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Characterisation of low-complexity regions in protein sequencesNava, Alain Coletta January 2009 (has links)
Low-complexity regions appear to be common in protein sequences and although funcons remain to be elucidated for most of them, they have been increasingly found to play crucial biological roles. Because most of these regions show poor conservation across protein families, it is difficult to compare them using common sequence analysis techniques. This study asks whether low-complexity regions play important roles in protein bind and if they are crucial to the overall functions of the proteins to which they belong.
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Nuclear transport of the alphaherpesvirus UL47 tegument proteinVerhagen, Janneke Martine January 2005 (has links)
No description available.
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Structural studies on C3 exoenzymes and Botulinum neurotoxinsEvans, Hazel January 2003 (has links)
No description available.
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Understanding the rheology of liquid protein formulationsDomingues Goncalves, Andrea January 2013 (has links)
The work described in this thesis had the main aim of understanding protein solution rheology. This was from a biopharmaceutical perspective, with account of the biophysical properties of proteins and in particular their level of aggregation. Molecular interactions influencing the rheology of a range of protein solutions were studied. Proteins were selected to relate directly to the diversity of protein types used in biopharmaceuticals. In addition, the roles of a surfactant formulation additive and synthetic amphiphilic polymers in the flow behaviour of protein solutions were studied. The effect of protein concentration on solution viscosity in a commercially available biopharmaceutical formulation of a recombinant albumin (rAlbumin) was studied. The effect of the level of protein aggregation, variation in protein concentration and its impact on solution viscosity was revealed. Theoretical models predicting the increase of viscosity with concentration were applied to these data. A recent model that accounts for multiple protein species in solution, predicted the experimental data best. The rAlbumin study, although a relatively simple system, represented a 'real-life' formulation with results highlighting the need to account for heterogeneity in the level of aggregation when addressing the increase of viscosity observed at high concentration of protein solutions. Beta-lactoglobulin (b-LG) excipient-free solutions were characterised by bulk and interfacial shear rheology. Solutions at various concentrations, characterised using conventional rheology instrumentation, evidenced an apparent yield stress behaviour at a low shear rate range (0.01 - 10 1/s), whilst showing constant viscosities throughout higher shear rates. Comparing interfacial shear rheology, air-water interface-free bulk rheology measurements, and tensiometry results, it was demonstrated that the complexity of this protein's solution rheology was due to the formation of a protein viscoelastic film at the air-water interface, as present in conventional rheometry. This is in agreement with literature. Further studies considered the effect of insoluble b-LG aggregates on the solutions' rheology, linking with their characterisation in size and quantication. The presence of insoluble proteinaceous particles was suggested to have an impact on the solution's flow behaviour, particularly at the lower shear rates. Excipient-free monoclonal antibody (mAb) solutions were studied with the aim of generating protein aggregates (soluble and insoluble) to explore their impact on solution rheology. mAb samples were subjected to thermal stress and were characterised for their purity, aggregate content and size. The change in species content did not alter the original protein's yield-stress behaviour at low shear rates. An increase in aggregate content was related to the increase of viscosities observed at high shear rates. Establishing a relationship between species content (in volume fraction) and viscosities, as for the rAlbumin study, was not possible due to this mAbs specific aggregation behaviour. However, from the b-LG and mAb case studies, our results highlight the importance of detailed characterisation of protein solutions with orthogonal biophysical techniques so as to better understand protein solution rheology. An additional study looking at the effect of polysorbate-80 upon protein rheology was made. In agreement with literature, this commonly used excipient in biopharmaceuticals was demonstrated to affect the rheological measurements of globular protein solutions. Amphiphilic brush-like poly(ethylene glycol) methacrylate polymers were also synthesised and tested as novel additives with b-LG and mAb solutions, for their potential effects on protein solution rheology, similar to those observed with polysorbate-80. Preliminary results showed that the effects of these polymers are likely related to competition for the air-water interface, between these and the proteins involved. This competition leads to changes in the yield-like behaviour at low shear rates.
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Production, characterization and use of isotopically enriched metalloproteins for the analysis of biological samples by species-specific isotope dilution ICP-MSDeitrich, Christian L. January 2009 (has links)
In this work, the chemical preparation and characterization of an isotopically enriched superoxide dismutase (SOD) is described. Its evaluation as a standard in species-specific isotope dilution analysis by HPLC coupled to ICP-MS is carefully evaluated. The proposed method involved the removal of the enzyme's metal co-factors under various conditions and their replacement with isotopically enriched ⁶⁵Cu and ⁶⁸Zn. SEC-ICP-MS showed that the prepared enriched enzymes had a different metal isotopic abundance compared to the wild-type enzyme. Isotopically enriched and wild-type SOD showed the same migration pattern in 1D-PAGE. An enzyme activity assay provided evidence that incorporated ⁶⁵Cu was bound to the correct SOD-binding motif, since the measured activity correlated directly with the amount of Cu present in the prepared enzyme. The addition of free Cu and Zn or a metal chelator did not result in any exchange or loss of metals from the enzyme at neutral pH. Striking experiments were undertaken to evaluate the use of isotopically enriched SOD in SS-IDMS. The chemical preparation study on SOD was further extended to prepare various other isotopically enriched metalloproteins, including carbonic anhydrase, ceruloplasmin, transferring and haemoglobin. Various enrichment procedures were conducted and their performances then evaluated, using SEC-ICP-MS and protein assays. A procedure for the quantification of SOD in tissue samples using an isotopically enriched SOD spike in combination with 2-dimensional HPLC and SS-IDMS was developed and assessed. The feasibility of employing isotopically enriched protein spikes for the speciation of metalloproteins by utilising gel electrophoresis and LA-ICP-MS was also investigated. Furthermore, the change of the iron speciation of the meat-containing protein myoglobin after various treatments was examined using a combination of SEC-ICP-MS and ESI-MS.
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Protein complexes in the gas phase : structural insights from ion mobility-mass spectrometry and computational modellingHall, Zoe Lauren January 2013 (has links)
Structure determination of macromolecular protein assemblies remains a challenge for well-established experimental methods. In this thesis, an emerging structural technique, ion mobility-mass spectrometry (IM-MS) is explored. An assessment of collision cross section (CCS) measurement accuracy using travelling-wave IM (TWIMS) instrumentation was carried out (Chapter 3). Through the collation of a protein complex CCS database and the development of a calibration framework for TWIMS, significant improvements to CCS measurement accuracy have been achieved. Next, the advantages and limitations of using IM-MS to generate restraints for structure characterisation were explored. Computational tools designed to exploit IM-MS data for structural modelling were developed and tested on a training set of systems (Chapter 4). These include two heteromeric protein complexes, and an oligomeric intermediate involved in beta-2-microglobulin aggregation. Further structural information can be attained by using gas-phase dissociation techniques, such as collision-induced dissociation (CID). The effects of charge state on CCS and the gas-phase dissociation pathway of complexes were investigated (Chapter 5). This highlighted the possibility of using CID in conjunction with supercharging to manipulate dissociation pathways to achieve more useful structural information. Finally, the gas-phase structures of globular and intrinsically disordered protein complexes were probed by IM-MS and molecular dynamics (MD) simulations (Chapter 6). Experimental observations were recapitulated remarkably closely by simulations, including gas-phase structural collapse and the ejection of monomer subunits when the energy of the system was increased sufficiently. Overall, this research has contributed to the IM-MS field by providing the framework for improved CCS measurements of large protein complexes and the use of restraints from IM-MS for structural modelling. Significantly, IM-MS has been used in combination with charge manipulation, CID and MD simulations to reveal further insights into the gas-phase structures, stabilities and dissociation pathways of multimeric protein complexes.
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Interfacial adsorption of proteins : a neutron reflectivity studyLatter, Edward Gareth January 2012 (has links)
Protein adsorption at the solid/liquid interface is of wide ranging importance in many different areas of science such as biomaterial design, the fate of nanoparticles and in the food industry. As a result, many studies have been undertaken with varying foci but there still remains a lack of agreement between many working in this field and fundamental questions regarding the adsorption of proteins at the solid/liquid interface. Neutron reflectivity is a powerful technique for probing the properties of adsorbed layers at interfaces due to its high structural resolution and the possibility of using isotopic substitution to distinguish between components of a mixture. In this work, neutron reflectivity has been used as the primary technique for the investigation of proteins adsorbed sequentially or from a binary mixture. Initially, the adsorption of four proteins (carbonic anhydrase II, lysozyme, human serum albumin and maltose binding protein) onto a clean silica surface was investigated which revealed the importance of electrostatic interactions and entropic contributions to the driving forces for adsorption. Most of the adsorbed layers were described by a 2-layer model with a thinner, denser layer adjacent to the surface and a thick, diffuse layer extending into the bulk solution. The presence of impurities is also shown to have a significant impact on the adsorption of HSA. A study of the HSA/myristic acid system shows that the presence of small amphiphiles can inhibit HSA adsorption and also remove a pre-adsorbed layer. A comparison was made between the protonated and deuterated forms of two proteins, HSA & MBP, showing the deuterated proteins to have a higher affinity for the surface with adsorption occurring in a 3:1 ratio when from a 1:1 mixture. Likewise, d-MBP displaced h-MBP more readily than vice versa in an investigation into the effect of incubation time on the properties of the protein layer. The extent of desorption into protein free buffer is not affected by incubation time but the extent to which d-MBP was displaced by h-MBP showed a clear trend of decreased exchange with increasing incubation time indicating an active exchange process was occurring. This was also observed to a lesser extent for the sequential adsorption of binary protein systems, HSA & LYS and HSA & MBP. When investigating binary protein mixtures the higher propensity for deuterated proteins to adsorb is observed. LYS dominates when adsorbed from a mixture with h-HSA but from a d-HSA & LYS mix both proteins were adsorbed. The marked difference between the adsorption characteristics of perdeuterated proteins and their protonated counterparts provides a good case study for testing the neutron reflectivity technique when investigating systems with more than one component. This thesis assesses the limitations of the methodology of contrast variation for investigating mixtures as well as using different solvent contrasts. A comparison of neutron reflectivity and dual polarisation interferometry (DPI) shows that the two techniques are similar and any small differences can be attributed to the small change in surface chemistry. This comparison also highlights the advantages of DPI; high throughput of samples and detailed information but the restriction to using a 1-layer model limits its use.
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Computational studies of protein dynamics and dynamic similarityMunz, Marton January 2012 (has links)
At the time of writing this thesis, the complete genomes of more than 180 organisms have been sequenced and more than 80000 biological macromolecular structures are available in the Protein Data Bank (PDB). While the number of sequenced genomes and solved three-dimensional structures are rapidly increasing, the functional annotation of protein sequences and structures is a much slower process, mostly because the experimental de-termination of protein function is expensive and time-consuming. A major class of in silico methods used for protein function prediction aim to transfer annotations between proteins based on sequence or structural similarities. These approaches rely on the assumption that homologous proteins of similar primary sequences and three-dimensional structures also have similar functions. While in most cases this assumption appears to be valid, an increasing number of examples show that proteins of highly similar sequences and/or structures can have different biochemical functions. Thus the relationship between the divergence of protein sequence, structure and function is more complex than previously anticipated. On the other hand, there is mounting evidence suggesting that minor changes of the sequences and structures of proteins can cause large differences in their conformational dynamics. As the intrinsic fluctuations of many proteins are key to their biochemical functions, the fact that very similar (almost identical) sequences or structures can have entirely different dynamics might be important for understanding the link between sequence, structure and function. In other words, the dynamic similarity of proteins could often serve as a better indicator of functional similarity than the similarity of their sequences or structures alone. Currently, little is known about how proteins are distributed in the 'dynamics space' and how protein motions depend on structure and sequence. These problems are relevant in the field of protein design, studying protein evolution and to better understand the functional differences of proteins. To address these questions, one needs a precise definition of dynamic similarity, which is not trivial given the complexity of protein motions. This thesis is intended to explore the possibilities of describing the similarity of proteins in the 'dynamics space'. To this end, novel methods of characterizing and comparing protein motions based on molecular dynamics simulation data were introduced. The generally applicable approach was tested on the family of PDZ domains; these small protein-protein interaction domains play key roles in many signalling pathways. The methodology was successfully used to characterize the dynamic dissimilarities of PDZ domains and helped to explain differences of their functional properties (e.g. binding promiscuity) also relevant for drug design studies. The software tools developed to implement the analysis are also introduced in the thesis. Finally, a network analysis study is presented to reveal dynamics-mediated intramolecular signalling pathways in an allosteric PDZ domain.
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Olefin metathesis for site-selective protein modificationLin, Yuya Angel January 2013 (has links)
Site-selective protein modification has become an important tool to study protein functions in chemical biology. In the preliminary work, allyl sulfides were found to be reactive substrates in aqueous cross-metathesis (CM) enabling the first examples of protein modification via this approach. In order to access the enhanced CM reactivity of allyl sulfide on proteins, facile chemical methods to install S-allyl cysteine on protein surface were developed. In particular, a cysteine-specific allylating reagent – allyl selenocyanate was used on protein substrate for the first time. The substrate scope of allyl sulfide-tagged proteins and factors that affect the outcome of CM was also investigated. A range of metathesis substrates containing different olefin tether of various lengths were screened; allyl ethers were found to be most suitable as CM partners. By reducing the steric hindrance around the allyl sulfide on protein surface through a chemical spacer, the rate and conversion of metathesis reaction on proteins was greatly enhanced. Moreover, allyl selenides were found to be more reactive than allyl sulfides in CM and enabled reactions with substrates that were previously impossible for the corresponding sulfur-analogue. Through this work, substrate selection guidelines for successful metathesis reaction on proteins were established. Rapid Se-relayed CM was further investigated through biomimetic chemical access to Se-allyl selenocysteine (Seac) via dehydroalanine. On-protein reaction kinetics revealed rate constants of Seac-mediated CM to be comparable or superior to off-protein rates of many current bioconjugations. This CM strategy was applied to histone proteins to install a mimic of acetylated lysine (K9Ac, an epigenetic marker). The resulting synthetic H3 was successfully recognized by antibody that binds natural H3-K9Ac. A Cope-type selenoxide elimination subsequently allowed the removal of such modification to regenerate dehydroalanine. Finally, preliminary research efforts towards metabolic incorporation of allyl sulfide-containing amino acid into proteins, and CM on cell surfaces were discussed.
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Rôle de la protéine Bcd1p/BCD1 dans les étapes précoces de la biogenèse des snoRNP à boîtes C/D eucaryotes / Functions of Bcd1p/BCD1 in the early steps of box C/D snoRNP biogenesisPaul, Arnaud 27 September 2018 (has links)
La biogenèse des ribosomes matures et fonctionnels est notamment dépendante de petites particules ribonucléoprotéiques composées d’ARN et de protéines, les snoRNP (small nucleolar RiboNucleoProteins). Celles-ci sont subdivisées en deux familles : les snoRNP à boîtes C/D et les snoRNP à boîtes H/ACA. Ces deux classes de snoRNP catalysent des modifications chimiques, respectivement de 2’-O-méthylation et de pseudouridylation, sur des positions spécifiques des ARN ribosomiques (ARNr), ou sont impliquées dans des clivages du long ARNr précurseur. Les snoRNP à boîtes C/D sont composées d’un snoARN à boîtes C/D servant de guide pour cibler la position à modifier, et d’un jeu invariant de quatre protéines : Snu13p/SNU13, Nop1p/Fibrillarine, Nop56p/NOP56 et Nop58p/NOP58 (levure/Homme). Ces snoRNP sont produites par la cellule grâce à la présence de plusieurs complexes protéiques constituant une machinerie pour leur assemblage. Outre plusieurs facteurs protéiques déjà connus dans la biogenèse de snoRNP à boîtes C/D comme les protéines Rsa1p/NUFIP, Hit1p/ZNHIT3 et les protéines du complexe R2TP, d’autres protéines pourraient compléter cette machinerie. Parmi ces facteurs additionnels, la protéine Bcd1p/ZNHIT6, pour Box C/D snoRNA protein 1, est essentielle pour maintenir spécifiquement la stabilité in vivo des snoARN à boîtes C/D, et des associations ont pu être identifiées entre Bcd1p/ZNHIT6 avec différents partenaires protéiques de la machinerie d’assemblage de ces particules. Toutefois, l’étape d’assemblage où Bcd1p/ZNHIT6 intervient et la fonction qu’elle y accomplit demeurent inconnues. L’utilisation d’outils in vivo et in vitro chez la levure S. cerevisiae et chez les mammifères nous ont permis de progresser dans la compréhension de la fonction de Bcd1p/ZNHIT6 dans l’assemblage des snoRNP à boîtes C/D. Bcd1p est un facteur d’assemblage recruté de manière co-transcriptionnelle sur les loci codant les snoARN à boîtes C/D et est requis pour le recrutement des complexes d’assemblage sur les snoARN en cours de transcription. Plus spécifiquement, Bcd1p affecte l’interaction de Nop58p avec le facteur d’assemblage Rsa1p, suggérant une fonction dans le recrutement de Nop58p dans une pré-snoRNP en cours d’assemblage. Ce travail a permis d’apporter des informations importantes permettant d’expliquer le caractère essentiel de Bcd1p dans la fonction et la biogenèse des snoRNP à boîtes C/D / Ribosome biogenesis is especially dependent on the action of small RNA/proteins complexes called small nucleolar ribonucleoproteins (snoRNPs). They are divided into two main families: the so-called box C/D snoRNPs and box H/ACA snoRNPs. Each category performs specific enzymatic processes, 2’-O-methylation and pseudouridylation, respectively, and induces target-specific chemical modification on rRNAs. Few snoRNPs are also essential for pre-rRNA processing. The box C/D snoRNPs are formed by the association of a box C/D snoRNA with a set of four invariant proteins: Snu13p/SNU13, Nop1p/Fibrillarine, Nop56p/NOP56 and Nop58p/NOP58 (yeast/Human). Biogenesis of these RNPs relies on the action of several proteins complexes which constitute a dedicated assembly machinery. Rsa1p/NUFIP, Hit1p/ZNHIT3, and components of the R2TP complex are the best characterized protein actors of this machinery. Additional protein factors probably participate in box C/D snoRNP biogenesis; Bcd1p/ZNHIT6 (Box C/D snoRNA protein 1) is such a candidate as it is essential for the in vivo stability of box C/D snoRNAs, and it was found associated with proteins involved in this machinery in yeast and Human. However, the mechanism governing the recruitment of this protein towards the biogenesis of box C/D snoRNP, and the step of the assembly process relying on the presence of Bcd1p are still unknown. In S. cerevisiae and Human, in vivo and in vitro tools allowed us to improve the understanding of the functions of Bcd1p/ZNHIT6 in box C/D snoRNP assembly. Bcd1p is an assembly factor that is recruited co-transcriptionally on box C/D snoRNA loci, and is required for the recruitment of assembly complexes on nascent snoRNAs. Bcd1p is important for Nop58p association with the assembly factor Rsa1p, which suggests that its primary function is to recruit Nop58p to nascent pre-snoRNPs. This work evidenced important information on the essential role of Bcd1p in C/D snoRNP biogenesis and function
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