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Though antibodies as therapeutics are limited to extracellular targets, their repertoire of molecular interactions has particular relevance to the many intracellular cellular proteins for which small molecule screening has reached impasse. For such proteins there is little recourse to theory, since molecular recognition is, in practical terms, still not well understood. Here I apply antibody discovery to the lysine demthylases KDM4D and PHF8, two proteins difficult to inhibit selectively due to the similarity of their binding pockets to those of the larger family. With a selective, picomolar affinity antibody, dependent on residues distal to the KDM4D active site, I present what is likely the first example of allosteric inhibition of a KDM4 lysine demethylase, demonstrating that there is opportunity outside active sites oversubscribed with pan inhibitors. Antibody discovery for PHF8, however, was plagued by a familiar problem: antibodies that bound when their antigen was immobilised directly to a surface, but barely bound at all when it was free in solution. The common explanation is that the partial denaturation that accompanies immobilisation reveals epitopes unavailable in solution, but examining the problem in detail for the Plant Homeodomain of PHF8 revealed a connection to its rarely sampled conformations. The prominence these antibodies in the immune responses to PHF8, and to some extent KDM4D, motivates two hypotheses on their origin: either the states are very immunogenic or there is a connection between states of irreversible damage and those sampled reversibly, but rarely, by a protein in solution.

Cette thèse traite de la RMN en phase liquide à champs multiples, pour la détermination de la structure et de la dynamique de petites molécules et de protéines. La dynamique ps-ns des chaînes latérales de la protéine ubiquitine a été étudiée par la relaxation du 13C des groupes méthyles δ1 des isoleucines, marqués sélectivement. Les vitesses de relaxation mesurées à plusieurs hauts champs magnétiques et les vitesses de relaxation longitudinale de 0.29 T à 9 T obtenues par relaxométrie haute résolution ont été analysées à l'aide du programme ICARUS, adapté à l’occasion pour les groupes méthyle. La matrice de relaxation a été calculée par un programme inédit, nommé RedKite. Un modèle de fonction de densité spectrale a été proposé pour prendre en compte les mouvements complexes des groupes méthyles. Nous avons ainsi pu accéder à une description de la dynamique des groupes méthyle sur trois ordres de grandeur d’échelles de temps. La spectroscopie RMN à deux champs magnétiques a été développée en collaboration avec Bruker. Le spectromètre à deux champs permet le contrôle des spins dans deux centres magnétiques avec une homogénéité suffisante et le transfert rapide de l’échantillon entre ces deux centres. Grâce à l'utilisation de cohérences à zéro-quantum, nous avons mesuré des spectres de corrélation homo- et hétéronucléaires à haute résolution dans lesquels les deux dimensions sont obtenues à deux champs très différents. Cette approche a été utilisée pour réduire considérablement la contribution de l’échange chimique à la relaxation transverse, permettant l’observation des signaux de noyaux en échange chimique invisibles à haut champ. / We present the development of multiple-field liquid-state NMR spectroscopy for the determination of the structure and dynamics of small molecules and proteins. Dynamics of proteins side-chains in the pico- to nanosecond range have been studied in the protein ubiquitin, by measuring the relaxation of carbon-13 nuclei in isoleucine-δ1 methyl groups, with site-specific isotope labelling. High-field relaxation rates and longitudinal relaxation rates obtained using high-resolution relaxometry have been analysed using a new version of the program ICARUS, adapted for methyl groups. The relaxation matrix has been calculated with a homemade program called RedKite. Models of spectral density function have been proposed to account for all motions of methyl groups. This unprecedented dataset allows for the description of motions in methyl groups over 3 orders of magnitudes of correlation times. Two-field NMR has been developed in collaboration with Bruker. The two-field NMR spectrometer allows for the control of nuclear spins in two magnetic centres with vastly different magnetic fields, coupled with a sample shuttle. Using zero-quantum coherences, homo and heteronuclear two-field high-resolution spectra have been obtained, where the two dimensions are acquired at very different magnetic fields. Such pulse sequences have been used to reduce the contribution of chemical exchange to transverse relaxation, even when this exchange makes signals invisible at high field. The reduced bandwidth of signals at low field has also been used to perform efficient isotropic mixing in a two-field TOCSY experiment. Correlations have been observed for carbon-13 signals separated by more than 150 ppm.

Recent calls in education have emphasized the critical need for curricula in the sciences to support student development of the general and disciplinary-specific practices that are relevant to modern scientific research and careers, as well as foundational scientific knowledge that reflects recent advances. In this regard, the life sciences, including biochemistry, have been under pressure to develop curricula that reflect current research knowledge and practices, and that develop student competence in areas such as experimentation and visualization. In contrast to these calls, biochemistry textbooks, and instruction based on them, seldom discuss how disciplinary knowledge is combined with experimental work or other disciplinary resources to investigate and communicate about biochemical phenomena. This is of great concern given that graduates entering life science careers must be able to reason with relevant disciplinary knowledge, utilize experimental research methods, and navigate data representations in order to solve research problems. It is therefore crucial for biochemistry instruction to expose students to the ways in which expert scientists navigate and reason with disciplinary resources in cutting-edge scientific research on topics such as protein folding and dynamics, the focus of this project. Thus, this dissertation aims to fill a gap in our understanding of how expert research scientists explain protein-folding and dynamics research, and how that research knowledge can be used to inform the development of instructional materials in this crucially important area of biochemistry. To address this goal, we explore three overarching research questions: How can we model experts’ explanations of their research related to protein folding and dynamics? (RQ1); How do experts use representations to explain their protein-folding and dynamics research? (RQ2); and How can we use expert research to inform the design and implementation of instructional materials aimed at developing biochemistry students’ understanding of protein-folding and dynamics? (RQ3). To address these research questions, we first collected and analyzed interview data from four experts to explore the nature of their research explanations. This data was used to develop a model (i.e. the MAtCH model) of how experts integrate theoretical knowledge with their research context, methods, and analogies when explaining how phenomena operate (RQ1). In doing so, we also established how the experts use and combine explanatory models depending on the phenomena discussed and their explanatory aims, as well as how they explain thermodynamic and kinetic concepts relevant to protein folding in ways that align with their experimental research methods. We then examined selected representations from the expert interviews to explore how experts use language and representations to create meaning when explaining their research (RQ2). In comparing these to representations from biochemistry textbooks, analysis of the data indicated that textbooks generally explain ‘what is known’ but seldom explain ‘how it is known,’ whereas the experts use a combination of language, multiple representations, and gestures to explain how experimental research methods can provide evidence for phenomena. From this analysis, suggestions were made regarding the design of instructional materials to support discussion of experimental research methods and student interpretation of representations in classroom activities. In the final study, these suggestions were used in combination with additional analysis of expert research to inform the development anticipated learning outcomes (ALOs) and the design of instructional materials aimed at developing biochemistry students’ understanding of protein folding and dynamics (RQ3). The materials focus on the use of hydrogen-deuterium exchange mass spectrometry (HDX-MS) to study changes in protein structure due to denaturation and interactions with other molecules. The instructional materials were piloted in an undergraduate biochemistry course for the health sciences, and the nature of students’ understandings were explored. Our findings suggest that research practice – including research context, experimental methods, and representations – influences reasoning and explanation, providing additional evidence of the importance of developing discursive literacy in science students. To that end, a major implication of this work is that student knowledge of experimentation and representation may be a critical component of developing functional scientific understanding. Each of the studies contained in this dissertation therefore suggests ways in which practitioners may use our findings to modify instruction and instructional materials so that they are more aligned with expert practices. In order to teach students how scientific research underpins factual knowledge in biochemistry, future research should continue to explore experts’ use of disciplinary resources and ways of thinking in order to inform teaching and learning strategies and materials that can support the development of students’ disciplinary literacy.

Motile microorganisms navigate through their environment using special molecular machinery in order to sense gradients of various signals: chemotaxis (reactions to chemical compounds) and phototaxis (to light) sensory cascades. Transmembrane receptors play a central role in these cascades as they receive input signals and transmit them inside the cell, where they modulate activity of the kinases CheA, which are tightly bound to their cytoplasmic domains. CheA further phosphorylates the response regulator protein CheY, which regulates the flagella. At the same time, CheA phosphorylates and, by means of this, activates another response regulator, CheB, which, along with the constantly active CheR protein, catalyzes two opposite reactions: methylation and demethylation of the specific glutamic acid residues located at the cytoplasmic domain of the receptors. The latter reactions establish the adaptation mechanism, which allows microbes to sense in a very broad range of the input signal intensities. Many functional, structural and dynamical aspects of the signal propagation through the prokaryotic receptors as well as a mechanism of the signal amplification remain still unclear. In the present thesis we have used various techniques of computational biophysics, chiefly molecular dynamics (MD) simulations, in order to approach these problems. In Chapter 3, we have carried out MD simulations of the isolated linker domain (HAMP) from the E. coli Tsr chemoreceptor. The MD simulations revealed highly dynamical nature of this domain, which allows for interconversion between several metastable states. These metastable states feature a number of structural and dynamical properties, which were previously reported for HAMP domains of various receptors obtained from different organisms. It allowed us to reconcile numerous experimental data and to hypothesize that different HAMP domains share similar mechanism of their action. In Chapter 4, we have performed MD simulations of the whole cytoplasmic domain of the Tsr chemoreceptor. The simulations revealed a mechanism for the inter-domain coupling between the HAMP domain and a part of the cytoplasmic domain adjacent to the HAMP, the adaptation subdomain, by means of the regulated unfolding of a short linker region termed the stutter. Also, we have found that the reversible methylation/demethylation of the cytoplasmic domain affects its flexibility and symmetry. Altogether, these findings suggest a mechanism of signal propagation at the level of an individual chemoreceptor dimer. In Chapter 5, we have built a model of the trimer-of-dimers of the archaeal phototaxis receptor complex (NpSRII:NpHtrII). Subsequent MD simulations revealed an important role of dynamics in signal transduction and, potentially, in the kinase activation. In Chapter 6, we have reconstructed a whole transmembrane lattice formed by the NpSRII:NpHtrII complexes. The concave shape of the obtained lattice naturally explains polar localization of the receptor arrays in prokaryotic cells. At the same time, additional MD simulations of an individual unit of this lattice (a dimer of the photosensor) revealed global motional modes in its transmembrane region, which presumably co-occur with its activation and can spread across the tightly packed transmembrane arrays allowing for the signal amplification.

Les chaperons moléculaires, une famille de protéines diverses en structure et taille, sont dédiés à accompagner, replier et protéger d’autres protéines afin qu’elles atteignent leur conformation finale et leur emplacement dans la cellule. Dans ce but, les chaperons moléculaires doivent être hautement spécialisés dans l’exécution de tâches spécifiques, telles que le repliement, le transport ou la désagrégation, et polyvalents dans leur motifs de reconnais- sance, afin de pouvoir interagir avec un grand nombre de protéines di érentes. Di érents chaperons moléculaires collaborent au sein de la cellule, formant ainsi un réseau complexe qui assure le contrôle de la qualité du protéome. Les interactions entre les di érents partenaires de ce réseau et entre les chap- erones et leurs substrats sont souvent dynamiques, ce qui rend leur obser- vation structurale particulièrement di cile pour les techniques de biologie structurale. Par conséquent, il y a à ce jour peu d’information sur les struc- tures et mécanismes d’interaction au sein des complexes chaperon-substrate. Dans cette thèse, je présente des études sur la structure, la dynamique et les interactions entre les substrats de deux chaperons moléculaires, en utilisant diverses méthodes biophysiques et in vivo.Dans la première partie, je montre que la chaperone TIM910, située dans l’espace inter-membranaire des mitochondries, lie ses substrats, des protéines membranaires destinées aux deux membranes mitochondriales, d’une manière très dynamique. Non seulement le complexe TIM910 est en échange constant entre les espèces monomèriques et hexameriques, mais aussi le substrat lié échange entre mulitples conformations à une échelle de millisecondes. Sur la base de la résonance magnétique nucléaire (RMN), de small-angle X-ray scat- tering (SAXS), de l’ultracentrifugation analytique (AUC) et des expériences mutationnelles in vivo et des tests fonctionnels d’import dans les mitochon- dries, je propose un modèle structurale de l’interaction entre le chaperon et la protéine membranaire. TIM910 lie ses substrats dans une poche hydrophobe à l’extérieur du chaperon. Cette interaction est modulaire et se fait avec un ou deux hexamères de TIM910, en fonction de la longueur du substrat.Dans la deuxième partie, nous avons étudié le comportement du récepteur N-terminal du unfoldase ClpC1 de M. tuberculosis en présence d’antibiotiques et de ligands di érents. Le domaine N-terminal de ClpC1 est le site de liai- son de divers antibiotiques nouveaux contre M. tuberculosis. L’antibiotique Cyclomarin A supprime complètement la dynamique induite par le ligand arginine-phosphate. Nous proposons que cette suppression de la dynamique soit le principe fondamental du mécanisme d’action de cet antibiotique.Dans les deux cas, les structures X-ray des chaperons dans leur état apo et la structure de ClpC-NTD liée à des antibiotiques étaient disponibles, mais ces structures statiques ne su sent pas pour expliquer le mécanisme d’action. La structure X-ray de TIM910 n’a pas fourni d’ indication sur l’endroit ou la façon dont les substrats sont liés. De même, les structures X-ray du domaine N-terminal de apo et de Cyclomarine A de ClpC1 ne présentent que des di érences de structure mineures. Les deux exemples montrent que les données structurelles statiques souvent ne permettent pas d’expliquer le fonctionnement d’un système moléculaire, donc la combinaison de di érentes techniques et le développement de nouvelles méthodes pour étudier les complexes chaperon-substrat sont primordiaux pour comprendre leur fonction. / The diverse group of molecular chaperones is dedicated to accompany, fold and protect other proteins until they reach their final conformation and loca- tion inside the cell. To this end, molecular chaperones need to be specialized in performing specific tasks, like folding, transport or disaggregation, and versatile in their recognition pattern to engage many di erent client pro- teins. Moreover, molecular chaperones need to be able to interact with each other and with other components of the protein quality control system in a complex network. Interactions between the di erent partners in this network and between the substrate and the chaperone are often dynamic processes, which are especially di cult to study using standard structural biology tech- niques. Consequently, structural data on chaperone/substrate complexes are sparse, and the mechanisms of chaperone action are poorly understood. In this thesis I present investigations of the structure, dynamics and substrate- interactions of two molecular chaperones, using various biophysical and in vivo methods.In the first part I show that the mitochondrial membrane protein chap- erone TIM910 binds its substrates in a highly dynamic manner. Not only is the TIM910 complex in constant exchange between monomeric and hex- americ species, but also the bound substrate samples multiple conformations on a millisecond timescale. Based on nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC) and in vivo mutational experiments I propose a structural model of the chap- erone/membrane protein interaction. TIM910 binds its substrates in a hy- drophobic pocket on the exterior of the chaperone in a modular fashion, where the number of TIM910 complexes bound depends on the length of the substrate.In the second part I studied the behavior of the N-terminal receptor do- main of the ClpC1 unfoldase from M.tuberculosis in the presence of di erent antibiotics and ligands. The N-terminal domain of ClpC1 is the binding site for various new antibiotics against M.tuberculosis. The antibiotic cyclomarin completely abolishes dynamics induced by the ligand arginine-phosphate. We propose that this suppression of dynamics is the underlying principle for the mechanism of action of this antibiotic.In both cases X-ray structures of the apo or antibiotic bound form were available, but not su cient to explain the mechanism of action. The X- ray structure of TIM910 provided no evidence on where or how substrates are bound. Likewise, X-ray structures of the apo and cyclomarin-bound N-terminal domain of ClpC1 show only minor di erences in structure.Both examples show that static structural data is often not enough to explain how a molecular system works, and only the combination of di er- ent techniques, including newly developed methods enable the atomic-level understanding of chaperone/substrate complexes.

Allosteric inhibition of kinases provides high selectivity and potency due to lower evolutionary pressure in conserving allosteric vs. orthosteric sites. The former are regions distinct from the kinase active site, yet, when perturbed through allosteric effectors, induce conformational and/or dynamical changes that in turn modulate kinase function. Protein kinases involved in cyclic nucleotide signalling are important targets for allosteric inhibition due to their association with diseases, from infections to Cushing’s syndrome. This dissertation specifically focuses on elucidating the molecular mechanism of allosteric inhibition in the cAMP-dependent protein kinase (PKA) and the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG), which are targets for a generalized tumor predisposition commonly referred to as Carney Complex and for malaria, respectively. In chapters 2 and 3, we focus on the agonism-antagonism switch (i.e. allosteric pluripotency) observed as the phosphorothioate analog of cAMP, Rp-cAMPS (Rp), binds to PKA. Utilizing Nuclear Magnetic Resonance (NMR), Molecular Dynamics (MD) simulations and Ensemble Allosteric Model (EAM), we determined that two highly homologous cAMP-binding domains respond differently to Rp, giving rise to a conformational ensemble that includes excited inhibition-competent states. The free energy difference between this state and the ground inhibition-incompetent state is tuned to be similar to the effective free energy of association of the regulatory (R) and catalytic (C) subunits, leading to allosteric pluripotency depending on conditions that perturb the R:C affinity. The general significance of these results is a re-definition of the concept of allosteric target to include not only the isolated allosteric receptor, but also its metabolic and proteomic sub-cellular environment. In chapter 4, we utilize a mutant that silences allosteric pluripotency to reveal that the agonism-antagonism switch of PKA not only arises from the mixed response of tandem domains, but also from the mixed response of allosteric regions within a single domain that mediates interactions with Rp. In chapter 5, the allosteric inhibition of PfPKG associated with malaria is induced through base-modified cGMP-analogs and the underlying inhibitory mechanism is determined. We show that, when bound to a PfPKG antagonist, the regulatory domain of PfPKG samples a mixed intermediate state distinct from the native inhibitory and active conformations. This mixed state stabilizes key cGMP-binding regions, while perturbing the regions critical for activation, and therefore it provides an avenue to preserve high affinity, while promoting significant inhibition. Overall, in this thesis, previously elusive mechanisms of allosteric inhibition were elucidated through the combination of NMR, MD, and EAM methods. Through this integrated approach, we have unveiled an emerging theme of inhibitory ‘mixed’ states, either within a single domain or between domains, which offer a simple but effective explanation for functional allostery in kinases. / Thesis / Candidate in Philosophy

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Macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT) are the two human members of the MIF superfamily, which are implicated in an array of autoimmune disorders, inflammatory diseases, and cancer via their pleiotropic functionality. Despite only sharing 34% sequence identity, MIF and D-DT have high structural homology and overlapping functional traits, including activation of the type II cell surface receptor CD74 and keto-enol tautomerase activity. The MIF and/or D-DT-induced activation of CD74 leads to signaling cascades pivotal for cell growth, proliferation, and inhibition of apoptosis. Such characteristics make MIF and D-DT attractive molecular targets for drug discovery. Currently, all small molecule antagonists targeting the MIF/D-DT-CD74 axis primarily bind to the catalytic sites of these proteins. Nevertheless, the precise interplay between the catalytic residues and those crucial for CD74 activation remains enigmatic. Notably, alterations of catalytic residues, particularly the catalytic residue Pro1, have been shown to impede CD74 activation. Leveraging molecular dynamics simulations and nuclear magnetic resonance (NMR) spectroscopy, we explored the dynamic coupling between the catalytically active N-terminus of MIF and surface residues pivotal for CD74 activation. Our investigation exposed previously unseen communication between the two sites and demonstrates the important role of MIF dynamics in the modulation of CD74 activation. The keto-enol tautomerization assay utilizing 4-hydroxyphenylpyruvate (4-HPP) as a substrate has been instrumental in screening and characterization of MIF and D-DT variants as well as small molecule inhibitors. However, discrepancies between inhibition constant (Ki) values and Michaelis-Menten parameters raised concerns about the accuracy of results from this assay and the conclusions made from them. Our rigorous analysis identified that impurities present in substrate samples impacted the kinetic parameters of wild-type (WT) MIF as well as the Ki values of ISO-1, a well-studied inhibitor. Our findings, which were validated with multiple proteins, underscore the pronounced influence of substrate impurities on enzymatic activity. Thereby emphasizing the imperative of meticulously controlled experimental conditions for robust data interpretation. While the majority of drug discovery efforts were focused on MIF, D-DT remains relatively underexplored in this regard. The identification of 4-(3-carboxyphenyl)-2,5- pyridinedicarboxylic acid (4-CPPC) as the first reversible and selective D-DT inhibitor opened new avenues of research for the protein. Structural analysis of D-DT – 4-CPPC revealed a ligand- induced conformational change of the C-terminal region that has mechanistic value. This observation is in stark contrast to MIF, which needs a rigid C-terminal for tertiary structure stability. In order to elucidate the impact of C-terminal conformational flexibility, we employed molecular dynamics simulations and NMR experiments. We found that while the binding of 4- CPPC did not alter the folding or thermostability of the protein, it drastically altered the protein’s dynamics, allowing for the formation of new, long-range intersubunit communications. Subsequent endeavors aimed at identifying highly selective D-DT inhibitors that did not cause a conformational change of the C-terminal region yielded 2,5-pyridinedicarboxylic acid (1). This molecule exhibits a low micromolar potency and a remarkable 79-fold specificity for D-DT over MIF. Crystallographic analysis of the D-DT-1 complex displayed that the C-terminal of D- DT was largely unperturbed by the binding of 1 and delineated structural disparities between D- DT and MIF active sites, underscoring the potential for rational drug design strategies. Further in vivo studies focusing on the cytokine activity of D-DT showed the efficacy of 1 as an inhibitor of D-DT induced activation of CD74. These findings show that 1 is a useful mechanistic tool for interrogating the pathophysiology of D-DT. Despite these exciting discoveries, the role of the C-terminal region in the enzymatic activity and conformational flexibility of D-DT required further investigation. In-depth interrogation of seventeen protein variants and WT D-DT uncovered a previously unknown functional role of the C-terminal region. These insights deepen our comprehension of protein structure-function relationships and provides an invaluable foundation for future drug discovery studies targeting D-DT-mediated pathological conditions. Overall, via our thorough experimental interrogations, we uncovered key structural and functional information about MIF and D-DT that will serve as the basis for future mechanistic and drug discovery projects.

No description available.