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Evolution des propriétés diélectriques, ferroélectriques et électromécaniques dans le système pseudo-binaire (1-x)BaTi0.8Zr0.2O3- xBa0.7Ca0.3TiO3 / Corrélations structures et propriétés / Evolution of the dielectric, ferroelectric and electromechanical properties in the pseudo-binary system (1-x)-BaTi0.8Zr0.2O3 xBa0.7Ca0.3TiO3 / structure-property correlationsBenabdallah, Feres 20 May 2013 (has links)
Ce travail de thèse a pour objectif la caractérisation des propriétés physico-chimiques descéramiques de composition (1-x) BaTi0.8Zr0.2O3-x Ba0.7Ca0.3TiO3 préparées par frittage conventionnelet frittage flash (SPS). Les études structurales réalisées au voisinage du point triple (x≈ 0.32) à l’aidede la diffraction des RX de haute résolution (synchrotron) sur poudre ont introduit des modificationsmajeures sur le diagramme de phase température-composition déjà proposé. La réponseélectromécanique géante mesurée est alors corrélée à la dégénérescence du profil de l’énergie libreinduite par les instabilités structurales. De plus, la flexibilité de la polarisation sous contraintesthermique et électrique est couplée à un assouplissement de la maille cristalline. Ces deuxcaractéristiques contribuent ensemble à une réponse électromécanique colossale via une forteactivité des murs de domaine. La dégradation des propriétés diélectriques, ferroélectriques etpiézoélectriques pour les céramiques BCTZ (x=0.32 et 0.5) élaborées par frittage flash estessentiellement attribuée aux fluctuations importantes de composition et à la stabilisation de laconfiguration des murs de domaines avec la diminution de la taille des grains. / The aim of this work is to make a full characterization of the structural, microstructural, dielectric,ferroelectric and piezoelectric properties of the perovskite-structured oxides (1-x) BaTi0.8Zr0.2O3-xBa0.7Ca0.3TiO3 prepared by a conventional solid-state reaction method (conventional sintering) andSPS fabrication technique. Using high-resolution synchrotron x-ray powder diffraction, the structuralinvestigations carried out close to the triple point (x≈ 0.32) have introduced significant corrections tothe previously published composition-temperature phase diagram. The colossal electromechanicalresponse was then correlated to a strongly degenerate free energy landscape caused by structuralinstabilities. Furthermore, the coupling between the high polarization flexibility under electric andthermal stresses and the ‘lattice softening’ gives rise to a giant electromechanical response due tohigh domain wall activities. The decrease of the dielectric, ferroelectric and piezoelectric propertiesof BCTZ ceramics (x=0.32 and 0.5) processed by SPS was essentially attributed to the largecompositional fluctuations and stable domain wall configurations as the grain size decreased.
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Downhill folders in slow motion:Mukhortava, Ann 23 October 2017 (has links) (PDF)
Die Proteinfaltung ist ein Prozess der molekularen Selbstorganisation, bei dem sich eine lineare Kette von Aminosäuren zu einer definierten, funktionellen dreidimensionalen Struktur zusammensetzt. Der Prozess der Faltung ist ein thermisch getriebener diffusiver Prozess durch eine Gibbs-Energie-Landschaft im Konformationsraum für die Struktur der minimalen Energie. Während dieses Prozesses zeigt die freie Enthalpie des Systems nicht immer eine monotone Abnahme; stattdessen führt eine suboptimale Kompensation der Enthalpie- und der Entropieänderung während jedes Faltungsschrittes zur Bildung von Freien-Enthalpie-Faltungsbarrieren. Diese Barrieren und damit verbundenen hochenergetischen Übergangszustände, die wichtige Informationen über Mechanismen der Proteinfaltung enthalten, sind jedoch kinetisch unzugänglich. Um den Prozess der Barrierebildung und die strukturellen Merkmale von Übergangszuständen aufzudecken, werden Proteine genutzt, die über barrierefreie Pfade falten – so genannte “downhill folder“. Aufgrund der geringen Faltungsbarrieren werden wichtige Interaktionen der Faltung zugänglich und erlauben Einblicke in die ratenbegrenzenden Faltungsvorgänge.
In dieser Arbeit vergleichen wir die Faltungsdynamiken von drei verschiedenen Varianten eines Lambda-Repressor-Fragments, bestehend aus den Aminosäuren 6 bis 85: ein Zwei-Zustands-Falter λWT (Y22W) und zwei downhill-folder-artige Varianten, λYA (Y22W/Q33Y/ G46,48A) und λHA (Y22W/Q33H/G46,48A). Um auf die Kinetik und die strukturelle Dynamik zu greifen zu können, werden Einzelmolekülkraftspektroskopische Experimente mit optische Pinzetten mit Submillisekunden- und Nanometer-Auflösung verwendet. Ich fand, dass die niedrige denaturierende Kraft die Mikrosekunden Faltungskinetik von downhill foldern auf eine Millisekunden-Zeitskala verlangsamt, sodass das System für Einzelmolekülstudien gut zugänglich ist.
Interessanterweise zeigten sich unter Krafteinwirkung die downhill-folder-artigen Varianten des Lambda-Repressors als kooperative Zwei-Zustands-Falter mit deutlich unterschiedlicher Faltungskinetik und Kraftabhängigkeit. Drei Varianten des Proteins zeigten ein hoch konformes Verhalten unter Last. Die modellfreie Rekonstruktion von Freien-Enthalpie-Landschaften ermöglichte es uns, die feinen Details der Transformation des Zwei-Zustands-Faltungspfad direkt in einen downhill-artigen Pfad aufzulösen. Die Auswirkungen von einzelnen Mutationen auf die Proteinstabilität, Bildung der Übergangszustände und die konformationelle Heterogenität der Faltungs- und Entfaltungszustände konnten beobachtet werden.
Interessanterweise zeigen unsere Ergebnisse, dass sich die untersuchten Varianten trotz der ultraschnellen Faltungszeit im Bereich von 2 μs in einem kooperativen Prozess über verbleibende Energiebarrieren falten und entfalten, was darauf hindeutet, dass wesentlich schnellere Faltungsraten notwendig sind um ein downhill Limit vollständig zu erreichen. / Protein folding is a process of molecular self-assembly in which a linear chain of amino acids assembles into a defined, functional three-dimensional structure. The process of folding is a thermally driven diffusive search on a free-energy landscape in the conformational space for the minimal-energy structure. During that process, the free energy of the system does not always show a monotonic decrease; instead, sub-optimal compensation of enthalpy and entropy change during each folding step leads to formation of folding free-energy barriers. However, these barriers, and associated high-energy transition states, that contain key information about mechanisms of protein folding, are kinetically inaccessible. To reveal the barrier-formation process and structural characteristics of transition states, proteins are employed that fold via barrierless paths – so-called downhill folders. Due to the low folding barriers, the key folding interactions become accessible, yielding insights about the rate-limiting folding events.
Here, I compared the folding dynamics of three different variants of a lambda repressor fragment, containing amino acids 6 to 85: a two-state folder λWT (Y22W) and two downhill-like folding variants, λYA (Y22W/Q33Y/G46,48A) and λHA (Y22W/Q33H/G46,48A). To access the kinetics and structural dynamics, single-molecule optical tweezers with submillisecond and nanometer resolution are used. I found that force perturbation slowed down the microsecond kinetics of downhill folders to a millisecond time-scale, making it accessible to single-molecule studies.
Interestingly, under load, the downhill-like variants of lambda repressor appeared as cooperative two-state folders with significantly different folding kinetics and force dependence. The three protein variants displayed a highly compliant behaviour under load. Model-free reconstruction of free-energy landscapes allowed us to directly resolve the fine details of the transformation of the two-state folding path into a downhill-like path. The effect of single mutations on protein stability, transition state formation and conformational heterogeneity of folding and unfolding states was observed.
Noteworthy, our results demonstrate, that despite the ultrafast folding time in a range of 2 µs, the studied variants fold and unfold in a cooperative process via residual barriers, suggesting that much faster folding rate constants are required to reach the full-downhill limit.
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Variable pressure NMR analyses to assess compressive motion in PETNR and catalytically germane PETNR:Ligand complexesGuerriero, Andrew January 2012 (has links)
The involvement of dynamical fluctuations in driving enzymatic processes is widely accepted. With respect to NQM tunnelling enzymes, the role of promoting motions in facilitating hydrogenic transfers is well studied. Few studies have however, specifically attributed, dedicated dynamical fluctuations characterised by their timescales and magnitudes, as a function of a reaction coordinate, to specific groups in a protein system. An effectively full suite of backbone resonance assignments were obtained for PETNR and on relevant ligand complexes. This provided an essential platform on which residue specific, backbone amide fluctuations were assessed. This thesis documents the application of pressure up to 1500 bar, in tandem with high resolution TROSY based NMR analysis, as a means of studying residue specific, conformer exchange perturbations. Residue specific amide compression profiles of the PETNR:FMN free enzyme system, and complexes with progesterone and tetrahydropyridine dinucleotides have been obtained. The binding of progesterone appears to induce conformational tightening of residues within the active site vicinity. The complexation of PETNR:FMN with tetrahydropyridine dinucleotides, appears to stimulate conformational shifts towards intermediate, and in some cases, slow exchange regimes in multiple residues about the active site vicinity. This is evidenced by extensive intensity attenuation of 1H-15N TROSY resonances, on the binding of tetrahydropyridine dinucleotides at 1 bar pressure, and on going from 1 bar to 1500 bar pressure. Multiple regions of sequence, spatially clustering about the active site vicinity within a 10 Å sphere of the FMN binding pocket, display appreciable sensitivity to ligand binding. Differential responses of residues to the application of high pressure between complexes was noted within segments of these regions. A region of sequence, named the β-hairpin flap displays significant differential compression profiles between the PETNR:FMN free enzyme system, and associated progesterone and tetrahydropyridine dinucleotide complexes. A role in mediating ligand engagement is proposed for R130 and R142 in the β-hairpin flap. A central hydrogen bonding network, perhaps constituting a putative proton wire in the active site of the PETNR:FMN:Progesterone complex, has been identified that could enable the shuttling of protons following catalytic protonation of oxidative substrate. The resonance response behaviour of G185 acts as a sensitive reporter on the formation of these interactions, revealed by an interrogation of the differences in chemical shift changes on progesterone binding, and in response to high pressure. The recruitment of high resolution crystallographic data sets readily supported a structural and dynamical interpretation of the observed chemical shift responses to ligand binding at 1 bar pressure, and on the application high pressure. A definitive atomistic identification of fast motion contribution to activation barrier compression was not obtained. Nevertheless, detailed, residue specific amide compression profiles, and shifts in backbone amide conformational exchange regimes in response to ground state ligand binding, and at high pressure, have been catalogued in the PETNR:FMN free enzyme system. These dynamical profiles in the free enzyme are contrasted against comparative, residue specific observations in analogue complexes of the oxidative and reductive half reactions of PETNR.
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Downhill folders in slow motion:: Lambda repressor variants probed by optical tweezersMukhortava, Ann 26 September 2017 (has links)
Die Proteinfaltung ist ein Prozess der molekularen Selbstorganisation, bei dem sich eine lineare Kette von Aminosäuren zu einer definierten, funktionellen dreidimensionalen Struktur zusammensetzt. Der Prozess der Faltung ist ein thermisch getriebener diffusiver Prozess durch eine Gibbs-Energie-Landschaft im Konformationsraum für die Struktur der minimalen Energie. Während dieses Prozesses zeigt die freie Enthalpie des Systems nicht immer eine monotone Abnahme; stattdessen führt eine suboptimale Kompensation der Enthalpie- und der Entropieänderung während jedes Faltungsschrittes zur Bildung von Freien-Enthalpie-Faltungsbarrieren. Diese Barrieren und damit verbundenen hochenergetischen Übergangszustände, die wichtige Informationen über Mechanismen der Proteinfaltung enthalten, sind jedoch kinetisch unzugänglich. Um den Prozess der Barrierebildung und die strukturellen Merkmale von Übergangszuständen aufzudecken, werden Proteine genutzt, die über barrierefreie Pfade falten – so genannte “downhill folder“. Aufgrund der geringen Faltungsbarrieren werden wichtige Interaktionen der Faltung zugänglich und erlauben Einblicke in die ratenbegrenzenden Faltungsvorgänge.
In dieser Arbeit vergleichen wir die Faltungsdynamiken von drei verschiedenen Varianten eines Lambda-Repressor-Fragments, bestehend aus den Aminosäuren 6 bis 85: ein Zwei-Zustands-Falter λWT (Y22W) und zwei downhill-folder-artige Varianten, λYA (Y22W/Q33Y/ G46,48A) und λHA (Y22W/Q33H/G46,48A). Um auf die Kinetik und die strukturelle Dynamik zu greifen zu können, werden Einzelmolekülkraftspektroskopische Experimente mit optische Pinzetten mit Submillisekunden- und Nanometer-Auflösung verwendet. Ich fand, dass die niedrige denaturierende Kraft die Mikrosekunden Faltungskinetik von downhill foldern auf eine Millisekunden-Zeitskala verlangsamt, sodass das System für Einzelmolekülstudien gut zugänglich ist.
Interessanterweise zeigten sich unter Krafteinwirkung die downhill-folder-artigen Varianten des Lambda-Repressors als kooperative Zwei-Zustands-Falter mit deutlich unterschiedlicher Faltungskinetik und Kraftabhängigkeit. Drei Varianten des Proteins zeigten ein hoch konformes Verhalten unter Last. Die modellfreie Rekonstruktion von Freien-Enthalpie-Landschaften ermöglichte es uns, die feinen Details der Transformation des Zwei-Zustands-Faltungspfad direkt in einen downhill-artigen Pfad aufzulösen. Die Auswirkungen von einzelnen Mutationen auf die Proteinstabilität, Bildung der Übergangszustände und die konformationelle Heterogenität der Faltungs- und Entfaltungszustände konnten beobachtet werden.
Interessanterweise zeigen unsere Ergebnisse, dass sich die untersuchten Varianten trotz der ultraschnellen Faltungszeit im Bereich von 2 μs in einem kooperativen Prozess über verbleibende Energiebarrieren falten und entfalten, was darauf hindeutet, dass wesentlich schnellere Faltungsraten notwendig sind um ein downhill Limit vollständig zu erreichen.:I Theoretical background 1
1 Introduction 3
2 Protein folding: the downhill scenario 5
2.1 Protein folding as a diffusion on a multidimensional energy landscape 5
2.2 Downhill folding proteins 7
2.2.1 Thermodynamic description of downhill folders 7
2.2.2 Identification criteria for downhill folders 8
2.3 Lambda repressor as a model system for studying downhill folding 9
2.3.1 Wild-type lambda repressor fragment λ{6-85} 10
2.3.2 Acceleration of λ{6-85} folding by specifific point mutations 11
2.3.3 The incipient-downhill λYA and downhill λHA variants 14
2.4 Single-molecule techniques as a promising tool for probing downhill folding dynamics 17
3 Single-molecule protein folding with optical tweezers 19
3.1 Optical tweezers 19
3.1.1 Working principle of optical tweezers 19
3.1.2 The optical tweezers setup 21
3.2 The dumbbell assay 22
3.3 Measurement protocols 23
3.3.1 Constant-velocity experiments 23
3.3.2 Constant-trap-distance experiments (equilibrium experiments) 24
4 Theory and analysis of single-molecule trajectories 27
4.1 Polymer elasticity models 27
4.2 Equilibrium free energies of protein folding in optical tweezers 28
4.3 Signal-pair correlation analysis 29
4.4 Force dependence of transition rate constants 29
4.4.1 Zero-load extrapolation of rates: the Berkemeier-Schlierf model 30
4.4.2 Detailed balance for unfolding and refolding data 31
4.5 Direct measurement of the energy landscape via deconvolution 32
II Results 33
5 Efficient strategy for protein-DNA hybrid formation 35
5.1 Currently available strategies for protein-DNA hybrid formation 35
5.2 Novel assembly of protein-DNA hybrids based on copper-free click chemistry 37
5.3 Click-chemistry based assembly preserves the native protein structure 40
5.4 Summary 42
6 Non-equilibrium mechanical unfolding and refolding of lambda repressor variants 45
6.1 Non-equilibrium unfolding and refolding of lambda repressor λWT 45
6.2 Non-equilibrium unfolding and refolding of incipient-downhill λYA and downhill λHA variants of lambda repressor 48
6.3 Summary 52
7 Equilibrium unfolding and refolding of lambda repressor variants 53
7.1 Importance of the trap stiffness to resolve low-force nanometer transitions 54
7.2 Signal pair-correlation analysis to achieve millisecond transitions 56
7.3 Force-dependent equilibrium kinetics of λWT 59
7.4 Equilibrium folding of incipient-downhill λYA and downhill λHA variants of lambda repressor 61
7.5 Summary 65
8 Model-free energy landscape reconstruction for λWT, incipient-downhill λYA and downhill λHA variants 69
8.1 Direct observation of the effect of a single mutation on the conformational heterogeneity and protein stability 71
8.2 Artifacts of barrier-height determination during deconvolution 75
8.3 Summary 76
9 Conclusions and Outlook 79 / Protein folding is a process of molecular self-assembly in which a linear chain of amino acids assembles into a defined, functional three-dimensional structure. The process of folding is a thermally driven diffusive search on a free-energy landscape in the conformational space for the minimal-energy structure. During that process, the free energy of the system does not always show a monotonic decrease; instead, sub-optimal compensation of enthalpy and entropy change during each folding step leads to formation of folding free-energy barriers. However, these barriers, and associated high-energy transition states, that contain key information about mechanisms of protein folding, are kinetically inaccessible. To reveal the barrier-formation process and structural characteristics of transition states, proteins are employed that fold via barrierless paths – so-called downhill folders. Due to the low folding barriers, the key folding interactions become accessible, yielding insights about the rate-limiting folding events.
Here, I compared the folding dynamics of three different variants of a lambda repressor fragment, containing amino acids 6 to 85: a two-state folder λWT (Y22W) and two downhill-like folding variants, λYA (Y22W/Q33Y/G46,48A) and λHA (Y22W/Q33H/G46,48A). To access the kinetics and structural dynamics, single-molecule optical tweezers with submillisecond and nanometer resolution are used. I found that force perturbation slowed down the microsecond kinetics of downhill folders to a millisecond time-scale, making it accessible to single-molecule studies.
Interestingly, under load, the downhill-like variants of lambda repressor appeared as cooperative two-state folders with significantly different folding kinetics and force dependence. The three protein variants displayed a highly compliant behaviour under load. Model-free reconstruction of free-energy landscapes allowed us to directly resolve the fine details of the transformation of the two-state folding path into a downhill-like path. The effect of single mutations on protein stability, transition state formation and conformational heterogeneity of folding and unfolding states was observed.
Noteworthy, our results demonstrate, that despite the ultrafast folding time in a range of 2 µs, the studied variants fold and unfold in a cooperative process via residual barriers, suggesting that much faster folding rate constants are required to reach the full-downhill limit.:I Theoretical background 1
1 Introduction 3
2 Protein folding: the downhill scenario 5
2.1 Protein folding as a diffusion on a multidimensional energy landscape 5
2.2 Downhill folding proteins 7
2.2.1 Thermodynamic description of downhill folders 7
2.2.2 Identification criteria for downhill folders 8
2.3 Lambda repressor as a model system for studying downhill folding 9
2.3.1 Wild-type lambda repressor fragment λ{6-85} 10
2.3.2 Acceleration of λ{6-85} folding by specifific point mutations 11
2.3.3 The incipient-downhill λYA and downhill λHA variants 14
2.4 Single-molecule techniques as a promising tool for probing downhill folding dynamics 17
3 Single-molecule protein folding with optical tweezers 19
3.1 Optical tweezers 19
3.1.1 Working principle of optical tweezers 19
3.1.2 The optical tweezers setup 21
3.2 The dumbbell assay 22
3.3 Measurement protocols 23
3.3.1 Constant-velocity experiments 23
3.3.2 Constant-trap-distance experiments (equilibrium experiments) 24
4 Theory and analysis of single-molecule trajectories 27
4.1 Polymer elasticity models 27
4.2 Equilibrium free energies of protein folding in optical tweezers 28
4.3 Signal-pair correlation analysis 29
4.4 Force dependence of transition rate constants 29
4.4.1 Zero-load extrapolation of rates: the Berkemeier-Schlierf model 30
4.4.2 Detailed balance for unfolding and refolding data 31
4.5 Direct measurement of the energy landscape via deconvolution 32
II Results 33
5 Efficient strategy for protein-DNA hybrid formation 35
5.1 Currently available strategies for protein-DNA hybrid formation 35
5.2 Novel assembly of protein-DNA hybrids based on copper-free click chemistry 37
5.3 Click-chemistry based assembly preserves the native protein structure 40
5.4 Summary 42
6 Non-equilibrium mechanical unfolding and refolding of lambda repressor variants 45
6.1 Non-equilibrium unfolding and refolding of lambda repressor λWT 45
6.2 Non-equilibrium unfolding and refolding of incipient-downhill λYA and downhill λHA variants of lambda repressor 48
6.3 Summary 52
7 Equilibrium unfolding and refolding of lambda repressor variants 53
7.1 Importance of the trap stiffness to resolve low-force nanometer transitions 54
7.2 Signal pair-correlation analysis to achieve millisecond transitions 56
7.3 Force-dependent equilibrium kinetics of λWT 59
7.4 Equilibrium folding of incipient-downhill λYA and downhill λHA variants of lambda repressor 61
7.5 Summary 65
8 Model-free energy landscape reconstruction for λWT, incipient-downhill λYA and downhill λHA variants 69
8.1 Direct observation of the effect of a single mutation on the conformational heterogeneity and protein stability 71
8.2 Artifacts of barrier-height determination during deconvolution 75
8.3 Summary 76
9 Conclusions and Outlook 79
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