Pentameriska ligandstyrda jonkanaler (pLGICs) är avgörande för omvandlingen av kemisk till elektrisk signalöverföring i djurs nervsystem. Dysfunktion i dessa kanaler har visat sig vara kopplad till flera sjukdomar inklusive epilepsi, schizofreni, Alzheimers och autism, vilket gör dem till en måltavla för en mängd olika läkemedel. Att studera eukaryota kanaler är dock mycket utmanande, så upptäckten av prokaryota homologer, som är mycket lättare att studera, har därmed bidragit mycket till förståelsen för struktur och funktion hos proteiner i denna familj. I detta projekt producerades och renades en prokaryotisk pLGIC kallad DeCLIC från Escherichia coli. Strukturell bestämning av kanalen genomfördes med användning av kryo-elektronmikroskopi vid lågt pH och i närvaro av kalcium. En elektrontäthet med 3.4 Å upplösning uppnåddes och jämfördes med tidigare bestämda strukturer vid olika förhållanden i ett försök att bestämma hur proteinets struktur moduleras av kalcium och pH. Resultaten visar flera skillnader i kanalens konformation i närvaro och frånvaro av kalcium såväl som vid olika pH-värden. Dessutom antyder analys av den bestämda elektrontätheten ett möjligt intermediärt tillstånd vid lågt pH i närvaro av kalcium. / Pentameric ligand-gated ion channels (pLGICs) are crucial for the conversion of chemical to electrical signaling in the nervous system of mammals. Dysfunction in these channels has been found to be connected to several diseases including epilepsy, schizophrenia, Alzheimer’s, and autism, making them the target of a wide variety of therapeutic agents. However, studying eukaryotic channels is challenging so the discovery of prokaryotic homologs that are much easier to study has thus greatly helped in the understanding of the structure and function in this family of proteins. In this project, a prokaryotic pLGIC called DeCLIC was produced and purified from Escherichia coli. Structural determination of the channel was pursued using cryo-electron microscopy at a low pH and in the presence of calcium. An electron density at 3.4 Å resolution was achieved and compared to previously determined structures at different conditions in an attempt to determine the structural modulation of calcium and pH. Results show multiple differences in channel conformation in the presence and absence of calcium as well as in different pH conditions. Furthermore, analysis of the determined electron density suggests a possible intermediate state at low pH in the presence of calcium.

The rapid pace of scientific advances is enabling a greater understanding of diseases at the molecular level. In turn, the process for researching and developing new medicines is growing in difficulty, costs, and length as a result of the scientific, technical, and regulatory challenges related to the development process. In light of these challenges, drug repositioning, the utilization of known drugs for a new medical indication, has emerged as an increasingly important strategy for the new drug discovery. Availability of prior knowledge regarding safety, efficacy and the appropriate administration route significantly reduces the development costs and cuts down the development time resulting in less effort to successfully bring a repositioned drug to market. In another aspect, a protein’s shape is closely linked with its function; thereby, the ability to predict this structure unlocks a greater understanding of what it does and how it works. Nowadays, more than 10,000 biologically relevant protein structures are yearly released and available to the scientific community. A number suspected to triple over the following years due to the recent breakthroughs in structure prediction techniques. This work introduces a novel structure-based drug repositioning approach, exploiting the similarities of drugs’ binding mode via identification and virtual screening of interaction patterns. Such patterns are uncovered with the use of PLIP, an automated tool for the in silico detection of non-covalent interactions defining the binding mode between drugs and their protein targets. Besides, the approach has been applied to a series of case studies with tangible results: the uncovering of an antimalarial drug as potential chemoresistance treatment, the explained binding mode of ibrutinib to the target VEGR2 as potential B-cells deactivator in autoimmune diseases, and three over the counter drugs with a proved anti-trypanocidal activity as treatments for Chagas disease. Overall the structure-based approach with interaction patterns proved to be a suitable framework for identifying novel repositioning candidates. The uncovered candidates were structurally unrelated to the currently available treatments, and experimental assays successfully demonstrated their inhibitory activity on the protein targets of interest. Furthermore, the approach represents a promising option for the 'in high demand' diseases and all rare and neglected diseases for which no reliable treatment has yet been found and for which the pharmaceutical industry makes only a little investment.

<p>  </p> <p>With the recent growth in demand for biologics across the globe, it remains critical to manufacture these biologics in solid-state to improve stability as well as to increase the ease of transportation across the world. To meet these increased demands, it is of paramount importance to use various processing methods that have shorter processing times. It is also important to understand the impact of the processing methods and various formulation components on the stability of the proteins.  In Chapter 1, a review of the various processing methods that are used in the industry along with additional processing methods that are being investigated will be discussed. The common drying methods such as lyophilization and spray drying along with the novel techniques as well as specific examples of processing parameters to improve the processing conditions that better suit the protein formulations will be mentioned. </p> <p>The studies in Chapter 2 examined the effects of processing methods (freeze drying and spray freeze drying) and the excipients on the protein structure and physical stability. Protein solids containing one of two model proteins (lysozyme or myoglobin) were produced with or without excipients (sucrose or mannitol) using freeze drying or spray freeze drying (SFD). The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), circular dichroism spectrometry (CD), size exclusion chromatography (SEC), BET surface area measurements, and solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS). ssFTIR and CD could identify little to no difference in the structure of the proteins in the formulation. ssHDX-MS was able to identify the population heterogeneity, which was undetectable by conventional characterization techniques of ssFTIR and CD. ssHDX-MS metrics such as Dmax and peak area showed a good correlation with the protein physical instability (loss of the monomeric peak area by size exclusion chromatography) in 90-day stability studies conducted at 40oC for lysozyme.  The higher specific surface area was associated with greater loss in monomer content for myoglobin-mannitol formulations as compared to myoglobin-only formulations. Spray freeze drying seems a viable manufacturing technique for protein solids with appropriate optimization of formulations. The differences observed within the formulations and between the processes using ssHDX-MS, BET surface area measurements, and SEC in this study provide an insight into the influence of drying methods and excipients on protein physical stability.</p> <p>Based on this work, it was identified that spray freeze drying can be a viable alternative to produce solid-state protein formulations with similar stability as the freeze drying process. However, due to the long processing times and scale-up issues involved in the spray freeze drying process, there is a necessity to explore additional drying processes. Chapter 3 focuses on using another novel technique known as electrostatic spray drying (ESD) to produce solid-state protein formulations at lower drying temperatures than conventional spray drying and its effect on protein stability. A mAb formulation was dried by either conventional spray drying or electrostatic spray drying with charge (ESD). The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), size exclusion chromatography (SEC), and solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). Particle characterizations such as BET surface area, particle size distribution, and particle morphology were also performed. Conventional spray drying of the mAb formulation at the inlet temperature of 70oC failed to generate dry powders due to poor drying efficiency; electrostatic spray drying at the same temperature at 5kV enabled the formation of powder formulation with satisfactory moisture contents. Deconvoluted peak areas of deuterated samples from the ssHDX-MS study showed a good correlation with the loss of the monomeric peak area measured by size exclusion chromatography in the 90-day accelerated stability study conducted at 40oC. Low-temperature (70oC inlet temperature) drying with an electrostatic charge (5kV) led to better protein physical stability as compared with the samples spray-dried at the high temperature (130oC inlet temperature) without charge.</p> <p>This study shows that electrostatic spray drying can produce solid monoclonal antibody formulation at a lower inlet temperature than traditional spray drying with better physical stability. While ESD can be a viable option for thermal-sensitive formulations, it is important to understand the impact of various formulation components on the stability of the proteins while using spray drying. Based on our previous studies, a good understanding of the effect of different sugars and the presence of surfactants on the spray-dried proteins has been established. However, the impact of the selection of buffer on protein stability has not been studied. In Chapter 4, the effect of buffer salts on the physical stability of spray dried and lyophilized formulations of a model protein, bovine serum albumin (BSA) were examined. BSA formulations with various buffers were dried by either lyophilization or spray drying. The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), powder X-ray diffraction (PXRD), size exclusion chromatography (SEC), solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS), and solid-state nuclear magnetic resonance spectroscopy (ssNMR). Particle characterizations such as BET surface area, particle size distribution, and particle morphology were also performed. Results from conventional techniques such as ssFTIR did not exhibit correlations with the physical stability of studied formulations. Deconvoluted peak areas of deuterated samples from the ssHDX-MS study showed a satisfactory correlation with the loss of the monomeric peak area measured by SEC (R2 of 0.8722 for spray-dried formulations and 0.8428 for lyophilized formulations) in the 90-day accelerated stability study conducted at 40oC. PXRD was unable to measure phase separation in the samples right after drying. In contrast, ssNMR successfully detected the occurrence of phase separation between the succinic buffer component and protein in the lyophilized formulation, which results in a distribution of microenvironmental acidity and the subsequent loss of long-term stability. In summary, this study demonstrated that buffer salts have less impact on physical stability for the spray-dried formulations than the lyophilized solids.</p> <p>The study in Chapter 5 looked at examining the physical stability of spray freeze dried (SFD) bovine serum albumin (BSA) solids produced using the radio frequency (RF)-assisted drying technique. BSA formulations were prepared with varying concentrations of trehalose and mannitol, with an excipient-free formulation as control. These formulations were produced using traditional spray freeze drying (SFD) or RF-assisted spray freeze drying (RFSFD). The dried formulations were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), Karl Fischer moisture content measurement, powder X-ray diffraction (PXRD), size exclusion chromatography (SEC), solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). Traditional characterization tools such as ssFTIR and moisture content did not have a good correlation with the physical stability of the formulations measured by SEC. ssHDX-MS metrics such as the maximum deuterium uptake (Dmax) (R2 = 0.791) and deconvoluted peak areas of the deuterated samples (R2 = 0.914) showed a satisfactory correlation with the SEC stability data. RFSFD improved the stability of formulations with 20 mg/ml of trehalose and no mannitol and had similar stability with all other formulations as compared to SFD. This study demonstrated that the RFSFD technique can significantly reduce the duration of primary drying cycle from 48 h to 27.5 h while maintaining or improving protein physical stability as compared to traditional lyophilization.</p> <p>Lastly, Chapter 6 consists of a summary of the conclusions formed from the work presented in this thesis. Furthermore, suggestions for future work are provided based on observations of results, less-explored areas of formulation and processing conditions as well as characterization tools to understand effects on protein physical stability.</p> <p><br></p>

<p>Proteins are significant components of living cells. They perform a diverse range of biological functions such as cell shape and metabolism. The functions of proteins are determined by their three-dimensional structures. Cryogenic-electron microscopy (cryo-EM) is a technology known for determining the structure of large macromolecular structures including protein complexes. When individual atomic protein structures are available, a critical task in structure modeling is fitting the individual structures into the cryo-EM density map.</p> <p>In my research, I report a new computational method, MarkovFit, which is a machine learning-based method that performs simultaneous rigid fitting of the atomic structures of individual proteins into cryo-EM maps of medium to low resolution to model the three-dimensional structure of protein complexes. MarkovFit uses Markov random field (MRF), which allows probabilistic evaluation of fitted models. MarkovFit starts by searching the conformational space using FFT for potential poses of protein structures, computes scores which quantify the goodness-of-fit between each individual protein and the cryo-EM map, and the interactions between the proteins. Afterwards, proteins and their interactions are represented using a MRF graph. MRF nodes use a belief propagation algorithm to exchange information, and the best conformations are then extracted and refined using two structural refinement methods. </p> <p>The performance of MarkovFit was tested on three datasets; a dataset of simulated cryo-EM maps at resolution 10 Å, a dataset of high-resolution experimentally-determined cryo-EM maps, and a dataset of experimentally-determined cryo-EM maps of medium to low resolution. In addition to that, the performance of MarkovFit was compared to two state-of-the-art methods on their datasets. Lastly, MarkovFit modeled the protein complexes from the individual protein atomic models generated by AlphaFold, an AI-based model developed by DeepMind for predicting the 3D structure of proteins from their amino acid sequences.</p>

No description available.

<p dir="ltr">Ion mobility spectrometry (IMS), with its unparalleled ability to separate and filter ions based on their overall size before channeling them into a Mass Spectrometer, has placed itself as a cornerstone of the modern Analytical Chemistry field. IMS provides an orthogonal separation, aiding in the identification and analysis processes of various compounds. While there have been many inventions for ion mobility (IM) devices with exponential growth in the separation capability in the past few years, there is very little emphasis on the theoretical explanation. For example, most modern IMS devices often use a high ratio of electric field to gas concentration (E/n) as it provides better separation capabilities. However, the interaction between ion and gas at such E/n cannot be explained by current IM theories as they ignore several critical factors such as the increase in ion’s energy due to energetic collisions, the energy loss/transferred in the internal degree of freedoms, and change in the ion’s structure, requiring empirical data to identify ions after separation. The thesis presented here contributes towards bridging this gap by elucidating the complex interplay of forces and interactions that govern the ion separation process, thereby explaining on how these mechanisms can be further exploited for refined separation and advancing the computational approach to identify the separated ion.</p><p dir="ltr">To explain the ion-gas interaction under high E/n, this research extends the Two-Temperature Theory (2TT) up to the fourth order approximation. The central idea of the 2TT is to solve moments of the Boltzmann equation for the ion’s velocity distribution involving ion-gas collisions. The research shows a decreasing error between each subsequent approximations, indicating convergence. This advancement is demonstrated through the development and application of our in-house program, IMoS, and validated against experimental data for small ions in monoatomic gases. This research also justifies the mechanisms of increasing and decreasing mobility as the electric field is increased by explaining the interplay between the interaction potential and the collision energy.</p><p dir="ltr">Subsequent chapters investigate the impact of internal degrees of freedom (rotational and vibrational) on ion mobility. This includes pioneering work with the Structures for Lossless Ion Manipulations (SLIM) device to separate isotopomers, alongside computational advancements in simulating these effects, leading to the development of IMoS 2.0. In IMoS 2.0 software an ion is placed in a virtual drift tube with electric field, where it is free to rotate and translate upon collision. The research notably uncovers the role of rotational degrees of freedom in isotopomer separation, a previously underexplored area.</p><p dir="ltr">To ascertain the effect of the vibrational DoF and differentiate from the ion’s structural expansion and heating resulting from energetic collisions, a combined simulation of ion mobility and molecular dynamics (IM-MD) was performed. This analysis revealed that structural expansion plays a dominant role for the cause of deviation at high E/n, to such an extent that the vibrational DoF (or inelastic collisions) can normally be disregarded. Moreover, the research also indicates that using a combination of IM-MD simulation, one can identify accurate gas-phase structure of the ion at any temperature from a pool of probable structures.</p><p dir="ltr">Guided by these conclusions, the research now takes a significant step forward by aiming to accurately characterize protein structures in the gas phase using IM-MD simulation. Traditional MD simulations provide larger structures since the force field is not optimized for the gas-phase simulation. To address this, a biasing force towards the center of the protein is applied, compressing it. This method efficiently explores multiple feasible configurations, including those obscured by energy barriers. This strategy generated structures that closely align with the experimental evidence.</p>

Kinks are functionally important structural features found in the alpha-helices of many proteins, particularly membrane proteins. Structurally, they are points at which a helix abruptly changes direction. Previous kink definition and identification methods often disagree with one another. Here I describe three novel methods to characterise kinks, which improve on existing approaches. First, Kink Finder, a computational method that consistently locates kinks and estimates the error in the kink angle. Second the B statistic, a statistically robust method for identifying kinks. Third, Alpha Helices Assessed by Humans, a crowdsourcing approach that provided a gold-standard data set on which to train and compare existing kink identification methods. In this thesis, I show that kinks are a feature of long -helices in both soluble and membrane proteins, rather than just transmembrane -helices. Characteristics of kinks in the two types of proteins are similar, with Proline being the dominant feature in both types of protein. In soluble proteins, kinked helices also have a clear structural preference in that they typically point into the solvent. I also explored the conservation of kinks in homologous proteins. I found examples of conserved and non-conserved kinks in both the helix pairs and the helix families. Helix pairs with non-conserved kinks generally have less similar sequences than helix pairs with conserved kinks. I identified helix families that show highly conserved kinks, and families that contain non-conserved kinks, suggesting that some kinks may be flexible points in protein structures.

Proteins are an important component of living organisms, composed of one or more polypeptide chains, each containing hundreds or even thousands of amino acids of 20 standard types. The structure of a protein from the sequence determines crucial functions of proteins such as initiating metabolic reactions, DNA replication, cell signaling, and transporting molecules. In the past, proteins were considered to always have a well-defined stable shape (structured proteins), however, it has recently been shown that there exist intrinsically disordered proteins (IDPs), which lack a fixed or ordered 3D structure, have dynamic characteristics and therefore, exist in multiple states. Based on this, we extend the mapping of protein sequence not only to a fixed stable structure but also to an ensemble of protein conformations, which help us explain the complex interaction within a cell that was otherwise obscured. The objective of this dissertation is to develop effective ab initio methods and tools for protein un/structure prediction by developing effective statistical energy function, conformational search method, and disulfide connectivity patterns predictor. The key outcomes of this dissertation research are: i) a sequence and structure-based energy function for structured proteins that includes energetic terms extracted from hydrophobic-hydrophilic properties, accessible surface area, torsion angles, and ubiquitously computed dihedral angles uPhi and uPsi, ii) an ab initio protein structure predictor that combines optimal energy function derived from sequence and structure-based properties of proteins and an effective conformational search method which includes angular rotation and segment translation strategies, iii) an SVM with RBF kernel-based framework to predict disulfide connectivity pattern, iv) a hydrophobic-hydrophilic property based energy function for unstructured proteins, and v) an ab initio conformational ensemble generator that combines energy function and conformational search method for unstructured proteins which can help understand the biological systems involving IDPs and assist in rational drugs design to cure critical diseases such as cancer or cardiovascular diseases caused by challenging states of IDPs.

Protéines sont largement utilisés dans la formulation dans le domaine pharmaceutique et de jouer un rôle important dans les fonctions biologiques. Il est bien connu que l'adsorption de protéines sur la surface solide est toujours observé pour un stockage à long terme, ce qui entraînera une réduction de la dose de substance active ou une perte de l'activité biologique. Dans certains cas, une courte période de contact avec la surface est suffisante pour modifier fortement la conformation des protéines : par exemple, l'insuline pertes 52% de son activité biologique après 5 minutes de contact avec la surface de verre, ainsi qu'une perte de 30% d’activité biologique du cétrorélix est observé après 2 heures de contact. Parmi tous les paramètres, la dénaturation des protéines est fortement liée à sa stabilité des propriétés de surface. La compréhension de l'adsorption de protéines est devenue une question cruciale dans l'industrie pharmaceutique.Pour mieux comprendre le comportement des protéines à la surface, la quantification des protéines adsorbées et sa conformation devrait être étudiée. L'objectif de notre recherche sera de comprendre les comportements des protéines sur différents surfaces de seringue pré - remplie classique.Le principal objectif de ce projet est de comprendre le comportement de plusieurs modèles de protéines comme la sérum d’albumine bovine (BSA), le lysozyme (LSZ) et la myoglobine (MGB) en contact avec des surfaces de seringues pré-remplie comme le verre et l’élastomère. Nous proposons d'utiliser la chromatographie liquide à haute performance (HPLC) pour la quantification de protéine adsorbée sur une surface plane en déterminant la déplétion des protéines en solution. La réflexion totale atténuée infrarouge à transformée de fourier (FTIR-ATR) spectroscopie est utilisée de suivre les changements structurels des protéines adsorbées sur des surfaces solides. [...] / Proteins are widely used in formulation in the pharmaceutical field and play a major role in biological functions. It is well known that protein adsorption on solid surface is always observed for a long-term storage, which will result in a reduced dose of active compound or a loss of biological activity. In some cases, only short time of contact are sufficient to drastically modify the protein conformation: for instance, insulin losses 52% of its biological activity after 5 minutes contacting with glass surface, as well as a loss of 30% of cetrorelix is observed after 2 hours. Among all parameters, the time frame of the denaturation process is strongly related to the protein stability and surface properties. The understanding of protein adsorption has therefore become a crucial issue in the pharmaceutical industry.To gain a better understanding of proteins’ behavior on the surface, adsorbed protein quantification and its conformation should be studied. The objective of our research in a first will be to understand proteins’ behaviors on various surfaces which composed a classical prefilled syringe.The main goal of this PhD project is to understand the behaviors of several model proteins like bovine serum albumin (BSA), lysozyme (LSZ) and myoglobin (MGB) in contact with the surfaces of prefilled syringes such as glass and elastomer. We propose to use the high performance liquid chromatography (HPLC) to quantify the amount of protein adsorbed on a flat surface by determining the depletion of the proteins in solution. Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy was as well as employed to follow the structural changes of adsorbed BSA on solid surface. [...]

Mass spectrometry has in recent years been established as the standard method for protein identification and characterization in proteomics with excellent intrinsic sensitivity and specificity. Fourier transform ion cyclotron resonance is the mass spectrometric technique that provides the highest resolving power and mass accuracy, increasing the amount of information that can be obtained from complex samples. This thesis concerns how useful information on proteins of interest can be extracted from mass spectrometric data on different levels of protein structure and how to obtain this data experimentally. It was shown that it is possible to analyze complex mixtures of protein tryptic digests by direct infusion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and identify abundant proteins by peptide mass fingerprinting. Coupling on-line methods such as liquid chromatography and capillary electrophoresis increased the number of proteins that could be identified in human body fluids. Protein identification was also improved by novel statistical methods utilizing prediction of chromatographic behavior and the non-randomness of enzymatic digestion. To identify proteins by short sequence tags, electron capture dissociation was implemented, improved and finally coupled on-line to liquid chromatography for the first time. The combined techniques can be used to sequence large proteins de novo or to localize and characterize any labile post-translational modification. New computer algorithms for the automated analysis of isotope exchange mass spectra were developed to facilitate the study of protein structural dynamics. The non-covalent interaction between HIV-inhibitory peptides and the oligomerization of amyloid β-peptides were investigated, reporting several new findings with possible relevance for development of anti-HIV drug therapies and understanding of fundamental mechanisms in Alzheimer’s disease.