Thèses sur le sujet « Proteins crowding »

This work aims to analytically understand the impact of two diametric opposite environments on protein structure and dynamics and compared them to the most common solvent on earth: water. The first environment is a popular denaturing solution (urea 8M), which has served for years in protein-science laboratories to investigate protein stability; still many open questions regarding its mechanism of action remained unclear. The second environment instead moves towards a more physiological representation of proteins. The cell interior, in fact, is a crowded solution highly populated prevalently by proteins, but studies on protein structure and dynamics have lead so far to confusing or even opposite observations. The lack of a consensus view in both phenomena possibly derives from the bias of the system under study. This work is an attempt of a comparative study using the most general systems: a diverse spectrum of proteins folds, different stages along the reaction path (early stages or end-point) and/or different protein force-fields. Our main objective was to derive common pattern and general rules valid at proteome level, focusing on three major aspects of proteins: the structure, the dynamic and the interactions with the solvent molecules. Molecular dynamics simulation appeared then as the most suitable tool because of its ability to i) analyze proteins at broad range of resolutions; ii) access the direct time-resolved dynamic of the system and iii) dissect the specific interactions that arise in the new settings. Specifically, the case of urea-induced unfolding needs a system for which is possible to clearly identify folded and unfolded state – globular proteins are then the most suitable ones. We extracted general rules on the folded/unfolded transition by studying independently the two end-points of folded/unfolded reaction. We simulated the urea-induced unfolded state of a model protein, ubiquitin to understand the energetics stabilizing unfolded structures in urea. We found that the unfolded ubiquitin in 8M urea is fully extend and flexible and capturing efficiently urea molecules to the first solvation shell. Dispersion, rather than electrostatic, appear the main energetic contribution to explain the stabilization of the unfolded state. We then simulated the early stages of urea-induced unfolding on a large dataset of folded proteins, which represent the major folds of globular proteins, aiming also to investigate the kinetic role of urea in triggering the protein unfolding. We found that partially unfolded proteins expose the apolar residues buried in the protein interior, mainly via cavitation. Similar to the unfolded state, it is the dispersion interactions that drive urea accumulation in the solvation shell but here urea molecules take advantage of microscopic unfolding events to penetrate the protein interior. Macromolecular crowding instead is a phenomenon that universally affects all the proteins. We simulated a system that included as crowding agents proteins with different conformational landscapes (a globular protein, an intrinsically disordered proteins and a molten globule) arranged to reach cell-like concentrations. We conclude that the universal effect of crowding, valid for all the proteins types, is exerted via the aspecific interactions and favors open and moderately extended conformations with higher secondary structure content. This phenomenon counterbalances the volume-exclusion, which prevails at higher crowding concentrations. The impact of crowding is proportional to the degree of disorder of the protein and for folded protein crowding favors structural rearrangements while unfolded structures experience a stronger stabilization and a higher secondary structures content. The synthetic crowder PEG doesn’t reproduce any of these effects, arising concerns about its employment in study cell-like environments.

Protein-protein interactions are central to understanding how cells carry out their wide array of functions and metabolic procedures. Conventional studies on specific protein interactions focus either on details of one-to-one binding interfaces, or on large networks that require a priori knowledge of binding strengths. Moreover, specific protein interactions, occurring within a crowded macromolecular environment, which is precisely the case for interactions in a real cell, are often under-investigated. A macromolecular simulation package, called BioSimz, has been developed to perform Langevin dynamics simulations on multiple protein-protein interactions at atomic resolution, aimed at bridging the gaps between structural, kinetic and crowding studies on protein-protein interactions. Simulations on twenty-seven experimentally determined protein-protein interactions, indicated that the use of contact frequency information of proteins forming specific encounters can guide docking algorithms towards the most likely binding regions. Further evidence from eleven benchmarked protein interactions showed that the association rate constant of a complex, kon, can be estimated, with good agreement to experimental values, based on the retention time of its specific encounter. Performing these simulations with ten types of environmental protein crowders, it suggests, from the change of kon, that macromolecular crowding improves the association kinetics of slower-binding proteins, while it damps the association kinetics of fast, electrostatics-driven protein-protein interactions. It is hypothesised, based on evidence from docking, kinetics and crowding, that the dynamics of specific protein-protein encounters is vitally important in determining their association affinity. There are multiple factors by which encounter dynamics, and subsequently the kon, can be influenced, such as anchor residues, long-range forces, and environmental steering via crowders’ electrostatics and/or volume exclusion. The capacity of emulating these conditions on a common platform not only provides a holistic view of interacting dynamics, but also offers the possibility of evaluating and engineering protein-protein interactions from aspects that have never been opened before.

Proteins function as worker molecules in the cell and their natural environment is crowded. How they fold in a cell-like environment and how they recognize their interacting partners in such conditions, are questions that underlie the work of this thesis. Two distinct subjects were investigated using a combination of biochemical- and biophysical methods. First, the unfolding/dissociation of a heptameric protein (cpn10) in the presence of the crowding agent Ficoll 70. Ficoll 70 was used to mimic the crowded environment in the cell and it has been used previously to study macromolecular crowding effects, or excluded volume effects, in protein folding studies. Second, the conformational changes upon interaction between the Mediator subunit Med25 and the transcription factor Dreb2a from Arabidopsis thaliana. Mediator is a transcriptional co-regulator complex which is conserved from yeast to humans. The molecular mechanisms of its action are however not entirely understood. It has been proposed that the Mediator complex conveys regulatory signals from promoter-bound transcription factors (activators/repressors) to the RNA polymerase II machinery through conformational rearrangements. The results from the folding study showed that cpn10 was stabilized in the presence of Ficoll 70 during thermal- and chemical induced unfolding (GuHCl). The thermal transition midpoint increased by 4°C, and the chemical midpoint by 0.5 M GuHCl as compared to buffer conditions. Also the heptamer-monomer dissociation was affected in the presence of Ficoll 70, the transition midpoint was lower in Ficoll 70 (3.1 μM) compared to in buffer (8.1 μM) thus indicating tighter binding in crowded conditions. The coupled unfolding/dissociation free energy for the heptamer increased by about 36 kJ/mol in Ficoll. Altogether, the results revealed that the stability effect on cpn10 due to macromolecular crowding was larger in the individual monomers (33%) than at the monomer-monomer interfaces (8%). The results from the interaction study indicated conformational changes upon interaction between the A. thaliana Med25 ACtivator Interaction Domain (ACID) and Dreb2a. Structural changes were probed to originate from unstructured Dreb2a and not from the Med25-ACID. Human Med25-ACID was also found to interact with the plant-specific Dreb2a, even though the ACIDs from human and A. thaliana share low sequence homology. Moreover, the human Med25-interacting transcription factor VP16 was found to interact with A. thaliana Med25. Finally, NMR, ITC and pull-down experiments showed that the unrelated transcription factors Dreb2a and VP16 interact with overlapping regions in the ACIDs of A. thaliana and human Med25. The results presented in this thesis contribute to previous reports in two different aspects. Firstly, they lend support to the findings that the intracellular environment affects the biophysical properties of proteins. It will therefore be important to continue comparing results between in vitro and cell-like conditions to measure the magnitude of such effects and to improve the understanding of protein folding and thereby misfolding of proteins in cells. Better knowledge of protein misfolding mechanisms is critical since they are associated to several neurodegenerative diseases such as Alzheimer’s and Parkinson's. Secondly, our results substantiate the notion that transcription factors are able to bind multiple targets and that they gain structure upon binding. They also show that subunits of the conserved Mediator complex, despite low sequence homologies, retain a conserved structure and function when comparing evolutionary diverged species.

Protein folding is the process during which an extended and unstructured polypeptide converts to its compact folded structure that is most often the functional state. The process has been characterized extensively in dilute buffer in-vitro during the last decades but the actual biological place for this process is the inside of living cells. The cytoplasm of a cell is filled with a plethora of different macromolecules that together occupy up to 40% of the total volume. This large amount of macromolecules restricts the available space to each individual molecule, which has been termed macromolecular crowding. Macromolecular crowding results in excluded volume effects and also increases chances for non-specific interactions. Macromolecular crowding should favor reactions that lead to a decrease in the total occupied volume by all molecules, such as folding reactions. Theoretical models have predicted that the stability of protein folded states should increase in presence of macromolecular crowding due to unfavorable effects on the extended unfolded state. To understand protein folding and function in living systems, we need to have a defined quantitative link between in-vitro dilute conditions (where most biophysical experiments are made) and in-vivo crowded conditions. An important question is thus how macromolecular crowding modifies the biophysical properties of a protein. The work underlying this thesis focused on how macromolecular crowding tunes protein equilibrium stability and kinetic folding processes. To mimic the crowded cellular environment, synthetic sugar-based polymers (Dextrans of different sizes and Ficoll 70) were used as crowding agents (crowders) in controlled in-vitro experiments. In contrast to previous studies which often have focused on one protein and one crowder at a time, the goal here was to make systematic analyses of how size, shape and concentration of the crowders affect both equilibrium and kinetic properties of structurally-different proteins. Three model proteins (cytochrome c, apoazurin and apoflavodoxin) were investigated under crowding by Ficoll 70 and different-size Dextrans, using various spectroscopic techniques such as far-UV circular dichroism and intrinsic tryptophan fluorescence. Thermodynamic models were applied to explain the experimental results. It was discovered that equilibrium stability of all three proteins increased in presence of crowding agents in a crowder concentration dependent manner. The stabilization effect was around 2-3 kJ/mol, larger for the various Dextrans than for Ficoll 70 at the same g/l, but independent of Dextran size (in the range 20 to 70 kDa). To further investigate the cause for the stabilization a theoretical crowding model was applied. In this model, Dextran and Ficoll were modeled as elongated rods and the protein was represented as a sphere, where the folded sphere representation was smaller than the unfolded sphere representation. It is notable that the observed stability changes could be reproduced by this model taking only steric interactions into account. This correlation showed that when using sugar-based crowding agents, excluded volume effects could be studied in isolation and there were no contributions from nonspecific interactions. Time-resolved experiments with apoazurin and apoflavodoxin revealed an increase in the folding rate constants while the unfolding rates were invariant in the presence of crowding agents. For apoflavodoxin and cytochrome c, the presence of crowding agents also altered the folding pathway such that it became more homogeneous (cytochrome c) and it gave less misfolding (apoflavodoxin). These results showed that macromolecular crowding restricts the conformational space of the unfolded polypeptide chain, makes the conformations more compact which, in turn, eliminates access to certain pathways. The results from kinetic and equilibrium measurements on three model proteins, together with available data from the literature, demonstrate that macromolecular crowding effects due to volume exclusion are in the order of a few kJ/mol. Considering the numerous concentration balances and cross-dependent reactions of the cellular machinery, small changes in energetics/kinetics of the magnitudes found here can still have dramatic consequences for cellular fitness. In fact local and transient changes in macromolecular crowding levels may be a way to tune biochemical reactions without invoking gene expression.

The pathogenesis of Alzheimer’s disease (AD), the most common senile dementia, is a complex process. A crucial event in AD is the aggregation of amyloid-β protein (Aβ), a cleavage product from the Amyloid Precursor Protein (APP). Aβ40, a common component in amyloid plaques found in patients, aggregates in vitro at concentrations, much higher than the one found in vivo. But in the presence of charged lipid membranes, aggregations occurs at much lower concentration in vitro compared to the membrane-free case. This can be understood due to the ability of Aβ to get electrostatically attracted to target membranes with a pronounced surface potential. This electrostatically driven process accumulates peptide at the membrane surface at concentrations high enough for aggregation while the bulk concentration still remains below threshold. Here, we elucidated the molecular nature of this Aβ-membrane process and its consequences for Aβ misfolding by Circular Dichroism Spectroscopy, Differential Scanning Calorimetry and Nuclear Magnetic Resonance Spectroscopy. First, we revealed by NMR that Aβ40 peptide does indeed interact electrostatically with membranes of negative and positive surface potential. Surprisingly, it even binds to nominal neutral membranes if these contain lipids of opposite charge. Combined NMR and CD studies also revealed that the peptide might be shielded from aggregation when incorporated into the membrane. Moreover, CD studies of Aβ40 added to charged membranes showed that both positively and negatively membranes induce aggregation albeit at different kinetics and finally that macromolecular crowding can both speed up and slow down aggregation of Aβ.

Understanding the mechanisms of virus capsid assembly has been an important research objective over the past few decades. Determining critical points along the pathways by which virus capsids form could prove extremely beneficial in producing more stable DNA vectors or pinpointing targets for antiviral therapy. The inability of current experimental technology to address this objective has resulted in a need for alternative approaches. Theoretical and computational studies offer an unprecedented opportunity for detailed examination of capsid assembly. The Schwartz Lab has previously developed a discrete event stochastic simulator to model virus assembly based upon local rules detailing the geometry and interaction kinetics of individual capsid subunits. Applying numerical optimization methods to learn kinetic rate parameters that fit simulation output to in vitro static light scattering data has been a successful avenue to understand the details of virus assembly systems; however, information describing in vitro assembly processes does not necessarily translate to real virus assembly pathways in vivo. There are a number of important distinctions between experimental and realistic assembly environments that must be addressed to produce an accurate model. This thesis will describe work expanding upon previous parameter estimation algorithms for more complex data over three model icosahedral virus systems: human papillomavirus (HPV), hepatitis B virus (HBV) and cowpea chlorotic mottle virus (CCMV). Then it will consider two important modifications to assembly environment to more accurately reflect in vivo conditions: macromolecular crowding and the presence of nucleic acid about which viruses may assemble. The results of this work led to a number of surprising revelations about the variability in potential assembly rates and mechanisms discovered and insight into how assembly mechanisms are affected by changes in concentration, fluctuations in kinetic rates and adjustments to the assembly environment.

Fluorescence-based techniques are widely used in bioscience, offering a high sensitivity and versatility. In this work, fluorescence electronic energy migration/ transfer is applied to measure intramolecular distances in two types of systems and under various conditions. The main part of the thesis utilizes the process of donor-acceptor energy transfer to probe distances within the ribosomal protein S16. Proteins are essential to all organisms. Therefore, it is of great interest to study protein structure and function in order to understand and prevent protein malfunction. Moreover, it is also important to try to study the proteins in an environment which resembles its natural habitat. Here two protein homologs were investigated; S16Thermo and S16Meso, isolated from a hyperthemophilic bacterium and a mesophilic bacterium, respectively. It was concluded that the chemically induced unfolded state ensemble of S16Thermo is more compact than the corresponding ensemble of S16Meso. This unfolded state compaction may be one reason for the increased thermal stability of S16Thermo as compared to S16Meso. The unfolded state of S16 was also studied under highly crowded conditions, mimicking the environment found in cells. It appears that a high degree of crowding, induced by 200 mg/mL dextran 20, forces the unfolded state ensemble of S16Thermo to become even more compact. Further, intramolecular distances in the folded state of five S16 mutants were investigated upon increasing amounts of dextran 20. We found that the probed distances in S16Thermo are unaffected by increasing degree of crowding. However, S16Meso shows decreasing intramolecular distances for all three studied variants, up to 100 mg/mL dextran. At higher concentrations, the change in distance becomes anisotropic. This suggests that marginally stable proteins like s16Meso may respond to macromolecular crowding by fine-tuning its structure. More stable proteins like S16Thermo however, show no structural change upon increasing degree of crowding. We also investigated the possibility of local specific interactions between the protein and crowding agent, by means of fluorescence quenching experiments. Upon increasing amounts of a tyrosine labelled dextran, a diverse pattern of fluorescence quantum yield and lifetime suggests that specific, local protein-crowder interactions may occur. In a second studied system, electronic energy migration between two donor-groups, separated by a rigid steroid, was studied by two-photon excitation depolarization experiments. Data were analysed by using recent advances, based on the extended Förster theory, which yield a reasonable value of the distance between the two interacting donor-groups. To the best of our knowledge, this is the first quantitative analysis of energy migration data, obtained from two-photon excited fluorescence.

Les cellules ont développé plusieurs voies de signalisation et de réponses transcriptionnelles pour réguler leur taille et coordonner leur croissance et leurs divisions cellulaires. L'intérieur des cellules est naturellement surchargé par des macromolécules. Cet encombrement macromoléculaire, appelé crowding, a été intensément étudié in vitro et est connu pour affecter la cinétique des réactions. Cependant, l'étude des effets d'encombrement in vivo est plus difficile en raison du haut niveau de complexité et d'hétérogénéité à l'intérieur d'une cellule. Au cours de cette thèse, nous nous sommes intéressés aux effets de changement du volume cellulaire sur la cinétique de réactions biochimiques chez la levure Saccharomyces cerevisiae. Pour cela, nous avons induit des stress osmotiques pour comprimer la cellule et étudier l'impact du crowding sur les cinétiques de signalisation. La réduction du volume cellulaire augmente la viscosité interne et peut retarder le fonctionnement de plusieurs voies de signalisation et de processus cellulaires. En augmentant progressivement le niveau de compression, on observe un ralentissement des processus biologiques jusqu'à un point où l'adaptation cellulaire est abolie. Ceci a été observé pour la translocation nucléaire de facteurs de transcription (Hog1, Msn2, Crz1, Mig1 et Yap1) ainsi que pour la mobilité des protéines Abp1 et Sec7. Nous montrons aussi que la compression altère la capacité de plusieurs protéines à diffuser dans le cytoplasme de différents types cellulaires. Nous proposons que ces altérations cinétiques induites par l'augmentation de la viscosité intracellulaire ne soient pas sans rappeler une transition vitreuse. Ces résultats suggèrent l'importance d'un encombrement macromoléculaire optimal permettant aux cellules de fonctionner correctement.

La drépanocytose, ou anémie falciforme, présente une variabilité interindividuelle considérable, conditionnée par de multiples facteurs, dynamiques et interactifs, depuis le niveau moléculaire jusqu’au niveau du patient. L’hémoglobine drépanocytaire, ou hémoglobine S (HbS, tétramère a2bS 2), est un mutant de l’hémoglobine A (a2b2) : elle possède à sa surface une valine (hydrophobe) substituant un acide glutamique natif (négativement chargé). Cette mutation entraîne l’agrégation de l’HbS désoxygénée en polymères, ainsi que l’altération des propriétés de l’érythrocyte -dont sa rhéologie et ses interactions avec les différentes cellules vasculaires. C’est pourquoi la polymérisation de l’HbS constitue un facteur étiologique clef, sinon le primum movens, de la drépanocytose, et une hypothèse thérapeutique (étayée par l’observation) postule que la réduction des fibres intra-érythrocytaires de HbS pourrait améliorer le statut clinique des patients en abaissant la fréquence et la sévérité des crises vasoocclusives. Dans l’optique de mieux comprendre et de mieux gérer la variabilité individuelle drépanocytaire, il apparaît donc indispensable de disposer, en premier lieu, d’une description réaliste de la polymérisation de l’HbS. L’objectif de ce travail de thèse est la mise en place et la validation d’un modèle mathématique de la polymérisation de l’HbS désoxygénée, en tant que processus cinétiquethermodynamique, sous l’influence de la concentration et de la température –les deux facteurs modulateurs les plus importants. A partir d’un modèle existant, mais linéaire et incomplet (Ferrone et al., 1985), nous avons procédé à son implémentation, à sa correction et à sa mise à jour, ainsi qu’à l’évaluation quantitative de ses performances dynamiques, par intégration complète et simulation numérique (Simulink©). Ceci nous a permis de réaliser un diagnostic et d’effectuer un certain nombre de raffinements, concernant en particulier (i) la voie de nucléation hétérogène (formation de néo-fibres sur les fibres préexistantes), (ii) la non-idéalité de la solution protéique de HbS, induite par le volume exclus des fibres polymères (coefficients d’activité calculé à partir de la « théorie des particules convexes »), ainsi que (iii) la structuration spatiale des polymères en domaines. Le modèle développé dans ce travail servira de base pour une description (i) de l’influence dynamique de l’oxygénation et des hémoglobines non-polymérisantes sur la polymérisation de HbS, puis (ii) des polymères de HbS sur les propriétés membranaires et rhéologiques de l’érythrocyte drépanocytaire
Sickle cell disease pathology exhibits a strong interindividual variability, which depends upon multiple, dynamic and interacting factors, from the molecular to the patient level. Sickle hemoglobin, hemoglobin S (HbS, a2bS 2 tetramer), is a mutant of HbA (a2b2), with a surface valine (hydrophobic) substituting a native glutamic acid (negatively charged). Such a mutation endows deoxygenated HbS with the propensity to agregate into polymers, altering erythrocyte properties –including its rheology and its interactions with vascular and circulatory cells. Thus HbS polymerization is a key etiological factor of sickle cell disease, if not the primum movens. Indeed, one therapeutical hypothesis (supported by observation) postulates that the reduction of intra-erythrocytic HbS fibers could improve patients clinical status by lowering the frequency and the severity of vasooclusive crisis. In order to better understand and manage sickle cell disease variability, it is essential to have a realistic description of HbS polymerization. This work aims at developing and validating a mathematical model of deoxygenated HbS polymerization, as a kinetic and thermodynamic process under the influence of concentration and temperature –the two most important modulators. Building on an existing, but linearized and uncomplete (Ferrone et al., 1985) model, we have implemented, corrected and updated, and quantitatively evaluated its dynamical performances: this was done by full numerical integration using Simulink©. This allowed us to make several improvements, related in particular to : (i) the heterogeneous nucleation pathway (seeding and formation of new fibers from pre-existing ones), (ii) the non-ideality of the HbS protein solution, caused by polymer fibers excluded volume (activity coefficients were calculated with the CPT, Convex Particle Theory), and (iii) the spatial organization of polymers into domains. The model developped in this work will ground the description of the dynamic influence (i) oxygenation and non-polymerizing hemoglobins, (ii) HbS polymers interactions with membrane and consequences upon rheological properties of sickle cell erythrocyte

“Solvation water role in driving structural conformation and self-assembly of peptides and proteins”, the title of the present thesis, summarizes the objective of my three years PhD course aimed to investigate the relationship existing between the structure of biomolecules in solution and their mutual influence with surrounding water molecules. The three year work has been mainly experimental and principally focused in analyzing the capability of vibrational spectroscopies and some non-linear spectroscopic techniques to disentangle various contributions to solvent-molecule interactions. However to gain a deeper insight in the comprehension of such a complex problem, different types of experimental methods and theoretical modeling have supplemented spectroscopic techniques. A general survey of UV Resonant Raman (UVRR) spectroscopy and picosecond transient grating (TG) technique is reported in Chapter 2. The specific interactions of water molecules with glutathione tripeptide dissolved in pure water and water/salts mixtures are investigated experimentally by UVRR spectroscopy and modeled by molecular dynamics simulations. The results are presented in Chapter 3 and focus on the peptide-solvent interactions at the peptide site of glutathione thanks to the high selectivity of UVRR spectroscopy. Spectra are collected and analyzed as a function of concentration, pH, temperature and ion nature. OH stretching and amide modes spectral regions result very sensitive to the variations of the experimental conditions. The output provides a picture of the hydrogen-bonding network around glutathione. The number and the strength of hydrogen bonds increase in the deprotonated form of the tripeptide that exhibits a more marked capacity in decreasing the intermolecular order of water in its hydration shell. Potassium salts and imidazolium based ionic liquids of the halogen series are used to investigate the ions effect on glutathione structure and hydration shell. UVRR spectra present specific features possessing a great dependence on the nature of the anion present in solution rather than that of the cation, suggesting a strong capacity of anions to modify the glutathione structure and its hydration shell. There is a strong evidence that chloride and bromide anions interact at the NH site of glutathione reducing the possibility to form hydrogen bonds with water molecules and making the environment more hydrophobic than in pure water. Instead, iodide anion increases the number of water molecules at the peptide site, creating a strong polar environment. The spectroscopic and computational data are in perfect agreement and their interpretation can be based on the peptide link resonance model. In all the studied solvation environments, a progressive reduction in the strength of hydrogen bond interactions on amide sites is probed upon the increment of temperature, accompanied by conformational changes involving also the trans-cis isomerization of glutathione. Chapter 4 deals with the results of the study on self-crowded lysozyme solutions characterized by different degrees of aggregation and networking. Lysozyme has been widely investigated, as a convenient model protein, due to its ability to form amyloid fibrils in acidic conditions at high temperatures. Most of these past studies involved rather diluted samples in which fibril assembly is relatively slow. More rarely the formation of amyloid aggregates was examined in concentrated conditions, despite their relevance in different fields, from cellular biology and medicine to biomaterial and food technologies. In the present study, thermal unfolding and aggregation of highly concentrated (>100 mg/ml) lysozyme solutions at pH=1.8 are investigated. A method is designed to form protein hydrogels in a few hours. Their properties can be easily modulated selecting the curing temperature. The whole gelation process was monitored in situ by Fourier transform infrared (FTIR) spectroscopy assisted by hydrogen/deuterium isotopic exchange, to probe conformational changes and amyloid structuring. Specific molecular conformations are put in relation to thermodynamic properties by calorimetric measurements, to structural information by small angle x-ray and neutron scattering and to viscoelastic properties by means of rheology and TG experiments. This multi-technique approach is necessary in order to obtain a consistent picture on structure-property correlation in self-crowded protein samples. Aggregates constituted by antiparallel cross β-sheet links grow up quickly (less than two hours) within the 45-60 °C temperature range, leading to temperature-dependent quasi-stationary level of amyloid structures, attributed to kinetically trapped oligomers. Upon subsequent cooling, hydrogels develop quickly through the formation of non-specific inter-oligomer contacts. Due to this supramolecular assembly, the hydrogel is transparent, thermo-reversible and rather weak from a mechanical point of view. Lysozyme solutions can be recovered back to a large extent, following a process of oligomer-to-monomer dissociation and refolding. Overall, evidence is given of the possibility of easily forming protein hydrogels in self-crowding conditions constituted by kinetically trapped amyloid oligomers, interconnected by weak interactions. This type of gels might be relevant in different fields, when concentrated protein systems experience denaturing conditions.

Proteins are the molecular machines that control and regulate most of the vital cellular functions in living organisms, and account for about half of the total dry mass of the cell. For a protein to function properly, it must attain its correct conformation and location within the crowded milieu of the cell. A complex set of molecular chaperone proteins assist polypeptides to acquire their native functional fold within the relevant biological timescale. However, the intricacy and diverse nature of the protein folding process and various environmental factors present numerous opportunities for error, which may lead to its misfolding and aggregation. Misfolded, aggregated, non-functional proteins and peptides within the cell are implicated with more than 55 pathological conditions including various neurodegenerative diseases such as Alzheimer’s,Parkinson’s and haemodialysis-related amyloidosis, which together affect more than 24 million people worldwide. Collectively, these disorders are classified as protein conformational or protein aggregation diseases. Over the past few years, significant progress has been made to understand the various types and processes of protein aggregation, identify the toxic species, structurally characterise various species generated during their assembly and comprehend the underlying mechanisms which regulate protein conformational diseases. This thesis has attempted to address some of the issues discussed above, to assist in advancing the understanding of the complexities of protein misfolding, aggregation and their mitigation. In this thesis, I have found that various fragments of mouse Acyl co-enzyme thioesterase 7 (mAcot7) and human full-length ACOT7 form amyloid fibrils under physiological conditions. Acot7 is a brain cytosolic protein, involved in fatty acid metabolism, and has a putative role during inflammation. Using various mouse Acot7 constructs, I demonstrated that mAcot7 undergoes nucleation-independent, multi-stranded polymerisation, leading to the formation of globular oligomeric species and subsequently amyloid fibrils. Arachidonoyl-CoA, the substrate of mAcot7 did not prevent the fibrillation of mAoct7, and the protein remained enzymatically active throughout the assembly process. Understanding the biological significance of fibrillar and other forms of Acot7 is one of the avenues to be explored in the future. After putting forth a unique molecular model of mouse Acot7 fibrillation, the next objective was to identify and characterise the heterogeneous population of protein aggregates formed during polymerisation. Bis(Triphenylphosphonium) tetraphenylethene (TPE-TPP), a novel aggregation-induced emission luminogen aided in detecting early-stage protein aggregates; the species that are considered to be the most toxic entities during the development and progression of protein conformational diseases. Compared to traditional amyloid binding fluorescent dyes such as Thioflavin T (ThT), TPE-TPP showed a broader applicability in monitoring the process of fibrillation in various conditions such as acidic pH, elevated temperature, presence of potential amyloid inhibitors, and its ability to detect variations in amyloid fibril morphologies. Once I could differentiate between the different species generated during protein aggregation, I attempted to explore ways to modify these entities. Utilising the intra- and extra-cellular molecular chaperones αB-crystallin (αB-C), clusterin, α2-macroglobulin and haptoglobin, I observed that αB-C and clusterin stabilise various forms of D76N β2-microglobulin (a potent amyloidogenic variant of β2-microglobulin) generated during the fibrillation process. It is likely that these chaperones prevent primary and secondary nucleation of D76N β2-microglobulin (D76N β2m) fibrillation in vivo and decelerate the proliferation of amyloid fibril plaques. Experiments performed in vitro are usually less confined compared to the crowded cellular environment, and such crowding conditions further confound the process of protein aggregation. Moreover, oligomers of various amyloid proteins such as α-synuclein (αS) are believed to be the primary cause of cellular damage, for example in Parkinson’s disease. Utilising the highly identical presynaptic α- and β-synuclein and crowding agents such as Ficoll 400, I analysed the impact of crowding on the association of the synuclein proteins. Under physiologically relevant conditions, I found that β-synuclein (βS) exists as positively charged oligomers, which gradually polymerise to form self-assembled, non-amyloid fibril-like structures. Furthermore, βS destabilises αS and induces its aggregation, which is significantly moderated under the crowded cell-like environment. The results documented in this dissertation have assisted in advancing our knowledge and understanding of the molecular, biophysical and biochemical mechanisms of amyloid fibril formation. In addition, this thesis determined the broader applicability of TPE-TPP compared to the standard amyloid dye (i.e. ThT) as a fluorescent probe to monitor amyloid fibril formation, detect early-stage aggregates, variations in fibril morphologies, and can be deemed useful in screening amyloid inhibitors. Part of the aim to identify and characterise various protein aggregate species was to develop a method for regulating potentially toxic entities. I showed that intra- and extra-cellular molecular chaperones were capable of acting on different stages of D76N β2m aggregation, thereby reducing the effects of undesirable, misfolded toxic protein forms. Furthermore, I demonstrated that there could be other potentially hazardous forms of unwanted protein aggregates (i.e. βS self-assembled structures) that are not necessarily amyloid fibrils. Greater comprehension of protein aggregation will not only shed light on protein conformational disorders and aid in the development of therapeutics against their toxic effects, but may also offer insights into opportunities for exploiting stable, non-toxic protein conformations to our advantage.

My thesis is divided into five chapters, starting with a general introduction in first chapter and sample preparation and protein-NMR assignment techniques in second chapter. The remaining three chapters focus on three different areas/projects that I have worked on. Chapter 1: Introduction to nanomaterials and all the experimental techniques This chapter reviews different kinds of nanomaterials and their application utilized for protein-nanomaterial interaction in our study, along with the introduction to different spectroscopy and microscopy techniques used for the interaction studies. Starting with introduction of nanomaterials and all the experimental techniques, which constitute the arsenal for structural studies of the protein-nanomaterial interaction, different steps enroute to structural and dynamic interaction are outlined in detail. Chapter 2: Preparation and Characterization of Proteins used for nanomaterial interaction studies Proteins are generally of three kinds- globular (structured), intrinsically disordered and membrane bound. These proteins have different functions in living organisms and play a major role to maintain metabolism and other important factors. To probe protein-nanomaterial interactions, we have chosen different protein/peptides. This chapter describes the protocol/procedure used for purifying the proteins. For studying a globular protein, ubiquitin was chosen. Nanomaterial-IDP interaction was investigated using the intrinsically disordered central linker domain of human insulin like growth factor binding protein-2 (L-hIGFBP2). The hydrophobic membrane interacting part of the prion protein was chosen as a representative membrane protein. The characterization of the proteins by NMR spectroscopy is also described. Chapter 3: A nanomaterial based novel macromolecular crowding agent Carbon quantum dots (CQD) are nanomaterials with size less than 10 nm, first obtained in 2004 during purification of single-walled carbon-nanotubes. Since then CQDs have been used in a wide range of applications due to their low cost of preparation and favorable properties such as chemical inertness, biocompatibility, non-toxicity and solubility in aqueous medium. One of the applications of CQDs has been their use for imaging and tracking proteins inside cells, based on their intrinsic fluorescence. Further, quantum dots exhibit concentration dependent aggregation while retaining their solubility. Fluorescent carbon quantum dots (CQD) induce macromolecular crowding making them suitable for probing the structure, function and dynamics of both hydrophilic and hydrophobic peptides/ proteins under near in-cell conditions. We have prepared hydrophilic and hydrophobic quantum dots to see the crowding effect. After characterization of CQD, we tested the property of proteins with CQD and found that CQD behaves as a macromolecular crowding agent by mimicking near in-cell conditions. In our study, we have chosen a globular protein, an intrinsically disordered protein (IDP) and one hydrophobic membrane peptide. We have also compared the crowding property of CQD with ficoll which is widely used commercial crowding agent. The overall study tells that the CQD acts like crowding agent and can be used for the study of macromolecular crowding effect. This makes them suitable for structural and functional studies of proteins in near in-cell conditions. Chapter 4: Ubiquitin-Graphene oxide interactions Described here is the interaction of human ubiquitin with GO using NMR spectroscopy and other techniques such as Fluorescence spectroscopy, isothermal titration calorimetry (ITC), UV-Visible spectroscopy, dynamic light scattering (DLS), zeta potential measurements and transmission electron microscopy (TEM). The globular protein ubiquitin interacts with GO and undergoes a dynamic and reversible association-dissociation in a fast exchange regimen as revealed by NMR spectroscopy. The conformation of the protein is not affected and the primary interaction is seen to be electrostatic in nature due to the polar functional groups present on the protein and GO sheet surface. For the first time we have shown that the interaction between ubiquitin and GO is dynamic in nature with fast and reversible adsorption/desorption of protein from the surface of GO. This insight will help in understanding the mechanistic aspects of interaction of GO with cellular proteins and will help in designing appropriate functionalized graphene oxide for its biological application. Chapter 5: Section A: Interaction of an intrinsically disordered protein (L-HIGFBP2) with graphene oxide The interaction between intrinsically disordered linker domain of human insulin-like growth factor binding protein-2 (L-hIGFBP2) with GO was studied using NMR spectroscopy and other techniques such as isothermal titration calorimetry (ITC), dynamic light scattering (DLS), zeta-potential measurements. The study revealed that the disordered protein L-hIGFBP2 interacts with GO through electrostatic interaction and undergoes a dynamic and reversible association-dissociation in a fast exchange regime. The conformation of the protein is not affected. Section B: Stability of an Intrinsically disordered protein through weak interaction with Silver nanoparticles Using NMR spectroscopy and other techniques we probed the mechanism of L-hIGFBP2–AgNP interactions which render the IDP stable. The study reveals a mechanism which involves a relatively fast and reversible association–dissociation of L-hIGFBP2 (dynamic exchange) from the surface of AgNP. The AgNP–L-hIGFBP2 complex remains stable for more than a month. The techniques employed in addition to NMR include UV-Visible spectroscopy, dynamic light scattering (DLS), zeta potential measurements and transmission electron microscopy (TEM) to probe the protein-AgNP interaction here in this section.

As molecular recognition players glycans mediate essential physiological events with high importance in Life. Galectin-3 (Gal-3) is a key regulator of the immune system and a potent effector in diverse cellular mechanisms inside the cell, at the cell surface and at extracellular matrix. Structurally Gal-3 FL is composed by a well-folded carbohydrate recognition domain (Gal-3 CRD) and a disorder tail. Gal-3 recognition events, including the dynamics and binding, are well characterized in diluted solutions. However, Gal-3 recognize galactose-unit in a complex biological medium that contains high concentrations of proteins, polysaccharides and metabolites. On this basis, the present project reports the consequences of distinct macromolecular crowders, two synthetic polymers (PEG 3350 and Ficoll 70) and two natural proteins (BSA and Lysozyme), into Gal-3/lactose recognition process combining NMR binding experiments from receptor to ligand viewpoint. 1H15N-HSQC experiments indicate no visible chemical shift perturbation of Gal-3 CRD signals after addition of crowders, noteworthy, the intensity of 1H15N-HSQC signals decrease for all crowders. Furthermore, the intensity of 1H15N-HSQC peaks of Gal-3 CRD recovered upon addition of a saturating amount of lactose. The solution viscosity accessed by water diffusion using diffusion experiments cannot explain the decrease of 1H15N-HSQC signals intensity in crowding conditions, as well as the raise of signals intensity upon addition of lactose. Hence, Gal-3 CRD/crowders interactions should take place in solution yielding an apparent large-sized complex that increases the global molecular tumbling of Gal-3 CRD with a dramatic broadening effect on 1H15N-HSQC Gal-3 CRD resonances. Preferential interactions occurred with the BSA, mainly due to its electrostatic protein surface and size. Crowding conditions reduce the diffusion coefficient of Gal-3 CRD in one order, accordingly to the literature data, and this value remains constant after addition of lactose. The dramatic line broadening effect precludes R2/R1 ratio determination of Gal-3 CRD in presence of BSA, noteworthy we were able to estimate R2/R1 ratio of Gal-3 CRD/lactose complex in presence of BSA. R2/R1 Gal-3 CRD/lactose values are thus different from that obtained in diluted solution mainly due to the raise in viscosity after addition of BSA. Gal-3 FL/crowder interactions were also exploited using BSA as the more physiological relevant crowder and ficoll as synthetic carbohydrate polymer. In contrast to Gal-3 CRD, no recovery of 1H15N-HSQC signal intensity was observed to Gal-3 FL after addition of lactose. Solution viscosity can prevent the detection of 1H15N-HSQC peaks in the case of larger Gal-3 FL by decreasing dramatically T2, or Gal-3 FL can self-associate in crowding conditions and potentiated by the presence of the unfolded tail. Noteworthy, STD-NMR experiment of lactose in presence of Gal-3 FL and BSA were performed indicating that lactose binding mode is conserved in crowding conditions. Finally, crowding conditions may have a biological implication contributing to co-localize Gal-3 at the cell surface favouring protein cell-adhesion and eventually promoting functional self-assembly of Gal-3.

During the past decade the effects of macromolecular crowding on reaction pathways is gaining in prominence. The stress is to move out of the realms of ideal solution studies and make conceptual modifications that consider non-ideality as a variable in our calculations. In recent years it has been shown that molecular crowding exerts significant effects on all in vivo processes, from DNA conformational changes, protein folding to DNA-protein interactions, enzyme pathways and signalling pathways. Both thermodynamic as well as kinetic parameters vary by orders of magnitude in uncrowded buffer system as compared to those in the crowded cellular milieu. Ignoring these differences will restrict our knowledge of biology to a “model system” with few practical understandings. The recent expansion of the genome database has stimulated a study on numerous previously unknown proteins. This has whetted our thirst to model the cellular determinants in a more comprehensive manner. Intracellular extract would have been the ideal solution to re-create the cellular environment. However, studies conducted in this solution will be contaminated by interference with other biologically active molecule and relevant statistical data cannot be extracted out from it. Recent advances in methodologies to mimic the cellular crowding include use of inert macromolecules to reduce the volume occupancy of target molecules and the use of immobilization techniques to increase the surface density of molecules in a small volumetric region. The use of crowding agents often results in non-specific interaction and side-reactions like aggregation of the target molecules with the crowding agents themselves. Immobilization of one of the interacting partners reduces the probability of aggregation and precipitation of bio-macromolecules by restricting their degrees of freedom. Covalent linkage of molecules on solid support is used extensively in research for creating a homogeneous surface of bound molecules which can be interrogated for their reactivity. However, when it comes to biomolecules, direct immobilization on solid support or use of organic linkers often results in denaturation. The use of bio-affinity immobilization techniques can help us overcome this problem. Since mild conditions are needed to regenerate such a surface, it finds universal applicability as bio-memory chips. This thesis focuses on our attempts to design a physiologically viable immobilization technique for following rotein-protein/protein-DNA interactions. The work explores the mechanism for biological interactions related to transcription process in E. coli. Chapter 1 deals with the literary survey of the importance and effects of molecular crowding on biological reactions. It gives a brief history of the efforts been made so far by experimentalists, to mimic macromolecular crowding and the methods applied. The chapter tries to project an all-round perspective of the pros and cons of different immobilization techniques as a means to achieve a high surface density of molecules and the advancements so far. Chapter 2 deals with the detailed technicality and applicability of the Langmuir-Blodgett method. It discusses the rationale behind our developing this technique as an alternate means of bio-affinity immobilization, under physiologically compatible conditions. It then goes on to describe our efforts to follow the sequence-specific and sequential assembly process of a functional RNA polymerase enzyme with one immobilized partner and also explore the role of omega subunit of RNAP in the reconstitution pathway. This chapter uses the assembly process of a multi-subunit enzyme to evaluate the efficiency of the LB system as a universal two-dimensional scaffold to follow sequence-specific protein-ligand interaction. Chapter 3 discusses the application of LB technique to quantitatively evaluate the kinetics and thermodynamics of promoter-RNA polymerase interaction under conditions of reduced dimensionality. Here, we follow the interaction of T7A1 phage promoter with Escherichia coli RNA polymerase using our Langmuir-Blodgett technique. The changes in mechanistic pathway and trapping of kinetic intermediates are discussed in detail due to the imposed restriction in the degrees of freedom of the system. The sensitivity of this detection method is compared vis-a-vis conventional immobilization methods like SPR. This chapter firmly establishes the universal application of LB technique as a means to emulate molecular crowding and as a sensitive assay for studying the effects of such crowding on vital biological reaction pathway. Chapter 4 describes the mechanistic pathway for the physical binding of MsDps1 protein with long dsDNA in order to physically protect DNA during oxidative stress. The chapter describes in detail the mechanism of physical sequestering of non-specific DNA strands and compaction of the genome under conditions where a kinetic bottleneck has been applied. The data obtained is compared with results obtained in the previous chapter for the sequence-specific DNA-protein interaction in order to understand the difference in recognition process between regulatory and structural proteins binding to DNA. Chapter 5 deals with the evaluation of the σ-competition model in E. coli for three different sigma factors (all belonging to the σ-70 family). Here again, we have evaluated the kinetic and thermodynamic parameters governing the binding of core RNAP with its different sigma factors (σ70, σ32and σ38) and performed a comparative study for the binding of each sigma factor to its core using two different non-homogeneous immobilization techniques. The data has been analyzed globally to resolve the discrepancies associated with establishing the relative affinity of the different sigma factors for the same core RNA polymerase under physiological conditions. Chapter 6 summarizes the work presented in this thesis. In the Appendix section we have followed the unzipping of promoter DNA sequence using Optical Tweezers in an attempt to follow the temporal fluctuations occurring in biological reactions in real time and at a single molecule level.

碩士
國立東華大學
化學系
95
Physiological fluid media contain macromolecules occupying a significant fraction (typically 20-30%) of the total volume. Biological macromolecules have evolved to function inside such crowded environments. It has been shown the natural and synthetic macromolecules can be used to mimic the crowding environments in cells. In this study, we applied NMR and other biophysical techniques to investigate the effect of crowding on the stability and conformation of ubiquitin and its mutants. Ubiquitin (Ub), a small protein with 76 residues, contained 5-stranded b-sheet and one a-helix. We have cloned and overexpressed wild type ubiquitin and its mutants, F4A/F45W, V26A/F45W, and I30A/F45W in E. coli. The wild type ubiquitin was in the folded state in the pH ranged from 2 to 10, however, these three mutants were unfolded at pH below 3. We found that the existence of dextran did not change the secondary structure content when ubiquitin was in the folded state. However, the crowding condition did induce a significant amount of secondary structure in partially unfolded ubiquitin. We also found that addition of anion (Cl-) to partially unfolded ubiquitin can drive the equilibrium from the unfolded state toward the native or native-like state. The NMR structures of F4A/F45W at neutral pH and at pH 2 in the presence of high concentration of Cl- were closely identical to the native structure of wild type ubiquitin. The tertiary structure of F4A/F45W at pH 2 in the presence of crowding agents was similar to the tertiary structure of F4A/F45W at pH 2 in the presence of high concentration of Cl-.

Gene expression, which connects genomic information to functional units in living cells, has received substantial attention since the completion of The Human Genome Project. Quantitative characterization of gene expression will provide valuable information for understanding the behavior of living cells, and possibilities of building synthetic gene circuits to control or modify the behavior of naturally occurring cells. Many aspects of quantitative gene expression have been studied, including gene expression dynamics and noise in E. coli. The gene expression process itself is stochastic, and modelling approaches have been broadly used to study gene expression noise; however, stochastic gene expression models are usually large and time intensive to simulate. To speed up simulations, we have developed a systematic method to simplify gene expression models with fast and slow dynamics, and investigated when we can ignore the gene expression from the background genome when modelling the gene expression from plasmids. When modelling the noise in gene expression, one usually neglected aspect is the slow maturation process of fluorescent proteins, necessary for the protein to give out fluorescence after it is produced. By modelling, we show that the maturation steps can bring large changes to both the mean protein number and the noise in the model. An unstudied aspect of gene expression dynamics is the time dependent gene expression behavior in E. coli batch culture. Contrary to the usual assumption, we have found, in E. coli batch culture gene expression, that there is no steady state in terms of both the mean number of proteins and the noise. Negative feedback is thought to be able to reduce the noise in a system, and experiments have shown that negative feedback indeed suppresses the noise in gene expression, but the modelling shows that negative feedback will increase the noise. We have found that the increase of noise by feedback is due to the exclusion of extrinsic noise from the model, and that negative feedback will suppress the extrinsic noise while increasing the intrinsic noise. Living cells are crowded with macromolecules, which will, predicted by modelling, make the reaction constant time dependent. Our experimental observation has confirmed this prediction.

Feng, Hui.
Thesis Ph.D. Chinese University of Hong Kong 2015.
Includes bibliographical references (leaves 91-102).
Abstracts also in Chinese.
Title from PDF title page (viewed on 05, January, 2017).

博士
國立清華大學
化學系
103
Spin-label electron spin resonance (ESR) spectroscopy has been extensively developed in the latest decade for studying problems in the fields of biology, physics, and chemistry. With the site-directed spin-labeling techniques, ESR can be employed to resolve the complexity of molecular dynamics, probing local environments of various molecular complexes such as protein, membrane, and macromolecular assemblies. In particular, continuous wave (cw) ESR and double electron-electron resonance (DEER) are among the most powerful ESR techniques. This dissertation demonstrates three biophysical applications of the ESR techniques that have never been reported. First, we describe how useful the ESR technique can be utilized to reveal details of molecular motions of spin-labeled biomolecules as confined in nanochannels. Specifically, we characterize the rotational dynamics of a long (14-residue) proline-based peptide (approximately 4 nm in length) under anisotropic nanoconfinement using spin-label ESR techniques as well as spectral simulations. We show by pulsed ESR experiments that the conformations of the peptide in several different nanochannels and a bulk solvent are retained. Parameters characterizing the dynamics of the peptide regarding temperature (200 ~ 300 K) and nanoconfinement are determined from nonlinear least-squares fits of theoretical calculations to the multifrequency (X- and Q-band) experimental spectra. Remarkably, we found that this long helical peptide undergoes a large degree of rotational anisotropy and orientational ordering inside the nanochannels, but not in the bulk solvent. The rotational anisotropy of the helical peptide barely changes with the nanoconfinement effects and remains substantial, as the nanochannel diameter is varied from 6.1 to 7.1 and 7.6 nm. This finding is advantageous for addressing purposes of anisotropic nanoconfinement and for advancing our understanding of the rotational dynamics of nano-objects as confined deeply inside the nanostructures of materials. In the second project presented in this dissertation, we report a ESR study of Bcl-2 associated X (BAX) protein. BAX protein plays a key role in the mitochondria-mediated apoptosis. However, it remains unclear by what mechanism BAX is triggered to initiate apoptosis. Here, we reveal the activation mechanism underlying the transformation from inactive to active BAX. An inactive BAX monomer was found to exhibit conformational heterogeneity and exist at equilibrium in two populations of conformation, one of which has never been reported. We show that upon apoptotic stimulus by BH3-only peptides, BAX can be induced to convert into either a ligand-bound monomer or an oligomer through a conformational selection mechanism. The kinetics of reaction is studied by means of time-resolved ESR, allowing a direct in-situ observation for the transformation of BAX from the native to the bound states. In vitro mitochondrial assays provide further discrimination between the proposed BAX states, thereby revealing a population-shift allosteric mechanism in the process. BAX′s apoptotic function is shown to critically depend on excursions between different structural conformations. In the third project, we apply the ESR techniques to investigate the effects of molecular crowding on protein stability. We carry out a comprehensive investigation on the conformational stability of T4 lysozyme (T4L) enzyme in varying crowding conditions, 300 − 500 g/L of crowders (including BSA protein, glycerol, Ficoll, and PVP polymers), using cw-ESR, circular dichroism, and Thermofluor spectroscopy methods. Double-labeled spectra were used to probe the local dynamical changes and distance distribution of T4L protein in the applied crowded and thermal conditions. ESR spectra were obtained from three T4L mutants to study the crowding effects on the tertiary structure (with mutant T4L-A), secondary structure (with mutant T4L-B), and hinge-bending activity (with mutant T4L-C) at temperatures 280 − 343 K. The results of the T4L-A and T4L-B show a decreased structural stability, in terms of conformational dynamics and free energy, with increasing concentration of the crowders. In contrast, the structural stability of the T4L-C mutant was found to increase with the crowder concentrations. This study indicates that structural domains or segments of a protein respond differently to molecular crowding effects. In summary, results presented in this dissertation have expanded the applications of spin-label ESR techniques one step further to resolving several important problems in the interdisciplinary field of biology, physics, and chemistry.

Ionic Liquids (ILs) consist in organic salts that are liquid at/or near room temperature. Since ILs are entirely composed of ions, the formation of ion pairs is expected to be one essential feature for describing solvation in ILs. In recent years, protein - ionic liquid (P-IL) interactions have been the subject of intensive studies mainly because of their capability to promote folding/unfolding of proteins. However, the ion pairs and their lifetimes in ILs in P-IL thematic is dismissed, since the action of ILs is therefore the result of a subtle equilibrium between anion-cation interaction, ion-solvent and ion-protein interaction. The work developed in this thesis innovates in this thematic, once the design of ILs for protein stabilisation was bio-inspired in the high concentration of organic charged metabolites found in cell milieu. Although this perception is overlooked, those combined concentrations have been estimated to be ~300 mM among the macromolecules at concentrations exceeding 300 g/L (macromolecular crowding) and transient ion-pair can naturally occur with a potential specific biological role. Hence the main objective of this work is to develop new bio-ILs with a detectable ion-pair and understand its effects on protein structure and stability, under crowding environment, using advanced NMR techniques and calorimetric techniques. The choline-glutamate ([Ch][Glu]) IL was synthesized and characterized. The ion-pair was detected in water solutions using mainly the selective NOE NMR technique. Through the same technique, it was possible to detect a similar ion-pair promotion under synthetic and natural crowding environments. Using NMR spectroscopy (protein diffusion, HSQC experiments, and hydrogen-deuterium exchange) and differential scanning calorimetry (DSC), the model protein GB1 (production and purification in isotopic enrichment media) it was studied in the presence of [Ch][Glu] under macromolecular crowding conditions (PEG, BSA, lysozyme). Under dilute condition, it is possible to assert that the [Ch][Glu] induces a preferential hydration by weak and non-specific interactions, which leads to a significant stabilisation. On the other hand, under crowding environment, the [Ch][Glu] ion pair is promoted, destabilising the protein by favourable weak hydrophobic interactions , which disrupt the hydration layer of the protein. However, this capability can mitigates the effect of protein crowders. Overall, this work explored the ion-pair existence and its consequences on proteins in conditions similar to cell milieu. In this way, the charged metabolites found in cell can be understood as key for protein stabilisation.

The characterization of molecules within a biologically relevant environment distinguishes nuclear magnetic resonance (NMR) spectroscopy from other molecular-based biophysical techniques, such as X-ray crystallography and cryo-electron microscopy. Due to its exceptional stability and reduced ability to interact in a specific manner with other cellular components, the GB1 protein represents the quintessential probe to investigate the physiochemical effects imposed by the crowded environment on the structure and dynamics of proteins, without simultaneously compromising the ability to obtain in-cell NMR spectra due to binding events. The general aim of this thesis was to investigate the possible interactions of the GB1 protein with the Escherichia coli lysate and intracellular milieu with the purpose of inferring the physiochemical effects imposed by these two crowded environments on the structure and dynamics of proteins. Thus, the experimental parameters critical for performing in-cell NMR experiments, including bacterial growth and protein overexpression within E. coli cells, were initially optimized. Subsequently, by monitoring proton and nitrogen chemical shifts of backbone amides and lysines side chains, as well as carbon and proton chemical shifts of side chains containing carbonyl groups, the preferential behaviour of GB1 was analysed in lysate and within cells, considering the pure protein in water as the reference state. Furthermore, interactions with the local environment were further examined by determining the overall translational motion of the protein through diffusion-ordered NMR spectroscopy (DOSY). The results obtained suggest that the intracellular environment is much more viscous than its artificially crowded counterpart and that GB1 exhibits a distinct behaviour in E. coli than in lysate. Specifically, residues at or near the more flexible and solvent-exposed loop regions of the protein display an increased preference for interaction with cellular components within cells compared to lysate. Finally, a comparison of diffusion coefficients obtained with DOSY and fluorescence correlation spectroscopy (FCS), the standard analytical technique for studying protein diffusion, was made.