Characterization of Structures and Dynamics of Intrinsically Disordered Domain and Proteins Using Time Resolved-Hydrogen Deuterium Exchange Mass Spectrometry

Proteins are inherently dynamic. Virtually all of the processes that underlie biological activity, including binding partner recognition, catalysis and allostery among many others, require transient adoption of specific high energy conformations. While many proteins contain highly dynamic regions or Intrinsically Disordered Domains (IDDs), Intrinsically Disordered Proteins (IDPs) are free of well-defined structural features altogether. These domains/proteins play integral roles in a wide variety of biological processes, but are often associated with neurodegenerative disease and cancer. Hence it is crucial to study these biomacromolecules to understand the structural underpinnings of their biological functions as well as the factors that drive disease pathology. However, many of the current high resolution structural techniques (like X-ray crystallography and NMR) are not feasible for such studies because of their reliance on the presence of a well-defined native conformation. An emerging structure labeling technique called Time Resolved-Hydrogen Deuterium Exchange (TR-HDX) can offer an alternative strategy. In this approach, structure-dependent hydrogen/deuterium labelling is measured on the millisecond timescale, providing an exquisitely sensitive picture of the weak hydrogen bonding networks that impart residual structure in IDDs and IDPs. In this work, TR-HDX was implemented onto microfluidic chip to achieve solution phase site-specific analysis of the structures and dynamics of p53 N-terminal transactivation domain and tau protein. Gas-phase TR-HDX was also implemented to study gas-phase protein structures that become populated during Differential Mobility Spectrometry (DMS) ion mobility analyses. Critical advances include the discovery and characterization of an amyloidogenic tau intermediate that may underlie Alzheimers disease pathology, a dynamics-based model for recruitment of co-factors to cancer protein p53 upon phosphorylation, a detailed account of how a set of anti-amyloid drug candidates affect tau structure and dynamics, and development of a method to predict solution-phase protein stability from DMS-HDX measurements

New Insights Into An Old Interaction: Developing A Model For PAI-1:VN Interactions

Active human Plasminogen Activator Inhibitor 1 (PAI-1) is most often found in complex with Vitronectin (VN), an ~62kDa glycoprotein. Research has shown PAI-1 and VN form higher order complexes in tissues, and our work indicates a 2:1 (PAI-1:VN) stoichiometry for these complexes. A logical model for PAI-1:VN interaction proposes that two PAI-1 molecules bind VN at separate sites. However, our small-angle neutron scattering (SANS) data suggest that there is a PAI-1: PAI-1:VN interaction, in which PAI-1 forms a dimer when in complex with VN. We tested this novel arrangement of PAI-1 within the complex by using a variety of biophysical methods. Through the use of VN binding deficient PAI-1 variants we were able to detect binding deficient PAI-1 in PAI-1:VN complexes, thus supporting the existence of a PAI-1:PAI-1:VN interaction. In addition to studying the PAI-1:VN complex assembly and macromolecular arrangement, we probed the disordered domain of VN in order to identify the effect of PAI-1 binding on the disordered nature of the domain. Additionally, we sought to examine the postulated binding site for PAI-1 in this domain. It is known that a class of proteins containing intrinsically disordered domains (IDDs) frequently undergoes a conformational change upon ligand binding. We present evidence that the disordered domain of VN can be classified as an IDD based on sequence composition and SANS data that demonstrate the IDD undergoes a disorder to order transition upon PAI-1 binding. Additionally, our SANS data support a model in which the IDD of VN interacts with the secondary binding site for VN on PAI-1. Overall, this work has greatly advanced the field, and has opened new paths of study for future research efforts in the Peterson lab

RNA-PROTEIN CONDENSATION PATTERNS THE CYTOSOLIC LANDSCAPE OF A SYNCYTIUM

To function properly and survive in differing environmental conditions, every cell must organize their cytoplasm in one form or another. This requirement is increasingly necessary in large, multinucleated cells. Traditionally, this has been thought to be mainly driven by membrane-bound compartments (i.e. organelles) that help eukaryotic life organize into distinct biochemical spaces. More recently, it’s become apparent that the continuous cytosol is also organized into distinct compartments, through entirely separate means. In the multinucleated Drosophila embryo, approximately 70% of mRNA transcripts were determined to be heterogeneously localized across the cell, and mRNA spatial organization in the multinucleate fungus Ashbya gossypii has implicated phase separating RNA binding proteins (RBPs), where transcript heterogeneity is critical for autonomous nuclear division and polarized growth in these syncytial cells. The ability of RNAs to condense into droplets is in many instances contributing to previously appreciated mRNA localization phenomena. Phase separation enables mRNAs to selectively and efficiently co-localize and be co-regulated allowing control of gene expression in time and space. The work presented here demonstrates that mRNA sequence not only drives the localized condensation of RNA-protein droplets in A. gossypii, but also, governs the identity of these specialized RNA granules. Work in this thesis provides evidence that the RNA binding protein, Whi3, exhibits differential phase-separation behavior depending on which RNA target it binds and that this differential behavior is specified by features within the mRNA sequence. In addition, this work investigates the possibility of an auto feedback mechanism by which Whi3 phase separates with its own mRNA to drive differential crowding within the cytosol to promote droplet condensation in crowded cytosolic regions, thus creating individual territories of cytosol within a common syncytial cytoplasm. These data suggest mechanisms by which cells can employ asymmetric RNA localization, specifically the localization of RNAs via phase-separating RNA binding proteins, to generate functionally distinct domains to achieve efficient, and timely cytosolic organization.Doctor of Philosoph

Rozponávání pomocí neuspořádaných oblastí proteinů

Intrinsic disorder is one of the many traits that can affect the functionality of multiple naturally occurring proteins in biological systems. This thesis reports on the latest findings on mechanisms that intrinsically disordered proteins or intrinsically disordered regions utilize in specific recognition at the molecular level. Here, the general characteristics of intrinsically disordered proteins are summarized, along with the extent of their abundance throughout different lifeforms and the variety of their molecular recognition mechanisms depicted on specific examples. Furthermore, this thesis focuses on protein transitions between ordered and disordered states induced by interaction with its' binding partner. In the last two chapters, characteristic features of intrinsically disordered proteins are described, and attention is paid to the way these features influence cellular signaling pathways such as interactional promiscuity, the role of signaling hubs, alternative splicing, and post- translational modification.Neuspořádanost je jedna z vlastností mnoha přirozeně se vyskytujících proteinů, která ovlivňuje jejich funkčnost v biologických systémech. Tato práce popisuje poznatky o mechanismech, kterými mohou neuspořádané proteiny nebo neuspořádané proteinové domény přispívat ke specifickému rozpoznávání na molekulární úrovni. Jsou zde shrnuty obecné charakteristiky neuspořádaných proteinů, míra jejich zastoupení napříč různými organismy a na konkrétních příkladech jsou zde představeny možné způsoby molekulárního rozpoznávání. Dále se tato práce zaměřuje na přechody mezi uspořádaným a neuspořádaným stavem vyvolané interakcí s vazebným partnerem. V posledních dvou kapitolách se věnuje charakteristickým rysům neuspořádaných proteinů ovlivňujících buněčnou signalizaci, kterými jsou vazebná promiskuita v podobě signaling hubs, alternativní splicing nebo post-translační modifikace.Katedra buněčné biologieDepartment of Cell BiologyFaculty of SciencePřírodovědecká fakult

Structural and Binding Studies of the Polar Organizing Protein Z (PopZ) Using NMR Spectroscopy

The polar organizing protein Z (PopZ) is an intrinsically disordered protein from Caulobacter crescentus that is necessary for the formation of three-dimensional microdomains at the cell poles, where it functions as a hub protein that recruits multiple regulatory proteins. Although a large portion of the protein is predicted to be disordered, PopZ can self-assemble into polymeric superstructure scaffolds that directly bind to at least ten different proteins. Here, we report the solution NMR structure of PopZΔ134–177, a truncated variant of PopZ that does not self-assemble but retains the ability to interact with heterologous proteins. We show that the unbound form of PopZΔ134–177 is unstructured in solution, with the exception of a small amphipathic α-helix encompassing residues M10-I17, which is included within a highly conserved region near the N-terminus. In applying NMR techniques to map the interactions between PopZΔ134–177 and one of its binding partners, RcdA, we find that the α-helix and neighboring residues extending to position E23 serve as the core of the binding motif. Consistent with this, a point mutation at position I17 perturbs the binding region and severely inhibits interaction with RcdA. Our results show that a partially structured Molecular Recognition Feature (MoRF) within an intrinsically disordered domain of PopZ contributes to the assembly of polar microdomains, revealing a structural basis for complex network assembly in Alphaproteobacteria that is analogous to those formed by intrinsically disordered hub proteins in other kingdoms

Deciphering mRNP – nuclear pore interactions : study of basket protein dynamics in budding yeast

The export of mRNAs from the nucleus to the cytoplasm is one of many steps along the gene expression pathway and is fundamental for mRNAs to meet with ribosomes for translation in the cytoplasm. Exchanges between nucleus and cytoplasm occur through the nuclear pore complex (NPC), which is a large multi-protein complex embedded in the nuclear membrane and assembled by 30 different proteins the nucleoporins. The nucleoplasmic side of the pore is believed to orchestrate many fundamental nuclear processes. Indeed, a growing body of evidence suggests that the nuclear pore is involved in a broad range of activities including modulation of DNA topology, DNA repair, epigenetic regulation of gene expression, and selective access to exporting molecules. The structural component required for orchestrating those nucleoplasmic functions is the basket, a ∼60- to 80-nm-long structure protruding into the nucleoplasm. The consensus view depicts the basket as a structure assembled by filamentous proteins, TPR (Translocated Promoter Region protein) in humans and by its two paralogues Mlp1 and Mlp2 (myosin-like proteins) in yeast, converging into a distal ring. In the first part of this thesis, we characterized the motion of specific mRNAs at the vicinity of the nuclear periphery. We observed that transcripts scan along the nuclear envelope, likely to find a nuclear pore to be exported. We also showed the scanning behavior was affected upon Mlp1 deletion or truncation as well as upon mutation of the nuclear poly(A) binding protein Nab2. These observations indicated that Mlp1 and hence baskets, as well as specific RNA binding proteins, facilitate the interaction of mRNA with the nuclear periphery. While the canonical structure of the NPC is well established, our understanding of the conditions and factors contributing to the assembly of a basket, as well as the stoichiometry of its components, remains incomplete. Although basket proteins have been implicated in the regulation of gene expression through gene anchoring to the nuclear periphery and in mRNA scanning before export, how this is mediated by Mlp1/2 is poorly understood. Moreover, the dynamics of basket proteins in yeast seem to obey different rules than those of other nucleoporins as their turnover at the pore is faster than any other NPC components. Furthermore, it has been observed that during heat shock Mlp1 and Mlp2 dissociate from nuclear pores and form intra-nuclear granules, sequestering mRNAs and RNA export factors. Yet the mechanism for the formation of these granules or their role during heat shock is poorly understood. In yeast, the nuclear baskets are not associated with all NPCs, as no baskets assemble on the pores adjacent to the nucleolus. Yet, how cells establish these basket-less pores and whether they represent specialized nuclear pores with different functions from basket-containing pores is still unknown. To understand the dynamics of basket assembly and the biological relevance of establishing distinct sets of pores, we dissected the biological processes leading to the formation of baskets. In addition, to highlight potential functional differences between the two types of pores, we identified the interactors of nuclear basket-containing and nucleolar basket-less pores. We showed that assembling a basket is not a default mode for a pore in the nucleoplasm and that active mRNA processing is required to maintain baskets integrity. While mRNA can be found associated with both types of pores, our results suggest that export kinetics may be different on basket-containing and basket-less pores. The eukaryotes organize their nucleus in discrete functional regions and the nuclear envelope has been envisioned as an organelle by and of itself. Our analyzes indicate that mRNAs and Mlp1 participate in an additional degree of nuclear compartmentalization by enabling the formation of a dynamic structure: the basket. Overall my project sheds new light on the nuclear organization and highlights the surprising entanglement between mRNA export and NPC plasticity.L'exportation des ARN messagers du noyau vers le cytoplasme est l'une des nombreuses étapes de la voie d'expression des gènes et est fondamentale pour que les ARNm rencontrent les ribosomes pour être traduits dans le cytoplasme. Les échanges entre le noyau et le cytoplasme se font par l'intermédiaire du complexe du pore nucléaire, qui est un grand complexe multiprotéique enchâssé dans la membrane nucléaire et assemblé par 30 protéines différentes, les nucléoporines. Le versant nucléoplasmique du pore orchestre de nombreux processus nucléaires fondamentaux. En effet, un nombre croissant d’études suggère que le pore nucléaire est impliqué dans un large éventail d'activités, notamment la modulation de la topologie de l'ADN, la réparation de l'ADN, la régulation épigénétique de l'expression des gènes et l'accès sélectif aux molécules candidates à l’export. Le composant structurel nécessaire pour orchestrer ces fonctions nucléoplasmiques est appelé le panier une structure de ∼60 à 80 nm de long faisant saillie dans le nucléoplasme. Une vision consensuelle dépeint le panier comme une structure assemblée par des protéines filamenteuses convergeant en un anneau distal, TPR (Translocated Promoter Region protein) chez l'homme et par ses deux paralogues Mlp1 et Mlp2 (myosin-like proteins) chez la levure. Dans la première partie de cette thèse, nous avons caractérisé le mouvement d'ARNm spécifiques au voisinage de la périphérie nucléaire. Nous avons observé que les transcrits scannent l'enveloppe nucléaire, probablement pour trouver un pore nucléaire afin d'être exportés. Nous avons également montré que ce comportement était affecté par la délétion ou la troncation de Mlp1 ainsi que par la mutation de la protéine de liaison aux queues poly(A) Nab2. Ces observations indiquent que Mlp1 et donc les paniers, ainsi que des protéines liant l’ARN, facilitent l'interaction des ARNm avec la périphérie nucléaire. Alors que la structure canonique du pore nucléaire est bien établie, notre compréhension des conditions et des facteurs contribuant à l'assemblage du panier, ainsi que de la stoechiométrie de ses composants, reste incomplète. Bien que les protéines du panier soient impliquées dans la régulation de l'expression des gènes par l'ancrage des gènes à la périphérie nucléaire et dans le recrutement des ARNm avant leur export, la manière dont le panier intervient dans ce processus est mal comprise. De plus, la dynamique des protéines du panier chez la levure semble obéir à des 6 règles différentes de celles des autres nucléoporines, car leur renouvellement (turn over) au niveau du pore est plus rapide que celui des autres composants du NPC. De plus, il a été observé que lors d'un choc thermique, Mlp1 et Mlp2 se dissocient des pores nucléaires et forment des granules intra-nucléaires, séquestrant les ARNm et les facteurs d'exportation d'ARN. Pourtant, le mécanisme de formation de ces granules ou leur rôle pendant le choc thermique est mal compris. Chez la levure, le panier nucléaire n'est pas associé à tous les pores nucléaires, et les paniers sont absents des pores adjacents au nucléole. La manière dont les cellules établissent ces pores sans paniers et s'ils représentent des pores nucléaires spécialisés ayant des fonctions différentes des pores contenant des corbeilles n’est pas connue. Pour comprendre la dynamique de l'assemblage des paniers et la pertinence biologique de former de deux types de pores distincts, nous avons disséqué les processus biologiques menant à la formation des paniers. De plus, afin de mettre en évidence les différences fonctionnelles potentielles entre les deux types de pores nous avons étudié les protéines associées aux pores contenant un panier nucléaire et des pores sans panier. Nous avons montré que l'assemblage d'un panier n'est pas un mode par défaut pour un pore dans le nucléoplasme et que la formation et la maturation des ARNm est nécessaire pour maintenir l'intégrité des paniers. Alors que l'ARNm peut être trouvé associé aux deux types de pores, nos résultats suggèrent que la cinétique d’export peut être différente sur les pores avec et sans panier. Les eucaryotes organisent leur noyau en régions fonctionnelles discrètes et l'enveloppe nucléaire a été envisagée comme pouvant être une organelle à part entière. Nos analyses indiquent que les ARNm et Mlp1 participent à un degré supplémentaire de compartimentation nucléaire en permettant la formation d'une structure dynamique : le panier. Mon projet apporte un nouvel éclairage sur l'organisation des compartiments nucléaire et met en évidence l'intrication surprenante entre l'export des ARNm et la plasticité des pores nucléaires

Knowledge-based identification of functional domains in proteins

The characterization of proteins and enzymes is traditionally organised according to the sequence-structure-function paradigm. The investigation of the inter-relationships between these three properties has motivated the development of several experimental and computational techniques, that have made available an unprecedented amount of sequence and structural data. The interest in developing comparative methods for rationalizing such copious information has, of course, grown in parallel. Regarding the structure-function relationship, for instance, the availability of experimentally resolved protein structures and of computer simulations have improved our understanding of the role of proteins' internal dynamics in assisting their functional rearrangements and activity. Several approaches are currently available for elucidating and comparing proteins' internal dynamics. These can capture the relevant collective degrees of freedom that recapitulate the main conformational changes. These collective coordinates have the potential to unveil remote evolutionary relationships between proteins, that are otherwise not easily accessible from purely sequence- or structure-based investigations. Starting from this premise, in the first chapter of this thesis I will present a novel and general computational method that can detect large-scale dynamical correlations in proteins by comparing different representative conformers. This is accomplished by applying dimensionality-reduction techniques to inter-amino acid distance fluctuation matrices. As a result, an optimal quasi-rigid domain decomposition of the protein or macromolecular assembly of interest is identified, and this facilitates the functionally-oriented interpretation of their internal dynamics. Building on this approach, in the second chapter I will discuss its systematic application to a class of membrane proteins of paramount biochemical interest, namely the class A G protein-coupled receptors. The comparative analysis of their internal dynamics, as encoded by the quasi-rigid domains, allowed us to identify recurrent patterns in the large-scale dynamics of these receptors. This, in turn, allowed us to single out a number of key functional sites. These were, for the most part, previously known -- a fact that at the same time validates the method, and gives confidence for the viability of the other, novel sites. Finally, for the last part of the thesis, I focussed on the sequence-structure relationship. In particular, I considered the problem of inferring structural properties of proteins from the analysis of large multiple sequence alignments of homologous sequences. For this purpose, I recasted the strategies developed for the dynamical features extraction in order to identify compact groups of coevolving residues, based only on the knowledge of amino acid variability in aligned primary sequences. Throughout the thesis, many methodological techniques have been taken into considerations, mainly based on concepts from graph theory and statistical data analysis (clustering). All these topics are explained in the methodological sections of each chapter

61st Annual Rocky Mountain Conference on Magnetic Resonance

Final program, abstracts, and information about the 61st annual meeting of the Rocky Mountain Conference on Magnetic Resonance, co-endorsed by the Colorado Section of the American Chemical Society and the Society for Applied Spectroscopy. Held in Copper Mountain, Colorado, July 25-29, 2022