Thesis advisor: Udayan Mohanty / The conformational dynamics plays a significant role in a wide range of biological systems, from small RNA molecules to the large-scale ribonucleoprotein assemblies, in which ions are found critical and have notable structural and functional impacts. In the glass-forming liquids, the structural dynamics also calls for further investigations and deeper understandings. To this end, using four distinct chapters, this dissertation discusses the ion-related conformational dynamics in various scales of biomolecular systems, as well as the fluctuation effects in the glass-forming liquids. In chapter 1, we investigate the dynamics of ions and water molecules in the outer solvation sphere of a widely studied 58-nucleotide rRNA fragment. Molecular dynamics (MD) simulations with explicit solvent molecules and atomic details are performed for the RNA fragment in ionic solution. We determine all of the association sites and spatial distributions of residence times for Mg2+, K+, and water molecules in those sites. In accordance to the analysis of the dynamics of the RNA fragment, we provide insights into how the dynamics of ions and water molecules are intricately linked with the kinetics of the RNA fragment. In addition, the long-lived sites for Mg2+ ions identified from the simulation agree with the metal ion locations determined in the X-ray structure. The excess ion atmosphere around the RNA fragment is calculated and compared with the experimental measures. The results from this study indicate that the 58-mer rRNA fragment in ionic solution forms a complex polymer that is encased by a fluctuating network of ions and water. In chapter 2, the conformational dynamics of a large-scale ribonucleoprotein assembly, ribosome, is studied with molecular dynamics simulations with a newly developed model that accounts for electrostatic and ionic effects on the biomolecules. In this study, an all-atom structure based model is constructed with explicit representations of non-hydrogen atoms from biomolecules and diffuse ions. Implicit treatment is applied to the solvent molecules with the solvation effect associated with diffuse ions described by effective potentials. Parameters in this model are refined against explicit solvent simulations and experimental measures. This model with refined parameters is able to capture the excess Mg2+ ions for prototypical RNA systems and their dependence on the Mg2+ concentrations. Motivated by this, we apply the model to a bacterial ribosome and find that the position of the extended L1 stalk region can be controlled by the diffuse ions. This simulation also indicated ion-induced long-range interactions between L1 stalk and tRNA, which provides insights into the impact of ions on the functional rearrangements of ribosome. In chapter 3, we focus on the dynamics of the glass-forming liquids. In this study, we generalized the Adam-Gibbs model of relaxation in glass-forming liquids and take into account the fluctuations in the number of molecules inside the cooperative rearranging region. We obtain the expressions of configurational fractions at the glass-transition temperature with and without the fluctuation effect in Adam-Gibbs model, and determine the configurational fraction for several glass-forming liquids at glass-transition temperature in the absence of fluctuation effects. A connection between the β Kohlrausch-Williams-Watts parameters and the configurational fraction at the glass-transition temperature is also reported in this study. In chapter 4, we apply the model developed in chapter 2 to a ribosome structure to investigate the effects of diffuse ions on the aminoacyl-tRNA (aa-tRNA) accommodation process. The aa-tRNA accommodation is a critical step in the tRNA selection process which serves the purpose of protein synthesis in the ribosome. Experimental and computational efforts were made to reveal the mechanism and the energetic properties of the accommodation process, while the effects from diffuse ions on this process remain elusive. To this end, we design and perform MD simulations of ribosome structure with different treatment of electrostatics and diffuse ions in the system. Simulations with various ionic concentrations are also performed to study the concentration effects. The simulation trajectories indicate that diffuse ions can facilitate the aa-tRNA accommodation process and stabilize the accommodated configurations. In addition, we observe that Mg2+ ions play critical roles in stabilizing the accommodated configurations and a few millimolar change of Mg2+ concentration can alter the tendency of the tRNA configurational change during the accommodation process. This result shed light on the investigations of suitable ionic environment for the tRNA selection in the ribosome. It will be fruitful to extend this strategy into the investigations of other conformational rearrangements in the ribosome, such as tRNA translocation and subunit rotation, which will provide us with deeper understanding about the functional mechanism of the ribosome. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

In this dissertation, I will review the history and general strategies for performing kinetic proteomics. I will then demonstrate that I have published an open source, user-friendly program for other scientists to use to perform kinetic proteomics data analysis, as well as publishing a novel discovery of key ribosomal subunits being replaced within the lifetime of the ribosome, which was discovered through use of kinetic proteomics. Finally, I will discuss work that is ongoing to improve my software tool for use in human subjects, and work being done to combine kinetic proteomics with other global analysis methods to make novel biological discoveries. Proteins are constantly synthesized and destroyed to ensure sufficient functioning proteins to meet cellular needs, a process called protein turnover. Synthesis and degradation are carefully balanced over time to ensure that average protein concentrations do not change drastically. The status quo of the cell, or protein homeostasis, is required for the health of the organism. If protein homeostasis breaks down, serious diseases, such as Alzheimers, can result when proteins aggregate instead of being degraded properly. Because protein turnover is the means to maintain protein homeostasis while keeping sufficient functioning proteins, measuring protein turnover is critical to understanding biological processes and disease states. Measuring protein turnover rates on a broad scale is possible using a method called kinetic proteomics, and the improvement of kinetic proteomics is where I have focused the work for this dissertation.

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[EN] Ribosome biogenesis is one of the most important and energy-consuming processes in the cell. However, the vast majority of the events and factors that are involved in the synthesis of ribosomal subunits are not well understood. Ribosome maturation comprises multiple steps of rRNA processing that require sequential association and dissociation of numerous assembly factors. These proteins establish a complex network of interactions that are essential for the pathway to continue. Extensive studies in Saccharomyces cerevisiae allowed to identify some of the genetic and functional correlations between the pre-ribosomal factors that could be organized into interdependent clusters or sub-complexes. A heterotrimer formed by Nop7, Erb1 and Ytm1 (PeBoW complex in mammals) is crucial for the proper formation of the 60S subunit. Depletion of any of the three proteins is inviable and certain truncations result in aberrant processing of 27SA2 rRNA thus impairing cell proliferation. Nop7 and Erb1 have been shown to bind RNA and are recruited to the pre60S before Ytm1. It is also known that the trimer has to be removed from the nascent particle in order to promote its normal maturation. Despite its relevance in the cell, the exact role of PeBoW is not clear and the interactions within the complex have been poorly characterized. In this study we carry out an extensive biochemical and structural analysis of Nop7-Erb1-Ytm1 trimer from S. cerevisiae and from a thermophilic fungus Chaetomium thermophilum. We have been able to reconstitute a stable complex in vitro that was then used in crystallographic trials. We have solved the structure of the C-terminal domain of Erb1 from yeast that folds into a seven-bladed ß-propeller. We prove that this part of the protein binds RNA in vitro, a property that might be important for its function. Moreover, in spite of previous reports suggesting that the ß-propeller domain of Erb1 would not be essential for ribosome biogenesis, we could solve the crystal structure of Ytm1 bound to the carboxy-terminal portion of Erb1 from C. thermophilum. That finding led us to redefine the macromolecular interactions that hold the complex together. First, we have verified that the N-terminal region of Nop7 interacts with Erb1. Furthermore, we have shown that a good affinity binding takes place in vitro between WD40 domain of Ytm1 and the ß-propeller of Erb1. Upon careful analysis of the interface involved in dimer formation we have designed a mutant of Erb1 that exhibits weaker association with Ytm1. We confirm our structural and biophysical data using S. cerevisiae. We prove that a point mutation that decreases the affinity between propellers of Erb1 and Ytm1 negatively affects growth in yeast because it interferes with 60S production. We show that a very conserved interface of protein-protein interaction could be targeted in order to hinder cell proliferation. / [ES] El ensamblaje de ribosomas es uno de los procesos más importantes y costosos energéticamente en una célula eucariota. A pesar de ello, se sabe relativamente poco acerca de la gran mayoría de los eventos y factores implicados en la síntesis de las subunidades ribosomales. La maduración de ribosomas comprende numerosos pasos de procesamiento del rRNA que requieren la asociación y disociación de más de doscientos factores de ensamblaje. Esas proteínas establecen una compleja red de interacciones que son esenciales para que el proceso pueda llevarse a cabo. Los estudios realizados en Saccharomyces cerevisiae han permitido la identificación de algunas correlaciones genéticas y funcionales entre los factores prerribosomales. Es el caso del heterotrímero formado por Nop7, Erb1 e Ytm1 (complejo PeBoW en mamíferos), que es imprescindible para la correcta formación de la subunidad 60S. La ausencia de cualquiera de las tres proteínas es inviable y también se conocen ciertas variantes truncadas que alteran el procesamiento del rRNA 27SA2 y de este modo afectan la proliferación celular. Se ha demostrado que Nop7 y Erb1 se asocian al rRNA y que su reclutamiento al pre60S ocurre antes de la unión a Ytm1. Además se sabe que el trímero tiene que separarse de la partícula prerribosomal emergente con el fin de favorecer su maduración. A pesar de su gran relevancia en la célula, no está claro el papel exacto del complejo PeBoW y tampoco se dispone de conocimientos suficientes acerca de las interacciones intermoleculares que lo mantienen. Durante el desarrollo de este proyecto se ha llevado a cabo un exhaustivo análisis bioquímico y estructural del trímero Nop7-Erb1-Ytm1 procedente de S. cerevisiae y del hongo termofílico Chaetomium thermophilum. En este trabajo hemos sido capaces de reconstituir el complejo estable in vitro que posteriormente se ha utilizado en los ensayos de cristalización, con los que hemos podido resolver la estructura del dominio carboxi-terminal de Erb1 de levadura, cuyo plegamiento corresponde a una hélice enrollada (ß-propeller) de siete hojas. Gracias a la información estructural, hemos demostrado que esa parte de la proteína es capaz de unir RNA in vitro, lo que puede ser una propiedad importante para su función. Además, a pesar de los estudios anteriores que sugerían que la hélice enrollada de Erb1 no era esencial en la biogénesis del ribosoma, hemos resuelto la estructura cristalina de la proteína Ytm1 unida al dominio C-terminal de Erb1 de C. thermophilum. Ese descubrimiento nos ha permitido redefinir las interacciones macromoleculares que mantienen el complejo. Inicialmente hemos confirmado que el extremo amino-terminal de Nop7 interacciona con Erb1. A continuación, hemos demostrado que el dominio WD40 de Ytm1 se une al ß-propeller de Erb1 con una buena afinidad. Después de un detallado análisis de la superficie involucrada en la formación del dímero, hemos sido capaces de diseñar una variante mutada de Erb1 que se asocia más débilmente con Ytm1. Los hallazgos estructurales y biofísicos se han confirmado in vivo usando S. cerevisiae donde hemos demostrado que una mutación puntual que disminuye la afinidad de unión entre los dominios C-terminales de Erb1 e Ytm1 manifiesta un efecto negativo sobre el crecimiento de levadura porque interfiere con la síntesis de 60S. Nuestros resultados establecen un buen ejemplo de una superficie conservada involucrada en interacciones proteína-proteína, que podría considerarse una buena diana para inhibir la proliferación celular eucariota. / [CA] L'ensamblatge de ribosomes és un dels processos més importants i energèticament costosos en una cèl·lula eucariota. Tot i això, es coneix relativament poc de la majoria dels factors implicats en la síntesi de les subunitats ribosomals. La maduració de ribosomes compren moltes etapes de processament del rRNA que requereix l'associació i dissociació de més de dos-cents factors d'ensamblatge. Aquestes proteïnes estableixen una complexa xarxa de interaccions que són essencials perquè el procés es pugi dur a terme. Els estudis realitzats en Saccharomyces cerevisiae han permès la identificació de algunes correlacions genètiques i funcionals entre els factors pre-ribosomals. Aquest és el cas del heterotrímer comprés per Nop7, Erb1 i Ytm1 (complex PeBoW en mamífers), que és imprescindible per a la correcta formació de la subunitat 60S. L'absència de qualsevol de les tres proteïnes és inviable i també és coneixen certes variants truncades que alteren el processament del rRNA 27SA3 i que d'aquesta manera afecten a la proliferació cel·lular. S'ha demostrat que Nop7 i Erb1 s'associen al rRNA i que el seu reclutament al pre60S té lloc abans de l'unió a Ytm1. A més a més, es sap que el trímer ha de separar-se de la partícula pre-ribosomal emergent per tal que es produeixi la seua maduració. Malgrat la seua rellevància en la cèl·lula, no s'ha aclarit el paper exacte del complex PeBoW i tampoc n'hi ha coneixements suficients de les interaccions intermoleculars que el mantenen. Durant el desenvolupament d'aquest projecte s'ha dut a terme un exhaustiu anàlisi bioquímic i estructural del trímer Nop7-Erb1-Ytm1 de S. cerevisiae i del fong termofílic Chaetomium thermophilum. En aquest treball hem estat capaços de reconstituir el complex estable in vitro que posteriorment s'ha utilitzat en el assajos de cristal·lització, amb els que hem pogut resoldre l'estructura del domini carboxi-terminal de Erb1 de llevat i que té un plegament corresponent a una hèlix enrotllada (ß-propeller) de set fulles. Gràcies a la informació estructural, hem pogut demostrar que aquesta part de la proteïna té la capacitat d'unir RNA in vitro, el que pot ser una propietat important per a la seua funció. A més a més, malgrat que els estudis anteriors suggerien que la hèlix enrotllada de Erb1 no era essencial en la biogènesis del ribosoma, hem pogut resoldre la estructura cristal·lina de la proteïna Ytm1 unida al domini C-terminal de Erb1 de C. thermophilum. Aquest descobriment ens ha permès redefinir les interaccions macromoleculars que mantenen el complex. Inicialment, hem confirmat que l'extrem amino-terminal de Nop7 interacciona amb Erb1. A continuació, hem demostrat que el domini WD40 de Ytm1 s'uneix al ß-propeller de Erb1 amb bona afinitat. Després d'un anàlisi detallat de la superfície involucrada en la formació del dímer, hem estat capaços de dissenyar una variant mutada de Erb1 que s'associa més dèbilment amb Ytm1. Les dades estructurals i biofísiques s'han confirmat in vivo utilitzant S. cerevisiae on hem demostrat que una mutació puntual que disminueix l'afinitat d'unió entre els dominis C-terminals de Erb1 i Ytm1 manifesta un efecte negatiu en el creixement del llevat perquè interfereix amb la síntesi del 60S. Els nostres resultats estableixen un bon exemple de una superfície conservada involucrada en interaccions proteïna-proteïna, que es podria considerar una bona diana per a inhibir la proliferació cel·lular eucariota. / Wegrecki, M. (2015). Structural, biophysical and functional characterization of Nop7-Erb1-Ytm1 complex and its implications in eukaryotic ribosome biogenesis [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/55941

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Insects and their natural enemies are engaged in a never-ending battle called the ‘co-evolutionary arms race.’ As a part of these contentious interactions, vulnerable insects evolve natural barriers that prevent successful attacks by their natural enemies. In response, natural enemies evolve strategies that overcome these barriers. Occasionally, microbial symbionts will also participate in these relationships by assisting their insect host in defense against natural enemies or by assisting the natural enemy in subduing its prey. Alternatively, microbial symbionts may become contenders themselves in the co-evolutionary arms race by becoming reproductive parasites of their hosts. To mediate successful outcomes in these relationships, microbial symbionts will often employ diverse protein toxins capable of manipulating and/or harming eukaryotic targets. In this dissertation, I study vertically transmitted Spiroplasma symbionts to address pressing questions about the evolution of symbiont protein toxins involved in insect manipulation and defense. In chapter II, I explore the genome of the first strain of Spiroplasma capable of inducing cytoplasmic incompatibility (CI) - a form of reproductive parasitism. I use bioinformatic techniques to look for potential protein effectors of CI and demonstrate that Spiroplasma evolved this intricate form of reproduction manipulation independent of other symbionts. In chapter III, I use bioinformatic approaches to characterize the expansion and diversification of multiple protein toxin families present in Spiroplasma. I identify dynamic evolutionary processes responsible for expanding and diversifying these toxin families and uncover a striking genus-wide association between protein toxin-associated domains in Spiroplasma and Spiroplasma transmission method. In chapter IV, I explore how protein expansion and diversification have influenced toxin function. Through molecular experiments with diverse Spiroplasma ribosome-inactivating protein (RIP) toxins, I implicate neofunctionalization as a common outcome in RIP toxin expansion. Lastly, in chapter V, I focus on the interactions between host and parasite by describing the first parasitoid wasp known to attack the adult stage of Drosophila hosts. This work introduces a new Drosophila-wasp study model for future novel studies into parasitoid-host interactions. Overall, this dissertation addresses broad questions about the evolution and origins of host, symbiont, and natural enemy interactions, and provides new tools and methods for future investigations.