The ParC/ParP system in the localization and segregation of chemotaxis signaling arrays in Vibrio cholerae and Vibrio parahaemolyticus

Chemotaxis proteins organize into large, highly ordered arrays. Particularly, in the enteric bacteria Vibrio cholerae and Vibrio parahaemolyticus, chemotaxis arrays are found at the cell pole, and their distribution follows a cell cycle dependent localization. The ParC/ParP system mediates this localization pattern and without either ParC or ParP, arrays are no longer positioned at the cell poles and fail to segregate upon division. Localization of arrays in these bacteria follow a hierarchical process, where arrays are tethered by ParP, which in turn links them to ParC, an ATPase that serves as a cell pole determinant in Vibrios. Here, we analyze the mechanism behind ParP’s ability to access the chemotaxis arrays and positions them at the cell pole. Furthermore, we show that even in the absence of histidine kinase CheA proteins, the arrays still exhibit the native spatial localization and the iconic hexagonal packing of the receptors. We show that the V. cholerae Cluster II array is versatile in respect of array composition for auxiliary chemotaxis proteins, such as ParP and that these arrays are structurally less stable due to their lower CheA occupancy in comparison to the ultrastable arrays found in E.coli. Additionally, we examine the dynamic localization of ParC and evaluate its influence in the overall localization of the arrays and ParP. We show that ParP’s C-terminus integrates into the core unit of signaling arrays through interactions with MCP proteins and the histidine kinase CheA. Our results indicate that ParP’s intercalation within the core units facilitates array formation, whereas its N-terminal interaction domain enables polar recruitment of arrays and promotes ParP’s own polar localization. Moreover, the data provides evidence that ParP serves as a critical nexus between the formation of the chemotactic arrays and their proper polar recruitment. Additionally, our data revealed that arrays in V. cholerae have the capacity to include several scaffolding proteins, displaying a previously uncharacterized variability. In turn, we demonstrate that this variability explains the high degree of structural instability shown by V. cholerae chemotaxis arrays. Finally, we show that ParC forms a protein gradient in V. parahaemolyticus cells. This protein gradient extends in a decreasing concentration from the cell pole towards mid-cell, and it is essential for ParC’s function in positioning ParP and consequently the chemosensory arrays. Similarly, gradient maintenance requires a continuous cycle of ParC between the cell pole and the cytoplasm, as well as ParC’s ability to associate with DNA and transition into different protein states in a nucleotide dependent manner. The data shows that ParC’s localization dynamics relies upon differential diffusion rates of its distinct protein states. Altogether, this work studies the complexity of the ParC/ ParP system and highlights the importance of each component in the correct placement of the chemotactic signaling arrays

Improvements of the Density-Functional Tight-Binding Parameters and Their Application in Histidine Kinase

Zweikomponentensysteme (Two Component Systems, TCS) sind einer der wichtigsten Signaltransduktionswege in Bakterien. Durch dieses System nehmen Bakterien ihre Umgebung wahr und regulieren ihre zellulären Aktivitäten. Histidinkinasen (HK) sind ein wichtiger Bestandteil von TCS. Obwohl TCS von einigen Eukaryoten genutzt werden, fehlen sie insbesondere im Tierreich. Aus diesem Grund und wegen ihrer Bedeutung für den bakteriellen Lebenszyklus sind Histidinkinasen ein vielversprechendes Ziel für die Entwicklung von Medikamenten zur Bekämpfung bakterieller Aktivitäten. Sie sind außerdem strukturell konserviert und weisen einige gemeinsame intermolekulare Funktionalitäten in jedem TCS auf, nämlich Autokinase-, Phosphotransfer- und Phosphatase-Aktivitäten. Diese Vorgänge sind für Biochemiker äußerst faszinierend, um bakterielle Aktivitäten zu verstehen. Das Hauptziel dieser Arbeit ist die Analyse der Histidin-Phosphorylierung, einem wichtigen Vorgang in TCS. Wir haben die chemischen Schritte der Autophosphorylierung in einer umfassenden QM/MM Hybrid Enhanced Sampling Simulation untersucht und den detaillierten Mechanismus aufgedeckt. Die darauffolgende Autophosphorylierung innerhalb der DHp-Domäne verläuft über einen pentakoordinierten Übergangszustand zu einem protonierten Phosphohistidin-Intermediat. Dieses wird anschließend durch eine geeignete benachbarte Base deprotoniert. Die Energetik der Reaktion wird durch den endgültigen Protonenakzeptor und die Anwesenheit eines Magnesiumkations gesteuert. Wir haben die DFTB3-Parameter für die Phosphor-Stickstoff-Wechselwirkung neu parametrisiert und an einer Krebsmedikamenten-Hydrolysereaktion gemessen. Anschließend wendeten wir diese Parameter auf unser Hauptziel, die Untersuchung der Reaktion in der Histidin-Kinase in einer QM/MM-Simulation, an. Des Weiteren haben wir ein künstliches neuronales Netz auf dieselbe Arzneimittelhydrolysereaktion angewendet, um die DFTB-Energien als alternative Methode quantitativ zu verbessern. Darüber hinaus haben wir auch die Schwefel-Schwefel-Abstoßungsparameter neu parametrisiert, um die Disulfid-Thiol-Austauschreaktion zu verbessern, und die potenzielle Energie des DFT(B3LYP)-Niveaus mit DFTB reproduziert. Dies wurde anschließend auf die Disulfid-Thiol-Austauschreaktion in QM/MM-Simulationen von Proteinen angewandt

Principles and theory of protein-based pattern formation

Biological systems perform functions by the orchestrated interplay of many small components without a "conductor." Such self-organization pervades life on many scales, from the subcellular level to populations of many organisms and whole ecosystems. On the intracellular level, protein-based pattern formation coordinates and instructs functions like cell division, differentiation and motility. A key feature of protein-based pattern formation is that the total numbers of the involved proteins remain constant on the timescale of pattern formation. The overarching theme of this thesis is the profound impact of this mass-conservation property on pattern formation and how one can harness mass conservation to understand the underlying physical principles. The central insight is that changes in local densities shift local reactive equilibria, and thus induce concentration gradients which, in turn, drive diffusive transport of mass. For two-component systems, this dynamic interplay can be captured by simple geometric objects in the (low-dimensional) phase space of chemical concentrations. On this phase-space level, physical insight can be gained from geometric criteria and graphical constructions. Moreover, we introduce the notion of regional (in)stabilities, which allows one to characterize the dynamics in the highly nonlinear regime reveals an inherent connection between Turing instability and stimulus-induced pattern formation. The insights gained for conceptual two-component systems can be generalized to systems with more components and several conserved masses. In the minimal setting of two diffusively coupled "reactors," the full dynamics can be embedded in the phase-space of redistributed masses where the phase space flow is organized by surfaces of local reactive equilibria. Building on the phase-space analysis for two component systems, we develop a new approach to the important open problem of wavelength selection in the highly nonlinear regime. We show that two-component reaction–diffusion systems always exhibit uninterrupted coarsening (the continual growth of the characteristic length scale) of patterns if they are strictly mass conserving. Selection of a finite wavelength emerges due to weakly broken mass-conservation, or coupling to additional components, which counteract and stop the competition instability that drives coarsening. For complex dynamical phenomena like wave patterns and the transition to spatiotemporal chaos, an analysis in terms of local equilibria and their stability properties provides a powerful tool to interpret data from numerical simulations and experiments, and to reveal the underlying physical mechanisms. In collaborations with different experimental labs, we studied the Min system of Escherichia coli. A central insight from these investigations is that bulk-surface coupling imparts a strong dependence of pattern formation on the geometry of the spatial confinement, which explains the qualitatively different dynamics observed inside cells compared to in vitro reconstitutions. By theoretically studying the polarization machinery in budding yeast and testing predictions in collaboration with experimentalists, we found that this functional module implements several redundant polarization mechanisms that depend on different subsets of proteins. Taken together, our work reveals unifying principles underlying biological self-organization and elucidates how microscopic interaction rules and physical constraints collectively lead to specific biological functions.Biologische Systeme führen Funktionen durch das orchestrierte Zusammenspiel vieler kleiner Komponenten ohne einen "Dirigenten" aus. Solche Selbstorganisation durchdringt das Leben auf vielen Skalen, von der subzellulären Ebene bis zu Populationen vieler Organismen und ganzen Ökosystemen. Auf der intrazellulären Ebene koordiniert und instruieren proteinbasierte Muster Funktionen wie Zellteilung, Differenzierung und Motilität. Ein wesentliches Merkmal der proteinbasierten Musterbildung ist, dass die Gesamtzahl der beteiligten Proteine auf der Zeitskala der Musterbildung konstant bleibt. Das übergreifende Thema dieser Arbeit ist es, den tiefgreifenden Einfluss dieser Massenerhaltung auf die Musterbildung zu untersuchen und Methoden zu entwickeln, die Massenerhaltung nutzen, um die zugrunde liegenden physikalischen Prinzipien von proteinbasierter Musterbildung zu verstehen. Die zentrale Erkenntnis ist, dass Änderungen der lokalen Dichten lokale reaktive Gleichgewichte verschieben und somit Konzentrationsgradienten induzieren, die wiederum den diffusiven Transport von Masse antreiben. Für Zweikomponentensysteme kann dieses dynamische Wechselspiel durch einfache geometrische Objekte im (niedrigdimensionalen) Phasenraum der chemischen Konzentrationen erfasst werden. Auf dieser Phasenraumebene können physikalische Erkenntnisse durch geometrische Kriterien und grafische Konstruktionen gewonnen werden. Darüber hinaus führen wir den Begriff der regionalen (In-)stabilität ein, der es erlaubt, die Dynamik im hochgradig nichtlinearen Regime zu charakterisieren und einen inhärenten Zusammenhang zwischen Turing-Instabilität und stimulusinduzierter Musterbildung aufzuzeigen. Die für konzeptionelle Zweikomponentensysteme gewonnenen Erkenntnisse können auf Systeme mit mehr Komponenten und mehreren erhaltenen Massen verallgemeinert werden. In der minimalen Fassung von zwei diffusiv gekoppelten "Reaktoren" kann die gesamte Dynamik in den Phasenraum umverteilter Massen eingebettet werden, wobei der Phasenraumfluss durch Flächen lokaler reaktiver Gleichgewichte organisiert wird. Aufbauend auf der Phasenraumanalyse für Zweikomponentensysteme entwickeln wir einen neuen Ansatz für die wichtige offene Fragestellung der Wellenängenselektion im hochgradig nichtlinearen Regime. Wir zeigen, dass "coarsening" (das stetige wachsen der charakteristischen Längenskala) von Mustern in Zweikomponentensystemen nie stoppt, wenn sie exakt massenerhaltend sind. Die Selektion einer endlichen Wellenlänge entsteht durch schwach gebrochene Massenerhaltung oder durch Kopplung an zusätzliche Komponenten. Diese Prozesse wirken der Masseumverteilung, die coarsening treibt, entgegen und stoppen so das coarsening. Bei komplexen dynamischen Phänomenen wie Wellenmustern und dem Übergang zu raumzeitlichen Chaos bietet eine Analyse in Bezug auf lokale Gleichgewichte und deren Stabilitätseigenschaften ein leistungsstarkes Werkzeug, um Daten aus numerischen Simulationen und Experimenten zu interpretieren und die zugrunde liegenden physikalischen Mechanismen aufzudecken. In Zusammenarbeit mit verschiedenen experimentellen Labors haben wir das Min-System von Escherichia coli untersucht. Eine zentrale Erkenntnis aus diesen Untersuchungen ist, dass die Kopplung zwischen Volumen und Oberfläche zu einer starken Abhängigkeit der Musterbildung von der räumlichen Geometrie führt. Das erklärt die qualitativ unterschiedliche Dynamik, die in Zellen im Vergleich zu in vitro Rekonstitutionen beobachtet wird. Durch die theoretische Untersuchung der Polarisationsmaschinerie in Hefezellen, kombiniert mit experimentellen Tests theoretischer Vorhersagen, haben wir herausgefunden, dass dieses Funktionsmodul mehrere redundante Polarisationsmechanismen implementiert, die von verschiedenen Untergruppen von Proteinen abhängen. Zusammengenommen beleuchtet unsere Arbeit die vereinheitlichenden Prinzipien, die der intrazellulären Selbstorganisation zugrunde liegen, und zeigt, wie mikroskopische Interaktionsregeln und physikalische Bedingungen gemeinsam zu spezifischen biologischen Funktionen führen

In vivo and in vitro analysis of RNases in Bacillus subtilis

RNA degradation is a key process in the control of gene expression in bacteria and is essential for the cell’s homeostasis of nucleotide pools. A key player is the so-called RNA degradosome, proposed to be a membrane-associated complex containing endo- and exoribunucleases, as well as glycolytic enzymes and a DEAD-box RNA helicase. It is believed that endonuclease RNase Y is central to the formation of the RNA degradosome in Bacillus subtilis, leading to recruitment of RNases PnpA, RNase J1, and RNase J2, RNA helicase CshA, as well as glycolytic enzymes enolase and phosphofructokinase occurs. RNase Y has a transmembrane helix, and is "quasi“ essential; it is also required for mRNA processing following transcription. RNase Y also interacts with the so-called Y‑complex, consisting of YaaT, YlbF, and, YmcA (RicT, RicF, RicA), which is important for RNase Y-mediated processing of mRNA. How RNase Y can operate in two different protein complexes and acts in RNA decay as well as transcription-associated processes, is unclear. In this work, I show that the RNA degradosome is quite dynamic, having RNase Y, the glycolytic enzyme enolase, and the RNA helicase CshA and PnpA as central parts strongly reacting to a block in transcription, and thus to loss of mRNA substrate, while RNase J1 and J2 as well as the glycolytic enzyme phosphofructokinase show a much weaker response and are thus likely more peripheral components. The Y-complex clearly shows diffusion within the cytosol, but also the formation of membrane-associated accumulations, dissociating when transcription is blocked. Single molecule tracking (SMT) shows that the loss of one component of the Y-complex does not strongly affect the dynamics of the other proteins, suggesting that the complex forms a flexible association rather than a 1:1:1 stoichiometry. Biochemical analyses suggest that YaaT forms a membrane-anchor for the Y-complex, although it also has a cytosolic, freely diffusing fraction. A model will be presented that the Y-complex could function as an adaptor between nucleoid-associated mRNA synthesis and membrane-associated processing and degradation. II My thesis also presents a protocol for the successful purification of membrane-associated RNase Y, as a basis for further biochemical characterization of the protein, and interaction studies. Another essential process in which RNases play a crucial role is DNA replication. In addition to the RNA primers, which are necessary for the placement of Okazaki fragments, DNA/RNA hybrids must be processed and RNA must be removed to ensure the stability of the DNA. A part of the thesis work shows that B. subtilis replication forks intimately employ two RNases of the „H“ family, DNA polymerase A and exonuclease ExoR in vivo. Recruitment appears to be based on substrate availability rather than on specific protein/protein interactions, involving redundant enzymatic activities

ASSESSING THE IMPACT OF SUBMERGED VEGETATION ON METHANE DYNAMICS IN A DISCONTINUOUS PERMAFROST LAKE SYSTEM, ABISKO, SWEDEN

Across the Arctic, postglacial lakes contribute a substantial amount of the total atmospheric methane (CH4), and their emissions are predicted to increase. However, there is still much uncertainty as to the contribution of northern water bodies to atmospheric CH4 emissions. This is mainly due to the spatiotemporal variability of the predominant pathway of emission from high latitude lakes: ebullition (bubbling). There are a myriad of factors that affect ebullition fluxes, including solar radiation input and atmospheric pressure, which make it difficult to model the impact on regional emissions. Very few studies have correlated sediment characteristics and submerged vegetation density with ebullition, to see what drives the variation across space and time. This study investigated the effect of submerged aquatic macrophyte (SAM) species distribution and abundance on CH4 dynamics in three postglacial lakes in Stordalen Mire, near Abisko, Sweden (68°21\u27N, 18°49\u27E). Submerged vegetation density maps developed from vegetation transects and sediment geochemistry derived from sediment cores were compared to ebullitive flux measured with bubble traps,. The source contribution of terrestrial and aquatic vegetation to the lake sediment carbon (C), the substrate for methanogens, was investigated using δ13C stable isotope analysis and organic carbon-to-nitrogen (C:N) elemental analyses. These data suggest that the organic C in postglacial subarctic lakes are a mixture of allochthonous and autochthonous inputs, with significant C being added by the in-situ decay of submerged vegetation, providing annual organic matter to the sediment. It was found that submerged vegetation density does not influence sediment CH4 concentrations, but rather, among shallow zone cores, the physical structure of the sediments drives most of the variation in ebullitive flux. Among shallow zones, the best predictor of overlying CH4 ebullitive efflux is the sediment porosity. It was also found that total sediment CH4 concentration has a strong negative relationship with ebullitive efflux, meaning that high sediment CH4 concentration is not an indication of high ebullition potential. Increased macrophyte density was not observed to ‘fertilize’ the sediment with organic C, nor did submerged vegetation density have any observed effect on sediment CH4 concentrations, downcore geochemistry, or ebullitive flux. Findings suggest that in a system that is not C-limited, it is perhaps the C quality and not the C quantity that drives the variability in methanogenesis. An investigation into which microbial communities exist in these sediments and in what abundance is required. These data also suggest that anaerobic oxidation of methane (AOM) might also be occurring even in freshwater lake environments, a finding that is implicative in terms of our understanding and modeling of CH4 ebullition and emissions across the Arctic, perhaps yielding new insights into how net emissions might change in the future

Probing structure, function and dynamics in bacterial primary and secondary transporter-associated binding proteins

Substrate binding proteins (SBPs) are ubiquitous in all life forms and have evolved to perform diverse physiological functions, such as in membrane transport, gene regulation, neurotransmission, and quorum sensing. It is quite astounding to observe such functional diversity among the SBPs even when they are restricted by their fold space. Therefore, the SBPs are an excellent set of proteins that can reveal how proteins evolution novel function in a structurally conserved/constrained fold. This study attempts to understand the phenomenon of affinity and specificity evolution in SBPs by combining a set of biochemical, biophysical, and structural studies on the SBPs involved in translocation of substrates across the membrane using ATP-binding cassette (ABC) transporters and tripartite ATP-independent periplasmic (TRAP) transporters in gram-negative bacteria Thermotoga maritima and Pseudomonas aeruginosa, respectively. Additionally, experimental, and computational methods were used in conjunction to highlight the variation in the dynamics of these SBPs. The results from this study highlight an intricate role of dynamics in complementing the structural alterations that are required for high-affinity ligand binding. Moreover, first ever neutron structure of a SBP was determined during my study to delineate the extensive network of water in the binding cavities of the SBPs that help stabilize larger substrates by forming water-mediated hydrogen bond interactions with the bound substrates. Furthermore, structures of two SBPs from T. maritima were determined in both substrate-free (apo) and substrate-bound (holo) forms and subsequently used for computational molecular dynamics simulation to determine the variation in dynamics due to substrate-binding. The novel TRAP SBP identified in P. aeruginosa was identified as a promiscuous binder of several tricarboxylic acid cycle (TCA) cycle intermediates. A total of six SBP structures were determined using X-ray crystallography and one SBP structure was determined using neutron crystallography. Finally, experimental neutron scattering was used to experimentally characterize the picosecond to nanosecond dynamics in SBPs and highlighted differences in the translational, rotational, and internal dynamical signatures of two SBP isoforms. Overall, the findings of this study can be broadly applied in biotechnology and biosensor development by artificially engineer affinity or specificity for a particular ligand

Uncovering the molecular basis of compartmentalization as a principle of neuronal organization

Cells are faced with coordinating countless, simultaneous, partly antagonistic biochemical reactions. This is especially true for neurons that must orchestrate the complex task of neurotransmission. One solution to this problem is the formation of specialized compartments. To understand the molecular mechanisms of such compartments this thesis investigates two system in neuronal cells: i. the plasma membrane and its underlying cytoskeleton and ii. synaptic vesicle clusters inside synaptic boutons. Towards this end, a combinatorial approach of computational modeling, single particle tracking and super-resolution microscopy is employed. A periodic array of actin rings in the neuronal axon initial segment has been known to confine membrane protein motion. Still, a local enrichment of ion channels offers an alternative explanation. Using computational modeling this thesis now shows that ion channels, in contrast to actin rings, cannot mediate confinement. Furthermore, by employing single particle tracking and super-resolution microscopy, this work shows that actin rings are close to the plasma membrane and that actin rings confine membrane proteins in several neuronal cell types. Further, it is shown that actin ring disruption leads to a reduction of membrane compartmentalization. Synaptic boutons in the axon of neurons are the location of synaptic vesicle release. Synaptic vesicles form dense clusters inside boutons, that are essential for pre-synaptic function. In vitro experiments have suggested that the soluble phosphoprotein synapsin 1 controls synaptic clustering via liquid liquid phase separation. However, the in vivo mechanism remains elusive. This work now shows via two-color single molecule tracking in live neurons that synapsin 1 drives synaptic vesicle clustering. Furthermore, using a synapsin knock-out model it is shown that synapsin 1 controls the mobility of synaptic vesicles through its intrinsically disordered region, which is responsible for phase separation. By studying the dynamics of compartmentalized systems in neuronal cells this work uncovers two molecular mechanisms: actin rings form membrane diffusion barriers and synapsin 1 controls synaptic vesicle clustering and mobility through liquid liquid phase separation. Thus, this thesis makes important strides towards deepening the understanding of neuronal function by uncovering how compartmentalization operates in both the plasma membrane and the cytosol of neuronal cells.Zellen müssen unzählige, gleichzeitige, teilweise gegensätzliche biochemische Reaktionen koordinieren. Dies gilt insbesondere für Neuronen, die die komplexe Aufgabe der Neurotransmission koordinieren. Eine Lösung für dieses Problem ist die Bildung spezialisierter Kompartimente. Um die molekulare Funktionsweise solcher Kompartimente zu verstehen, werden in dieser Arbeit zwei Systeme in neuronalen Zellen untersucht: i. die Plasmamembran und das darunterliegende Zytoskelett und ii. synaptische Vesikel-Cluster in Boutons. Zu diesem Zweck, wurde ein kombinatorischer Ansatz aus Computermodellierung, Einzelpartikelverfolgung und superauflösender Mikroskopie verwendet. Periodische Aktinringe im neuronalen Axoninitialsegment schränken die Bewegung von Membranproteinen ein. Jedoch liefert eine lokale Anreicherung von Ionenkanälen eine alternative Erklärung. Durch Computermodellierung wird in dieser Arbeit nun gezeigt, dass Ionenkanäle keine Einschränkung der Membranmolekülbewegung bewirken. Darüber hinaus wird durch Einzelpartikelverfolgung und superauflösende Mikroskopie gezeigt, dass Aktinringe nahe der Plasmamembran sind und dass Aktinringe Membranproteine in verschiedenen neuronalen Zelltypen in ihrer Bewegung einschränken. Weiterhin wird gezeigt, dass die Zerstörung der Aktinringe Membrankompartmentalisierung reduziert. Synaptische Boutons im Axon sind der Ort der Freisetzung synaptischer Vesikel. Synaptische Vesikel bilden dichte Cluster in Boutons, welche für die Funktion der Präsynapse essenziell sind. In vitro Experimente haben gezeigt, dass das lösliche Phosphoprotein Synapsin 1 das Clustern durch Flüssig-Flüssig-Phasentrennung steuert, der Mechanismus in vivo ist jedoch unklar. Diese Arbeit zeigt nun mittels Zweifarben-Einzelmolekülverfolgung in lebenden Neuronen, dass Synapsin 1 das Clustern synaptischer Vesikel steuert. Anhand eines Synapsin-Knock-out-Modells wird gezeigt, dass Synapsin 1 die Mobilität synaptischer Vesikel durch seine intrinsisch ungeordnete Region kontrolliert, die für die Phasentrennung verantwortlich ist. Durch Untersuchungen der Dynamik kompartmentalisierter Systeme in neuronalen Zellen deckt diese Arbeit zwei molekulare Mechanismen auf: Aktinringe bilden Membrandiffusionsbarrieren und Synapsin 1 steuert Clustern und Mobilität synaptischer Vesikel durch Flüssig-Flüssig-Phasentrennung. Somit macht diese Arbeit wichtige Fortschritte zum Verständnis der Funktionsweise neuronaler Zellen, indem sie aufdeckt, wie die Kompartmentalisierung der Plasmamembran und des Zytosols gesteuert wird

The Role of Mutations in Protein Structural Dynamics and Function: A Multi-scale Computational Approach

abstract: Proteins are a fundamental unit in biology. Although proteins have been extensively studied, there is still much to investigate. The mechanism by which proteins fold into their native state, how evolution shapes structural dynamics, and the dynamic mechanisms of many diseases are not well understood. In this thesis, protein folding is explored using a multi-scale modeling method including (i) geometric constraint based simulations that efficiently search for native like topologies and (ii) reservoir replica exchange molecular dynamics, which identify the low free energy structures and refines these structures toward the native conformation. A test set of eight proteins and three ancestral steroid receptor proteins are folded to 2.7Å all-atom RMSD from their experimental crystal structures. Protein evolution and disease associated mutations (DAMs) are most commonly studied by in silico multiple sequence alignment methods. Here, however, the structural dynamics are incorporated to give insight into the evolution of three ancestral proteins and the mechanism of several diseases in human ferritin protein. The differences in conformational dynamics of these evolutionary related, functionally diverged ancestral steroid receptor proteins are investigated by obtaining the most collective motion through essential dynamics. Strikingly, this analysis shows that evolutionary diverged proteins of the same family do not share the same dynamic subspace. Rather, those sharing the same function are simultaneously clustered together and distant from those functionally diverged homologs. This dynamics analysis also identifies 77% of mutations (functional and permissive) necessary to evolve new function. In silico methods for prediction of DAMs rely on differences in evolution rate due to purifying selection and therefore the accuracy of DAM prediction decreases at fast and slow evolvable sites. Here, we investigate structural dynamics through computing the contribution of each residue to the biologically relevant fluctuations and from this define a metric: the dynamic stability index (DSI). Using DSI we study the mechanism for three diseases observed in the human ferritin protein. The T30I and R40G DAMs show a loss of dynamic stability at the C-terminus helix and nearby regulatory loop, agreeing with experimental results implicating the same regulatory loop as a cause in cataracts syndrome.Dissertation/ThesisPh.D. Physics 201

THE BALANCING ACT OF CYTOKININ IN ENVIRONMENTAL STRESS TOLERANCE

Cytokinin, long known as a phytohormone that regulates plant growth and development, has been recently recognized as an important regulator of stress responses. However, our current knowledge about the mechanisms by which cytokinin regulates stress responses is fragmentary, as many of the studies in this field yielded conflicting results. Most of the work described here has focused on analyses of the molecular mechanisms of cytokinin-dependent regulation of growth and development under stress conditions, with an emphasis on the role of cytokinin-dependent regulation of protein synthesis in development and stress tolerance. One of the important contributions of this study is the finding that cytokinin- dependent induction of protein synthesis requires both the canonical cytokinin signaling pathway and isoforms RPL4A and RPL4D of the RIBOSOMAL PROTEIN L4. Further analyses investigated the role of the cytokinin-dependent increase in protein synthesis in stress responses, and this research underlined the importance of balanced regulation of cytokinin-induced protein synthesis. For example, Arabidopsis lines in which cytokinin action is increased have increased protein synthesis but are growth-retarded and have decreased osmotic stress tolerance. Both the osmotic stress hypersensitivity and plant growth retardation of these cytokinin gain-of-function lines can be reversed to the wild- type level by lowering their protein synthesis levels. These cytokinin gain-of-function lines, on the other hand, are more tolerant to heat and oxidative stress, indicating that optimal cytokinin action represents a balancing act in maintaining tolerance levels to a range of abiotic stresses

Accurate and Robust Mechanical Modeling of Proteins

Through their motion, proteins perform essential functions in the living cell. Although we cannot observe protein motion directly, over 68,000 crystal structures are freely available from the Protein Data Bank. Computational protein rigidity analysis systems leverage this data, building a mechanical model from atoms and pairwise interactions determined from a static 3D structure. The rigid and flexible components of the model are then calculated with a pebble game algorithm, predicting a protein\u27s flexibility with much more computational efficiency than physical simulation. In prior work with rigidity analysis systems, the available modeling options were hard-coded, and evaluation was limited to case studies. The focus of this thesis is improving accuracy and robustness of rigidity analysis systems. The first contribution is in new approaches to mechanical modeling of noncovalent interactions, namely hydrogen bonds and hydrophobic interactions. Unlike covalent bonds, the behavior of these interactions varies with their energies. I systematically investigate energy-refined modeling of these interactions. Included in this is a method to assign a score to a predicted cluster decomposition, adapted from the B-cubed score from information retrieval. Another contribution of this thesis is in new approaches to measuring the robustness of rigidity analysis results. The protein\u27s fold is held in place by weak, noncovalent interactions, known to break and form during natural fluctuations. Rigidity analysis has been conventionally performed on only a single snapshot, rather than on an entire trajectory, and no information was made available on the sensitivity of the clusters to variations in the interaction network. I propose an approach to measure the robustness of rigidity results, by studying how detrimental the loss of a single interaction may be to a cluster\u27s rigidity. The accompanying study shows that, when present, highly critical interactions are concentrated around the active site, indicating that nature has designed a very versatile system for transitioning between unique conformations. Over the course of this thesis, we develop the KINARI library for experimenting with extensions to rigidity analysis. The modular design of the software allows for easy extensions and tool development. A specific feature is the inclusion of several modeling options, allowing more freedom in exploring biological hypotheses and future benchmarking experiments