilibrium pulling experiments with feedback protocols. Such FT has been studied experimentally using two cases and numerical simulations to analyze the information-to-energy conversion and find the optimal conditions to reduce dissipation during these experiments. Based on the obtained results, a new concept has been introduced, which we have named Feedback Strategy. The feedback strategy has been studied using different numerical simulations. In addition, we have extended a methodology used in single-molecule experiments known as CEBA (an acronym for Continuous Effective Barrier Approach) that allows characterizing the effective barrier that mediates transitions between the native and denatured state for DNA/RNA and protein molecules. In particular, the folding of DNA hairpins presenting intermediate states has been studied as a simple model to demonstrate the benefits of the extended methodology. Moreover, two small DNA hairpins folding with and without intermediate states have been studied over a wide temperature range to derive the temperature response of the effective barrier. Finally, the interaction of DNA with ligands that bind weakly to the DNA double-helix has been studied using non-equilibrium experiments at different ligand concentrations. Specifically, the drug known as Netropsin has been used to demonstrate the benefits of the method proposed in this part. This ligand has the particularity that it can bind to DNA via two modes: a specific mode that links Netropsin to the 5'- AATT-3' motif or a non-specific mode that links Netropsin to rich-AT areas. The kinetic properties of both binding modes and their binding energy have been determined using the experiments mentioned above. |
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2022 |
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2022 |
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info:eu-repo/semantics/doctoralThesis info:eu-repo/semantics/publishedVersion |
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doctoralThesis |
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publishedVersion |
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https://hdl.handle.net/2445/185906 http://hdl.handle.net/10803/674311 |
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https://hdl.handle.net/2445/185906 http://hdl.handle.net/10803/674311 |
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Inglés |
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Inglés |
| dc.rights.none.fl_str_mv |
cc by-nc-sa (c) Rico Pastó, Marc, 2022 http://creativecommons.org/licenses/by-nc-sa/3.0/es/ info:eu-repo/semantics/openAccess |
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cc by-nc-sa (c) Rico Pastó, Marc, 2022 http://creativecommons.org/licenses/by-nc-sa/3.0/es/ |
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openAccess |
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application/pdf |
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Universitat de Barcelona |
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Universitat de Barcelona |
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Tesis Doctorals - Facultat - Física reponame:Dipòsit Digital de la UB instname:Universidad de Barcelona |
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Universidad de Barcelona |
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Dipòsit Digital de la UB |
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Dipòsit Digital de la UB |
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1869407577158189056 |
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Calorimetric force spectroscopy experiments in DNA and protein foldingRico Pastó, MarcTermodinàmicaBiomolèculesMecànica estadísticaThermodynamicsBiomoleculesStatistical mechanics[eng] In the present thesis, single-molecule experiments have been carried out using optical tweezers with a temperature controller. Employing equilibrium and non-equilibrium experiments, the free energy, enthalpy, and entropy during the molecular folding of the protein barnase and different DNA hairpins have been determined as a function of temperature. From these measurements, we derived the heat capacity change per base pair during the DNA hybridization reaction. In addition, with a new analysis developed in this thesis, the data obtained have allowed us to characterize the thermodynamic properties of the transition state in the free energy landscape governing molecular folding. These results have revealed the nature of the transition state. In particular, the results have shown that the transition state exhibits the thermodynamic characteristics predicted by the molten globule model developed to explain molecular folding in the 1980s-90s. Characterizing this state is highly difficult in bulk experiments, where many molecules are studied simultaneously. However, the single-molecule experiments presented in this Ph.D. thesis have allowed characterizing all the thermodynamic potentials with unprecedented accuracy. In parallel, a fluctuation theorem (FT) has been derived to investigate non-equilibrium pulling experiments with feedback protocols. Such FT has been studied experimentally using two cases and numerical simulations to analyze the information-to-energy conversion and find the optimal conditions to reduce dissipation during these experiments. Based on the obtained results, a new concept has been introduced, which we have named Feedback Strategy. The feedback strategy has been studied using different numerical simulations. In addition, we have extended a methodology used in single-molecule experiments known as CEBA (an acronym for Continuous Effective Barrier Approach) that allows characterizing the effective barrier that mediates transitions between the native and denatured state for DNA/RNA and protein molecules. In particular, the folding of DNA hairpins presenting intermediate states has been studied as a simple model to demonstrate the benefits of the extended methodology. Moreover, two small DNA hairpins folding with and without intermediate states have been studied over a wide temperature range to derive the temperature response of the effective barrier. Finally, the interaction of DNA with ligands that bind weakly to the DNA double-helix has been studied using non-equilibrium experiments at different ligand concentrations. Specifically, the drug known as Netropsin has been used to demonstrate the benefits of the method proposed in this part. This ligand has the particularity that it can bind to DNA via two modes: a specific mode that links Netropsin to the 5'- AATT-3' motif or a non-specific mode that links Netropsin to rich-AT areas. The kinetic properties of both binding modes and their binding energy have been determined using the experiments mentioned above.[cat] En aquesta tesi s'han realitzat experiments de molècula individual en equilibri i no-equilibri mitjançant unes pinces òptiques amb controlador de temperatura per determinar l'energia lliure, entalpia i entropia en funció de la temperatura durant el plegament de la proteïna barnasa i diferents forquetes d'ADN. A partir d'aquestes mesures, s'ha derivat el canvi de capacitat calorífica per parell de base en la reacció d'hibridació de l'ADN. A més, amb una metodologia desenvolupada en aquesta tesi, les dades obtingudes han permès caracteritzar les propietats termodinàmiques de l'estat de transició. Els resultats obtinguts han demostrat que l'estat de transició presenta les característiques termodinàmiques predites pel model de proteïna fosa desenvolupat en els anys 80-90 per explicar el plegament molecular. Caracteritzar aquest estat presenta grans complicacions en experiments clàssics de granel on un gran número de molècules són estudiades alhora. En canvi, els experiments de molècula individual presentats en aquesta tesi doctoral han permès caracteritzar amb una precisió sense precedents tots els potencials termodinàmics d'aquest estat. Paral·lelament, s'ha derivat un teorema de fluctuació (TF) per estudiar experiments de no-equilibri aplicant un protocol de retroalimentació. Dit TF ha estat estudiat experimentalment i mitjançant simulacions numèriques per a estudiar la conversió d'informació en energia i trobar les condicions òptimes per a reduir la dissipació. Basat en els resultats obtinguts, s'ha introduït un nou concepte en el camp, que hem denominat Estratègia de Retroalimentació. Finalment, s'han realitzat dues col·laboracions, una per estendre una metodologia coneguda com a CEBA (acrònim de Continus Effective Barrier Aproach en anglès) que permet caracteritzar la barrera efectiva que mitjana transicions entre l'estat natiu i desnaturalitzat per a molècules d'ADN/RNA i proteïnes. En segon lloc, s'ha estudiat la interacció de l'ADN amb lligands que s'uneixen de manera feble a la doble-hèlix d'ADN mitjançant experiments de no-equilibri a diferents concentracions de lligant.Universitat de BarcelonaRitort Farran, FèlixUniversitat de Barcelona. Facultat de Física2022info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://hdl.handle.net/2445/185906http://hdl.handle.net/10803/674311Tesis Doctorals - Facultat - Físicareponame:Dipòsit Digital de la UBinstname:Universidad de BarcelonaIngléscc by-nc-sa (c) Rico Pastó, Marc, 2022http://creativecommons.org/licenses/by-nc-sa/3.0/es/info:eu-repo/semantics/openAccessoai:diposit.ub.edu:2445/1859062026-05-27T06:46:51Z |
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15,301603 |