Дисертації: "Proteins self-assembly"

1

Edwards, Todd Criswell. "Self-assembly of proteins at interfaces and two-dimensional protein crystallization /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/8093.

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2

Wason, Akshita. "Investigation of lsm proteins as scaffolds in bionanotechnology." Thesis, University of Canterbury. School of Biological Sciences, 2014. http://hdl.handle.net/10092/10065.

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Self-assembling materials have gained attention in the field of nanotechnology due to their potential to be used as building blocks for fabricating complex nanoscale devices. The biological world is abundant with examples of functional self-assembling biomolecules. Proteins are one such example, found in a variety of geometries and shapes. This research is focussed on the use of ring-shaped self-assembling proteins, called Lsm proteins, as componentary for applications in bionanotechnology. Lsm proteins were used because of their spontaneous association into stable rings, tolerance to mutations, and affinity to RNA. This thesis primarily focussed on the thermophilic Lsmα (from Methanobacterium. thermoautotrophicum) that assembles as heptameric rings. The oligomeric state of the heptameric protein, and hence the diameter of its central cavity, was manipulated by judiciously altering appropriate residues at the subunit interface. Lsmα presented a complex set of interactions at the interface. Out of the mutations introduced, R65P yielded a protein for which SEC and SAXS data were consistent with a hexameric state. Moreover, key residues, L70 and I71, were identified that contribute to the stability of the toroid structure. Covalent linking of rings provided nanotubular structures. To achieve this, the surface of the Lsmα ring scaffold was modified with Cys residues. This approach led to the formation of novel Lsmα nanotubes approximately 20 nm in length. Importantly, the assembly could be controlled by changing the redox conditions. As an alternative method to manipulate the supramolecular assembly, His6-tags were attached at the termini of the Lsmα sequence. The higher-order organisation of the constructs was influenced by the position of the His6-tag. The N-terminally attached His6-tag version of Lsmα showed a metal-dependent assembly into cage-like structures, approximately 9 nm across. This organisation was highly stable, reproducible, and reversible in nature. The results presented in this thesis aid the understanding of generating complex nanostructures via in vitro self-assembly. The Lsmα rings were assembled into higher-order architectures at the quaternary level by employing protein engineering strategies. Future work is necessary to functionalise these supramolecular structures; however, this study confirms the potential role of Lsmα proteins as a molecular building block in bionanotechnology.

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3

Santonicola, Mariagabriella. "Molecular self-assembly and interactions in solutions of membrane proteins and surfactants." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 248 p, 2007. http://proquest.umi.com/pqdweb?did=1257806151&sid=6&Fmt=2&clientId=8331&RQT=309&VName=PQD.

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Thesis (Ph.D.)--University of Delaware, 2006.
Principal faculty advisors: Eric W. Kaler, College of Engineering; and Abraham M. Lenhoff, Dept. of Chemical Engineering. Includes bibliographical references.

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4

Mille, Christian. "Templating and self-assembly of biomimetic materials." Doctoral thesis, Stockholms universitet, Institutionen för material- och miljökemi (MMK), 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-80459.

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This thesis focuses on the use of biomolecular assemblies for creating materials with novel properties. Several aspects of biomimetic materials have been investigated, from fundamental studies on membrane shaping molecules to the integration of biomolecules with inorganic materials. Triply periodic minimal surfaces (TPMS) are mathematically defined surfaces that partition space and present a large surface area in a confined space. These surfaces have analogues in many physical systems. The endoplasmic reticulum (ER) can form intricate structures and it acts as a replica for the wing scales of the butterfly C. rubi, which is characterized by electron microscopy and reflectometry. It was shown to contain a photonic crystal and an analogue to a TPMS. These photonic crystals have been replicated in silica and titania, leading to blue scales with replication on the nanometer scale. Replicas analyzed with left and right handed polarized light are shown be optically active. A macroporous hollow core particle was synthesized using a double templating method where a swollen block copolymer was utilized to create polyhedral nanofoam. Emulsified oil was used as a secondary template which gave hollow spheres with thin porous walls. The resulting material had a high porosity and low thermal conductivity. The areas of inorganic materials and functional biomolecules were combined to create a functional nanoporous endoskeleton. The membrane protein ATP synthase were incorporated in liposomes which were deposited on nanoporous silica spheres creating a tight and functional membrane. Using confocal microscopy, it was possible to follow the transport of Na+ through the membrane. Yop1p is a membrane protein responsible for shaping the ER. The protein was purified and reconstituted into liposomes of three different sizes. The vesicles in the 10-20 nm size range resulted in tubular structures. Thus, it was shown that Yop1p acts as a stabilizer of high curvature structures.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Submitted. Paper 4: Submitted. Paper 5: Submitted.

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5

Rodon, Fores Jennifer. "Localized protein-assisted self-assembly : from mechanism to applications." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAE017.

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La cellule est un système chimique complexe qui a bénéficié de milliards d’années d’évolution pour se perfectionner, et représente une machinerie très bien organisée ne laissant rien au hasard. Pour assurer son rôle, elle contrôle un ensemble de processus d’auto-assemblage où des composants isolés interagissent spontanément entre eux pour conduire à la formation de structures organisées et fonctionnelles telles que les microtubules, le collagène ou les fibres d’actine. En m’inspirant de l’organisation cellulaire, mon projet doctoral consiste en la conception de systèmes chimiques artificiels basés sur l’auto-assemblage de peptides originaux. Ces édifices donnent naissance à des hydrogels supramoléculaires d’intérêts dans le domaine des biomatériaux. Je m’intéresse à la fois à des aspect fondamentaux concernant la compréhension de l’initiation des processus d’auto-assemblage en présence de biomacromolécules, mais aussi à des problématiques plus appliquées consistant à élaborer des stratégies pour contrôler le lieu mais aussi le moment où ces édifices moléculaires auto-assemblés prennent naissance. Enfin, je m’intéresse à l’émergence des différentes propriétés apparaissant lors de la formation de certains auto-assemblages comme la catalyse ou l’auto-catalyse
The cell is a complex chemical system that has benefited from billions of years of evolution to perfect itself, and represents a very well organized machinery leaving nothing to chance. To ensure its role, it controls a set of self-assembly processes where isolated components interact spontaneously with each other to lead to the formation of organized and functional structures such as microtubules, collagen or actin fibers. Inspired by cellular organization, my doctoral project involves the design of artificial chemical systems based on the self-assembly of original peptides. These buildings give rise to supramolecular hydrogels of interest in the field of biomaterials. I am interested at the same time in fundamental aspects concerning the comprehension of the initiation of the processes of self-assembly in the presence of biomacromolecules, but also with more applied problems of elaborating strategies to control the place but also the moment where these self-assembled molecular structures originate. Finally, I am interested in the emergence of the different properties appearing during the formation of certain self-assemblies such as catalysis and auto-catalysis

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6

Skoda, Maximilian W. A. "Interaction of proteins with oligo(ethylene glycol) self-assembled monolayers." Thesis, University of Oxford, 2007. http://ora.ox.ac.uk/objects/uuid:e36c47f8-1afc-4655-a84a-05bd06d0e45f.

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The aim of this thesis is the study of protein resistant oligo(ethylene glycol) (OEG) self-assembled monolayers (SAMs) using in situ techniques, such as neutron reflectivity (NR), polarisation modulation infrared spectroscopy (PMIR) and small-angle x-ray scattering (SAXS). In order to elucidate the mechanisms that lead to the nonfouling properties of these SAMs, the SAM-water, protein-protein and protein-SAM interactions have been studied separately. NR measurements, focused on the solid-liquid interface between OEG SAMs and water, show clear evidence of an extended layer with reduced density water. The reduction in density is up to 10% compared to the bulk value, and extends up to 5 nm into the bulk. The effective area (density reduction x length) of this reduced density water layer did not significantly change when the temperature was reduced to 5°C. In a complementary study, the interaction of water with protein-resistant HS(CHV₂)₁₁(OCH₂CH₂)₃OMe monolayers was examined using in and ex situ PMIR. In particular, shifts in the position of the characteristic C-O-C stretching vibration were observed after the monolayers had been exposed to water. The shift in frequency increased when the SAM was observed in direct contact with a thin layer of water. It was found that the magnitude of the shift also depended on the surface coverage of the SAM. These results suggest a rather strong interaction of oligo(ethylene glycol) SAMs with water and indicate the penetration of water into the upper region of the monolayer. These findings indicate the presence of a tightly bound water layer at the SAM-water interface. Further NR studies of the interface between OEG SAMs and a highly concentrated protein solution revealed an oscillating protein density profile. A protein depleted region of about 4-5 nm close to the SAM was followed by a more densely populated region of 5-6 nm. These oscillations were then rapidly damped out until the bulk value was reached. The influence of temperature and salt concentration on the protein density profile was small, indicating a rather minor contribution of electrostatic interactions to the protein repulsive force. SAXS measurements of OEG coated gold colloids mixed with proteins in solution did also not show any pronounced salt concentration dependence of the colloid-protein interaction. The strong association of water with the SAM and the layer of tightly bound water, together with the lack of electrostatic repulsion, suggest that the adsorption of proteins is energetically hindered by the presence of a strongly bound hydration layer.

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7

Williamson, Alexander James. "Methods, rules and limits of successful self-assembly." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:9eb549f9-3372-4a38-9370-a9b0e58ca26b.

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The self-assembly of structured particles into monodisperse clusters is a challenge on the nano-, micro- and even macro-scale. While biological systems are able to self-assemble with comparative ease, many aspects of this self-assembly are not fully understood. In this thesis, we look at the strategies and rules that can be applied to encourage the formation of monodisperse clusters. Though much of the inspiration is biological in nature, the simulations use a simple minimal patchy particle model and are thus applicable to a wide range of systems. The topics that this thesis addresses include: Encapsulation: We show how clusters can be used to encapsulate objects and demonstrate that such `templates' can be used to control the assembly mechanisms and enhance the formation of more complex objects. Hierarchical self-assembly: We investigate the use of hierarchical mechanisms in enhancing the formation of clusters. We find that, while we are able to extend the ranges where we see successful assembly by using a hierarchical assembly pathway, it does not straightforwardly provide a route to enhance the complexity of structures that can be formed. Pore formation: We use our simple model to investigate a particular biological example, namely the self-assembly and formation of heptameric alpha-haemolysin pores, and show that pore insertion is key to rationalising experimental results on this system. Phase re-entrance: We look at the computation of equilibrium phase diagrams for self-assembling systems, particularly focusing on the possible presence of an unusual liquid-vapour phase re-entrance that has been suggested by dynamical simulations, using a variety of techniques.

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8

Wan, Fan. "Biomimetic Surface Coatings from Modular Amphiphilic Proteins." Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31639.

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Engineering of biofunctional scaffolds to precisely regulate cell behavior and tissue growth is of significance in regenerative medicine. Protein-based biomaterials are attractive candidates for functionalization of scaffold surfaces since the ability to precisely control protein sequence and structure allows for fine-tuning of cell-substrate interactions that regulate cell behavior. In this thesis, a series of de novo proteins for bio-functionalization of interfaces was designed, synthesized, and studied. These proteins are based on a diblock motif consisting of a surface-active, amphiphilic block β-sheet domain linked to a disordered, water-soluble block with a terminal functional domain. Several types of functional domains were investigated, including sequences that act as ligands for cell surface receptors and sequences that act as templates for the growth of inorganic particles. Under moderate temperature and pH conditions, the amphiphilic β-sheet block was shown to have a strong affinity to a variety of scaffold materials and to form stable protein coatings on hydrophobic materials by self-assembly. Moreover, the surface adsorption of the proteins was shown to have minimal impact on the presentation of the functional end domains in the soluble block. For the case of diblocks with the RGDS integrin binding sequence, the capability for mediating cell attachment and spreading was demonstrated via control over ligand density on hydrophobic polymer surfaces. The case of diblock proteins with templating domains for inorganic materials was investigated for two systems. First, hydroxyapatite-binding domains were ligated to the end terminus of the water-soluble block to develop proteins for possible bone regeneration applications. It was demonstrated that the hydroxyapatite-binding domain had strong affinity to hydroxyapatite nanoparticles and was able to induce calcium phosphate mineralization on the surfaces coated with diblock proteins from dilute solutions with Ca2+.and PO43-. Next, a silver-binding domain was ligated to the end terminus to create a diblock protein for potential antimicrobial surface applications. The silver-binding domain was shown to accumulate and reduce silver ions, resulting in the formation of silver nanoparticles on the surfaces functionalized by the protein.

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9

Kurland, Nicholas. "Design of Engineered Biomaterial Architectures Through Natural Silk Proteins." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/571.

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Silk proteins have provided a source of unique and versatile building blocks in the fabrication of biomedical devices for addressing a range of applications. Critical to advancing this field is the ability to establish an understanding of these proteins in their native and engineered states as well as in developing scalable processing strategies, which can fully exploit or enhance the stability, structure, and functionality of the two constituent proteins, silk fibroin and sericin. The research outlined in this dissertation focuses on the evolution in architecture and capability of silks, to effectively position a functionally-diverse, renewable class of silk materials within the rapidly expanding field of smart biomaterials. Study of the process of building macroscopic silk fibers provides insight into the initial steps in the broader picture of silk assembly, yielding biomaterials with greatly improved attributes in the assembled state over those of protein precursors alone. Self-organization processes in silk proteins enable their aggregation into highly organized architectures through simple, physical association processes. In this work, a model is developed for the process of aqueous behavior and aggregation, and subsequent two-dimensional behavior of natural silk sericin, to enable formation of a range of distinct, complex architectures. This model is then translated to an engineered system of fibroin microparticles, demonstrating the role of similar phenomena in creating autonomously-organized structures, providing key insight into future “bottom up” assembly strategies. The aqueous behavior of the water-soluble silk sericin protein was then exploited to create biocomposites capable of enhanced response and biocompatibility, through a novel protein-template strategy. In this work, sericin was added to the biocompatible and biodegradable poly(amino acid), poly(aspartic acid), to improve its pH-dependent swelling response. This work demonstrated the production of a range of porous scaffolds capable providing meaningful response to environmental stimuli, with application in tissue engineering scaffolds and biosensing technologies. Finally, to expand the capabilities of silk proteins beyond process-driven parameters to directly fabricate engineered architectures, a method for silk photopatterning was explored, enabling the direct fabrication of biologically-relevant structures at the micro and nanoscales. Using a facile bioconjugation strategy, native silk proteins could be transformed into proteins with a photoactive capacity. The well-established platform of photolithography could then be incorporated into fabrication strategies to produce a range of architectures capable of addressing spatially-directed material requirements in cell culture and further applications in the use of non-toxic, renewable biological materials.

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10

Valkov, Eugene. "Design and analysis of self-assembling protein systems." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670100.

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11

Zeng, Like. "SELF-ASSEMBLY OF SILK-ELASTINLIKE PROTEIN POLYMERS INTO THREE-DIMENSIONAL SCAFFOLDS FOR BIOMEDICAL APPLICATIONS." Diss., The University of Arizona, 2014. http://hdl.handle.net/10150/325002.

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Production of brand new protein-based materials with precise control over the amino acid sequences at single residue level has been made possible by genetic engineering, through which artificial genes can be developed that encode protein-based materials with desired features. As an example, silk-elastinlike protein polymers (SELPs), composed of tandem repeats of amino acid sequence motifs from Bombyx mori (silkworm) silk and mammalian elastin, have been produced in this approach. SELPs have been studied extensively in the past two decades, however, the fundamental mechanism governing the self-assembly process to date still remains largely unresolved. Further, regardless of the unprecedented success when exploited in areas including drug delivery, gene therapy, and tissue augmentation, SELPs scaffolds as a three-dimensional cell culture model system are complicated by the inability of SELPs to provide the embedded tissue cells with appropriate biochemical stimuli essential for cell survival and function. In this dissertation, it is reported that the self-assembly of silk-elastinlike protein polymers (SELPs) into nanofibers in aqueous solutions can be modulated by tuning the curing temperature, the size of the silk blocks, and the charge of the elastin blocks. A core-sheath model was proposed for nanofiber formation, with the silk blocks in the cores and the hydrated elastin blocks in the sheaths. The folding of the silk blocks into stable cores - affected by the size of the silk blocks and the charge of the elastin blocks - plays a critical role in the assembly of silk-elastin nanofibers. The assembled nanofibers further form nanofiber clusters on the microscale, and the nanofiber clusters then coalesce into nanofiber micro-assemblies, interconnection of which eventually leads to the formation of three-dimensional scaffolds with distinct nanoscale and microscale features. SELP-Collagen hybrid scaffolds were also fabricated to enable independent control over the scaffolds' biochemical input and matrix stiffness. It is reported herein that in the hybrid scaffolds, collagen provides essential biochemical cues needed to promote cell attachment and function while SELP imparts matrix stiffness tunability. To obtain tissue-specificity in matrix stiffness that spans over several orders of magnitude covering from soft brain to stiff cartilage, the hybrid SELP-Collagen scaffolds were crosslinked by transglutaminase at physiological conditions compatible for simultaneous cell encapsulation. The effect of the increase in matrix stiffness induced by such enzymatic crosslinking on cellular viability and proliferation was also evaluated using in vitro cell assays.

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12

Korkmaz, Nuriye. "Self-assembly and Structure Investigation of Recombinant S-layer Proteins Expressed in Yeast for Nanobiotechnological Applications." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2011. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-64317.

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In numerous Gram-negative and Gram-positive bacteria as well as in Archaea SL proteins form the outermost layer of the cell envelope. SL (glyco)monomers self-assemble with oblique (p2), tetragonal (p4), or hexagonal (p3, p6) symmetries [12]. SL subunits interact with each other and with the underlying cell surface by relatively weak non-covalent forces such as hydrogen-bonds, ionic bonds, salt-bridges or hydrophobic interactions. This makes them easy to isolate by applying chaotropic agents like urea and guanidine hydrochloride (GuHCl), chelating chemicals, or by changing the pH of the environment [10]. Upon dialysis in an ambient buffer monomers recrystallize into regular arrays that possess the forms of flat sheets, open ended cylinders, or spheres on solid substrates, at air-water intefaces and on lipid films, making them appealing for nanobiotechnological applications [3, 18]. The aim of this study was to investigate the structure, thermal stability, in vivo self-assembly process, recrystallization and metallization of three different recombinant SL proteins (SslA-eGFP, mSbsC-eGFP and S13240-eGFP) expressed in yeast S. cerevisiae BY4741 which could be further used in nanobiotechnological applications. In order to fulfill this aim, I investigated the in vivo expression of SL proteins (SslA, SbsC, S13240) tagged with eGFP (SL-eGFP) in the yeast S. cerevisiae BY4141. First, I characterized the heterologous expression of SL fusion constructs with growth and fluorescence measurements combined with Western blot analyses. Fluorescence microscopy investigations of overnight grown cultures showed that SslA-eGFP fusion protein was expressed as fluorescent patches, mSbsC-eGFP as tubular networks, and S13240-eGFP as hollow-like fibrillar network structures, while eGFP did not show any distinct structure Thermal stability of in vivo expressed SL-eGFP fusion proteins were investigated by fluorescence microscopy and immunodetection. In vivo self-assembly kinetics during mitosis and meiosis was the second main issue. In parallel, association of in vivo mSbsC-eGFP structures with the cellular components was of interest. A network of tubular structures in the cytosol of the transformed yeast cells that did not colocalize with microtubules or the actin cytoskeleton was observed. Time-resolved analysis of the formation of these structures during vegetative growth and sporulation was investigated by live fluorescence microscopy. While in meiosis ascospores seemed to receive assembled structures from the diploid cells, during mitosis surface layer structures were formed de novo in the buds. Surface layer assembly always started with the appearance of a dot-like structure in the cytoplasm, suggesting a single nucleation point. In order to get these in vivo SL assemblies stably outside the cells (in situ), cell distruption experiments were conducted. The tubular structures formed by the protein in vivo were retained upon bursting the cells by osmotic shock; however their average length was decreased. During dialysis, monomers obtained by treatment with chaotropic agents recrystallized again to form tube-like structures. This process was strictly dependent on calcium ions, with an optimal concentration of 10 mM. Further increase of the Ca2+ concentration resulted in multiple non-productive nucleation points. It was further shown that the lengths of the S-layer assemblies increased with time and could be controlled by pH. After 48 hours the average length at pH 9.0 was 4.13 µm compared to 2.69 µm at pH 5.5. Successful chemical deposition of platinum indicates the potential of recrystallized mSbsC-eGFP structures for nanobiotechnological applications. For example, such metalized protein nanotubes could be used in conductive nanocircuit technologies as nanowires.

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13

Jha, Kshitij Chandra. "Polarization and Self-Assembly at Metal-Organic Interfaces: Models and Molecular-Level Processes." University of Akron / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=akron1333644685.

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14

Herrera, Rodríguez Ana María [Verfasser], and Ulrich S. [Akademischer Betreuer] Schwarz. "The role of flow in the self-assembly of dragline spider silk proteins / Ana María Herrera Rodríguez ; Betreuer: Ulrich Schwarz." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://nbn-resolving.de/urn:nbn:de:bsz:16-heidok-283289.

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15

Herrera, Rodríguez Ana María [Verfasser], and Ulrich [Akademischer Betreuer] Schwarz. "The role of flow in the self-assembly of dragline spider silk proteins / Ana María Herrera Rodríguez ; Betreuer: Ulrich Schwarz." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1210926873/34.

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16

Altamura, Lucie. "Bio-inspired protein nanowire : electrical conductivity and use as redox mediator for enzyme wiring." Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GRENY006.

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Nous avons développé un nano-fil conducteur, constitué uniquement de protéines et bio-inspiré des nano-fils bactériens conducteurs. Pour cela, une protéine chimère a été créée par l'association d'une protéine prion capable de s'auto-assembler en fibre et d'une métalloprotéine, une rubrédoxine, capable d'effectuer des transferts d'électrons. Comme montré par des techniques de microscopies et de spectroscopies (absorbance UV-visible et RPE), la protéine chimère est capable de former des fibres à la surface desquelles on retrouve les rubrédoxines. Les propriétés électroniques des nano-fils ont été caractérisées par des mesures courant-tension sur des échantillons secs et par électrochimie. Les mesures courant-tension ont montré que la conduction se faisait par plusieurs mécanismes. Les acides aminés aromatiques présents au centre du domaine prion semblent impliqués dans un des mécanismes de conduction. Les mesures électrochimiques ont quant à elles montré une conduction par sauts entre rubrédoxines. De plus, nous avons utilisé les nano-fils comme interface entre une enzyme, la laccase, et une électrode. Un courant électrocatalytique dû à la réduction de l'oxygène a été obtenu prouvant ainsi la capacité de nos nano-fils à agir comme médiateurs d'électrons. Les nano-fils conducteurs faits de protéines sont une structure intéressante pour comprendre le transport de charges dans les systèmes biologiques et sont également très prometteurs pour le développement de la bioélectronique et plus particulièrement de biocapteurs et de biopiles enzymatiques
The discovery of bacterial nanowires able to transport electrons on long range within biofilms and transfer them to electrodes is very promising for the development of bioelectronics and bio-electrochemical interfaces. However, their assembling process, their molecular composition and the electron transport mechanism are not fully understood yet. We took inspiration from bacterial nanowires to design conductive protein nanowires. We fused the sequence of a rubredoxin, an electron transfer iron-sulfur protein, to the sequence of HET-s(218-289), a prion domain that forms amyloid fibril by self-assembling under well-defined conditions. The resulting chimeric protein forms amyloid fibrils and displays redox proteins organized on the surface as shown by microscopy techniques and UV-Vis and EPR spectroscopy. Electron transfer mechanisms were studied in “dry state” current-voltage (I-V ) measurements and as hydrated film by electrochemistry. Dry state measurements allowed to evidence several conduction pathways with a possible role of aromatic residues in the conduction. Electrochemistry revealed electron transport by hopping between adjacent redox centers. This property allowed the use of our protein as mediator between a multicopper enzyme (laccase) and an electrode for electrocatalytic reduction of oxygen. These protein nanowires are interesting structures for the study of charge transport mechanisms in biological systems but are also very promising for the design of biosensors and enzymatic biofuel cells

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17

Bonnet, Nelly. "Trifluoro alkyl oligo(ethylene glycol)-terminated alkanethiol self-assembled monolayers : synthesis, characterisation, and protein adsorption properties." Thesis, University of St Andrews, 2010. http://hdl.handle.net/10023/2127.

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Self-assembled monolayers have been proven to be well-ordered and to give stable ultrathin films. They show a remarkably high diversity with respect to their functionalisation giving rise to many possible applications. This thesis is focused on the potential use of these molecular thin films in life sciences. The reproduction of a membrane-like environment with these tightly packed and organized unimolecular layers has led to important breakthroughs in their nanotechnological application as biomaterials. Their straightforward modification allows the chemical and physical properties of biological interfaces to be altered. In particular, Oligo(ethylene glycol) based alkanethiol self-assembled monolayers were intensively studied as biointerfaces for their ability to resist the non specific adsorption of proteins. The electrostatic repulsion which originates from these monolayers was seen as one of the possible factors causing this protein repulsion. On the other hand proteins adsorb on alkanethiol self-assembled monolayers. This can be partially attributed to an attractive hydrophobic interaction between the biomolecules and the surface. As a result of the understanding of these two driving forces which are relevant for non-specific protein adsorption/repulsion, novel self-assembling molecules were tailored in an attempt to adjust the adsorption of proteins at the SAM-liquid interface. This was conceivable with these newly designed SAMs since they allow a combination of these forces. We have chosen the ionic strength of the liquid environment as the external parameter which could act on the amount of adsorbed proteins because the electrostatic force created by oligo(ethylene glycol) groups depends on it. In addition to the synthesis of six new molecules, the preparation and characterisation of the novel self-assembled monolayers are reported in this thesis. The density of the monolayers was estimated by X-ray photoelectron spectroscopy and ellipsometry, and the wettability properties were studied by measuring the contact angle. The total force acting on proteins from the SAMs was studied with an atomic force microscope, equipped with a tip mimicking proteins, by measuring force-distance curves. An in-situ technique was investigated in order to study the influence of the variation of this total force on the quantity of adsorbed proteins by varying the ionic strength.

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18

Reeh, Philipp. "Dynamic Multivalency For The Recognition Of Protein Surfaces." Doctoral thesis, Universitat Rovira i Virgili, 2014. http://hdl.handle.net/10803/283236.

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Анотація:

En esta tesis doctoral el concepto de multivalencia en el reconocimiento de proteínas (lectinas) con azúcares se combinó con la idea de la química dinámica combinatoria. Esto se aplicó, no sólo para sacar ventaja del efecto de la mejor afinidad de tales sistemas multivalentes, sino también para dotar al sistema con una mayor variedad de constituciones y geometrías. La determinación de las afinidades relativas de los miembros de la biblioteca dinámica dio una visión de los requisitos necesarios para la unión entre azúcares – lectina. El primer enfoque para acceder a los sistemas multivalentes para el reconocimiento de lectinas (presentado en el capítulo 2 de la tesis), está basado en estrategias bien conocidas. Enlaces covalentes reversibles son usados para acceder a bibliotecas dinámicas combinatorias (DCLs). En esta parte del trabajo se confirmó la viabilidad del procedimiento analítico elegido. Especies diméricas, similares a las que se había conocido desde experimentos de otros grupos, mostraban buena analisabilidad de los DCLs formados. El método analítico elegido (HPLC MS) permitió la detección de las afinidades y selectividades relativas de tales constituyentes de la respectiva biblioteca. Para la elaboración de las bibliotecas dinámicas, varios intentos fueron realizados basados en el mismo concepto: Una subunidad central con múltiples puntos de conexión para favorecer interacciones reversibles. Formación de una librería dinámica basada en un conector central. Específicamente, se evaluaron los puentes di sulfuro y la formación de imina. Algunos de estos estudios resultaron ser complicados por problemas secundarios, tales como solubilidad en agua y las interacciones secundarias no deseadas de unidades centrales, debido principalmente a reacciones intramoleculares. Sin embargo, finalmente se obtuvo una biblioteca combinatoria dinámica multivalente y se analizó con éxito mediante técnicas de HPLC-MS. El DCL, está basado en el intercambio de sulfuro para formar puentes di-sulfuro entre las diferentes unidades de azúcar. Esto fue posible gracias a la solubilidad en agua de las subunidades carboxilato y al uso de enlaces cortos entre los puntos de conexión del tiol y de la estructura central, evitándose la formación de enlaces intramoleculares. Formación de una librería dinámica. Las partes se conectan a través de enlaces disulfuros. Sin embargo, incluso cuando se controlaron los problemas mencionados anteriormente, la formación fiable y estable de los miembros de la librería era difícil debido a la desintegración sustancial durante la etapa de análisis. Por lo tanto, la posterior comparación de las afinidades de los miembros de la DCL no era posible. No obstante, los enfoques presentados ofrecen oportunidades para nuevos experimentos, que con una cuidadosa elección de las condiciones pueden conducir al éxito. Desafortunadamente, el marco temporal de esta tesis no lo permitió estudiar en detalle; había que seguir otras pistas más prometedoras. La coordinación de ligando metal, especialmente con ligandos de tipo bipiridina coordinados a centros de FeII, evitó la mayoría de problemas encontrados anteriormente (parte desarrollada en el Capítulo 3). Observándose que en las condiciones necesarias para trabajar con la proteína elegida (ConA lectina) la formación de complejos era muy fiable. Como primera prueba de concepto para un comportamiento de dinámica combinatoria, se evaluaron DCLs simples que no contenían azúcares sobre la base coordinativa bipyridina. Una librería dinámica basada en el intercambio de ligandos de un centro metálico. Después, DCLs que contenían azúcares fueron sintetizadas y fueron comprobadas con la proteína. Ligandos con sustituyentes azúcar fueron usados como bloques prefabricados, obteniéndose, mediante síntesis sencillas y con buenos rendimientos. Ligandos con sustituyentes azúcares y basados en bipyridina que pueden formar complejos hexavalentes. Métodos de HPLC bien elegidos permitieron el análisis de los DCLs, así como la determinación de las afinidades relativas con la lectina ConA. La cuantificación de las entidades con más afinidad apoyó el concepto de multivalencia para sistemas que intercambian dinámicamente múltiples unidades de reconocimiento. A partir de este estudio básico, se desarrollaron otras DCLs que incorporaron componentes de diferentes geometrías. Las afinidades relativas de estos complejos compararon y revelaron que algunos miembros de la biblioteca contienen disposiciones tridimensionales más afines para la interacción con la lectina. La proteína tiene más afinidad a un único miembro de la biblioteca dinámica Por otra parte, los miembros de la librería de forma esférica parecen mostrar mayor afinidad a la proteína, en acuerdo con la teoría de “statistical rebinding”. Una biblioteca dinámica de geometrías diferentes. En resumen, DCLs basadas en la coordinación con metal (en contraste con enlaces covalentes dinámicos) han demostrado que constituyen una manera fácil de acceder a los procesos de intercambio multivalentes, proporcionando nuevas perspectivas para desentrañar las reglas de interacciones multivalentes de azúcares - lectina.

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19

Fernandez, Maxence. "Auto-assemblage de nanoparticules métalliques et semi-conductrices dirigé par reconnaissance entre protéines artificielles." Thesis, Rennes 1, 2019. http://www.theses.fr/2019REN1S129.

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L’auto-assemblage de nanoparticules dirigé par des biomolécules constitue une approche prometteuse pour la mise au point de nanomatériaux structurés présentant des propriétés optiques collectives originales. L’objet de cette thèse concerne l’auto-assemblage de nanoparticules métalliques et semi-conductrices dirigé par des protéines artificielles appelées α-Repeat. Dans cette optique, des nanocristaux semi-conducteurs (CdSe/ZnS ou CdSe/CdS) et des nanoparticules d’or sphériques ou anisotropes ont été préparés. Ces nanoparticules ont été fonctionnalisées avec des ligands peptidiques PEGylés, qui leur confère une stabilité colloïdale satisfaisante tout en conservant leurs propriétés optiques. Une stratégie de fonctionnalisation basée sur des étiquettes d’affinité poly-cystéine et poly-histidine a permis de greffer les protéines sur la surface des nanoparticules inorganiques. Les nanoparticules ainsi fonctionnalisées avec les protéines artificielles ont ensuite été utilisées pour l’auto-assemblage de nanoparticules semi-conductrices et l’auto-assemblage hybride entre des nanoparticules semi-conductrices et des nanoparticules métalliques. L’étude structurale des ensembles obtenus a montré, dans certains cas, une interdistance bien définie et inférieure à 10 nm. Finalement, l’étude des propriétés optiques a révélé des transferts d’énergie non radiatifs entre nanoparticules semi-conductrices et nanoparticules métalliques, qui témoignent d’interactions exciton—plasmon très fortes induites par l’auto-assemblage
Nanoparticles self-assembly driven by biomolecules is a promising approach for developing nanostructured materials with new optical properties. The purpose of this work is the self-assembly of metal and semiconductor nanoparticles directed by artificial proteins called α-Repeat. For this purpose, semiconductor nanocrystals (CdSe/ZnS or CdSe/CdS) and spherical or anisotropic gold nanoparticles have been prepared. These nanoparticles have been functionalized with PEGylated peptide ligands providing them adequate colloidal stability while maintaining their optical properties. A functionalization strategy based on polycysteine and poly-histidine tags has allowed the proteins to be grafted onto the surface of inorganic nanoparticles. Nanoparticles functionalized with artificial proteins were then used for the self-assembly of semiconductor nanoparticles and hybrid self-assembly between semiconductor nanoparticles and metal nanoparticles. The structure study of self-assembled nanostructures has shown, in some cases, a very well defined sub-10 nm interparticle distance. Finally, the study of optical properties revealed very strong exciton-plasmon interactions induced by self-assembly. This self-assembling process strongly affected the emission properties of the semiconductor nanoparticles in hybrid ensembles

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20

Ridgley, Devin Michael. "Self-Assembly of Large Amyloid Fibers." Diss., Virginia Tech, 2014. http://hdl.handle.net/10919/48186.

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Анотація:

Functional amyloids found throughout nature have demonstrated that amyloid fibers are potential industrial biomaterials. This work introduces a new 'template plus adder' cooperative mechanism for the spontaneous self-assembly of micrometer sized amyloid fibers. A short hydrophobic template peptide induces a conformation change within a highly α-helical adder protein to form β-sheets that continue to assemble into micrometer sized amyloid fibers. This study utilizes a variety of proteins that have template or adder characteristics which suggests that this mechanism may be employed throughout nature. Depending on the amino acid composition of the proteins used the mixtures form amyloid fibers of a cylindrical (~10 μm diameter, ~2 GPa Young's modulus) or tape (5-10 μm height, 10-20 μm width and 100-200 MPa Young's modulus) morphology. Processing conditions are altered to manipulate the morphology and structural characteristics of the fibers. Spectroscopy is utilized to identify certain amino acid groups that contribute to the self-assembly process. Aliphatic amino acids (A, I, V and L) are responsible for initiating conformation change of the adder proteins to assemble into amyloid tapes. Additional polyglutamine segments (Q-blocks) within the protein mixtures will form Q hydrogen bonds to reinforce the amyloid structure and form a cylindrical fiber of higher modulus. Atomic force microscopy is utilized to delineate the self-assembly of amyloid tapes and cylindrical fibers from protofibrils (15-30 nm width) to fibers (10-20 μm width) spanning three orders of magnitude. The aliphatic amino acid content of the adder proteins' α-helices is a good predictor of high density β-sheet formation within the protein mixture. Thus, it is possible to predict the propensity of a protein to undergo conformation change into amyloid structures. Finally, Escherichia coli is genetically engineered to express a template protein which self-assembles into large amyloid fibers when combined with extracellular myoglobin, an adder protein. The goal of this thesis is to produce, manipulate and characterize the self-assembly of large amyloid fibers for their potential industrial biomaterial applications. The techniques used throughout this study outline various methods to design and engineer amyloid fibers of a tailored modulus and morphology. Furthermore, the mechanisms described here may offer some insight into naturally occurring amyloid forming systems.
Ph. D.

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21

Thomas, Carla S. (Carla Stephanie). "Self-assembly of globular protein-polymer diblock copolymers." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/87528.

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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2014.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Self-assembly of protein-polymer block copolymers provides a simple bottom-up approach towards protein nanopatteming for the fabrication of more effective and efficient bioelectronic and biocatalytic devices. Changes in shape and surface chemistry between proteinpolymer conjugates and classical coil-coil block copolymers result in significant differences between the self-assembly of these two classes of molecules. A model material is used to explore the self-assembly behavior of globular protein-polymer block copolymers as well as investigate protein functionality, stability, and secondary structure in the resulting nanostructured materials. Across a wide range of polymer coil fractions from 0.21 to 0.82, a variety of morphologies including hexagonally packed cylinders, lamellae, perforated lamellae, weakly ordered nanostructures and a disordered phase are observed. Surprisingly, a lyotropic re-entrant order-disorder transition is observed in all materials between 30 and 70 wt% indicating the solvent-mediated effective interaction potential is non-monotonic with concentration. Solid state materials are prepared through evaporation of aqueous solvent, which leads to the formation of kinetically determined nanostructured morphologies. The type of nanostructure is strongly determined by the solvent quality for the polymer block. Good solvents produce well-ordered nanostructures similar to those observed in coil-coil block copolymers, while poor solvents produced an aggregated micellar structure. Importantly, protein secondary structure remains largely unaltered, even in a completely dehydrated environment. As much as 80% of the protein solution functionality is retained in these solid state materials. This quantity depends primarily on the processing conditions, but also the polymer fraction, with ambient temperatures and materials composed of 45-60% polymer retaining the highest levels of protein functionality. Interestingly, there exists some fraction of protein functionality which is reversibly lost in the solid state and regained upon rehydration. The addition of small molecule osmolytes is demonstrated to eliminate this reversible loss and improve protein functionality retention up to 100% in the solid state. Osmolytes with a high glass transition temperature are capable of increasing the thermal stability of dehydrated films by 15 °C, while those with a low glass transition temperature decrease it.
by Carla S. Thomas.
Ph. D.

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22

Bellaiche, Mathias Moussine Jacques. "Molecular mechanisms of protein self-assembly and aggregation." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/277621.

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In this thesis, we investigate the mechanisms driving the self-assembly of peptides and proteins using computational and theoretical tools, always validating our results with experimental measures when possible. In the first part, Chapters 2-5, we focus on the Aβ system, a peptide whose aggregation is intimately linked with the development of Alzheimer's Disease. We begin by simulating the major alloforms of the peptide, Aβ_40 and Aβ_42, demonstrating that the two populate similar disordered ensembles and matching experimental data. Next we investigate how disordered Aβ_42 monomers interact with each other, finding that oligomerisation into amorphous aggregates is driven largely by hydrophobic, non-specific forces. We then move on to probing the aggregation of Aβ_42 into amyloid structures using a native-centric coarse-grained model, and explain the results with a novel Markov state analysis from which we are able to extract structural, kinetic and thermodynamic information on elongation reactions. Finally, we probe the interactions of Aβ_42 monomers with Aβ_42 fibrillar surfaces using a specially designed enhanced sampling scheme, which allows us to obtain enthalpy-driven binding thermodynamics consistent with experiments and to propose major polar binding modes. In the second part of the thesis, Chapters 6 and 7, we model the aggregation of two other self-assembling systems, viruses and a truncated form of the molecular chaperone Hsp70. We first develop a data analysis platform to extract information on the microscopic mechanisms of viral capsid self-assembly from experimental data, synthesising the results from several different systems to draw general evolutionary conclusions about the assembly mechanism. Finally, we model the oligomerisation of Hsp70 thermodynamically and kinetically, showing that its self-assembly is a highly cooperative reaction that is under strong structural constraints.

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23

Huang, Aaron. "Predicting self-assembly in globular protein-polymer bioconjugates." Thesis, Massachusetts Institute of Technology, 2018. https://hdl.handle.net/1721.1/121893.

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Анотація:

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2018
Cataloged from PDF version of thesis.
Includes bibliographical references.
Globular proteins offer powerful solutions for addressing challenges in the fields of medicine, industry, defense, and energy. Enzymes perform reactions with high efficiency and specificity, allowing for minimal generation of undesired side products even while exhibiting rapid turnover-traits difficult to replicate in synthetic catalysts. These targets make proteins attractive tools for immobilization to form functional catalysts and sensors. Nevertheless, there are many challenges in creating these advanced materials. The activity of the protein must be retained, and control over the structure of the material is desirable. Protein-polymer block copolymers offer an attractive solution to these issues. These materials have been shown to selfassemble into ordered nanodomains while retaining protein activity. However, the phase behavior of these materials is not fully understood due to the complex nature of anisotropic interactions between the proteins.
Within this thesis, a method for creating highly-active thin-film catalysts from myoglobin-PNIPAM bioconjugates is established by flow-coating these materials onto solid supports and then cross-linking them with glutaraldehyde. These catalysts exhibit considerable stability and perform reactions 5-10 times more efficiently than catalysts formed using other common immobilization techniques. However, the self-assembly and structural control of this catalyst was observed to be poor, and it was hypothesized that the poor self-assembly relative to mCherry and EGFP systems could be a consequence of the protein shape. In order to probe the effect of protein shape on self-assembly, a panel of mCherry bioconjugates with differing conjugation sites was studied using small-angle x-ray scattering.
The self-assembly behavior of these conjugation site variants was observed to be robust with only minor differences in phase boundaries and observed phases resulting from the changes in conjugation site. However, observed changes in the domain spacing signaled that modifications to conjugation site offer control over protein orientation within the domains. Based on studies showing that polymer chemistry in bioconjugates has a significant effect on self-assembly, an attempt to quantify these protein-polymer interactions was made using contrast-variation small-angle neutron scattering on mCherry and polymer blends. This technique allows for decomposition of the scattering intensity into its component parts corresponding to correlations between the 3 different pairs of the 2 species in the blends. From this analysis, it was determined that the best ordering bioconjugates have primarily repulsive interactions that can be described using a depletion layer model.
Lastly, the effect of protein properties was screened using a large library of bioconjugates made from 11 different proteins. The primary observed trend was that order increases as molecular weight increases, but a narrow region around 28-30 kDa was observed where bioconjugate ordering was significantly enhanced and additional nanostructures were accessible.
by Aaron Huang.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemical Engineering

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24

Giasuddin, Abul Bashar Mohammad. "Silane Modulation of Protein Conformation and Self-Assembly." DigitalCommons@USU, 2018. https://digitalcommons.usu.edu/etd/7029.

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This research focused on development of nanoparticle- based therapeutics against amyloid fibrils. Amyloid fibrils are associated with various diseases such as Parkinson’s, Huntington’s, mad cow disease, Alzheimer’s, and cataracts. Amyloid fibrils develop when proteins change their shape from a native form to a pathogenic “misfolded” form. The misfolded proteins have the ability to recruit more native proteins into the pathogenic forms, which self-assemble into amyloid fibrils that are hallmarks of the various protein-misfolding diseases listed above. Amyloid fibrils are highly resistant to degradation, which may contribute to the symptoms of amyloid diseases. Synthetic drugs, natural compounds, and antibodies are widely explored for potential to stop pathogenic protein assembly or to promote fibril degradation and clearance, but to date have had little success in relieving symptoms in clinical trials. In this research, I have synthesized fluorine-containing silica nanoparticles (NPs), and tested their fibril-inhibiting activity against amyloid fibrils formed by a non-pathogenic protein, β-lactoglobulin (BLG). These fluoro-silica NPs prevented BLG amyloid formation, whereas non-fluorinated nanoparticle analogs did not inhibit fibrillation under the same reaction conditions. The fluoro-silica NPs interacted with the BLG protein in a manner that prevented the protein from adopting a form that could self-assemble into fibrils. Additional applications of the NPs were explored as small-molecule drug-delivery systems; such that multiple functionalities could be introduced into a single nano- therapeutic.

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25

Dai, Jianhua. "Simulation of Multiobject Nanoscale Systems." University of Akron / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=akron1239154185.

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26

Du, Weiwei. "Electrostatic Self-Assembly of Biocompatible Thin Films." Thesis, Virginia Tech, 1999. http://hdl.handle.net/10919/10106.

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Анотація:

The design of biocompatible synthetic surfaces is an important issue for medical applications. Surface modification techniques provide good approaches to control the interactions between living systems and implanted materials by modifying the surface characteristics. This thesis work demonstrates the feasibility and effectiveness of the novel and low-cost electrostatic self-assembly (ESA) technique for the manufacturing of biocompatible thin film coatings. The ESA process is based on the alternating adsorption of molecular layers of oppositely charged polymers/nanoparticles, and can be applied in the fabrication of well-organized multilayer thin films possessing various biocompatible properties. ESA multilayer assemblies incorporating various biomaterials including metal oxides and polymers were fabricated, the uniformity, thickness, layer-by-layer linearity, and surface morphology of the films were characterized by UV/vis spectroscopy, ellipsometry, and AFM imaging. Preliminary biocompatibility testing was conducted, concentrating on contact angle surface characterization and the in vitro measurements of protein adsorption. The use of Fourier Transform Infrared Reflection-Absorption Spectroscopy (FT-IRAS) for the investigation of the protein adsorption behavior upon the ESA multilayer films is presented.
Master of Science

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27

Stevens, Marryat. "Exploiting the assembly of designed self-assembling protein structures." Thesis, University of Sussex, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.426313.

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28

Yao, Helen Ph D. Massachusetts Institute of Technology. "Driving forces of self-assembly in protein-polymer bioconjugates." Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/130592.

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Анотація:

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, September, 2020
Cataloged from the official PDF of thesis.
Includes bibliographical references.
Protein-polymer bioconjugates have shown great promise as high-performance biomaterials, with a diverse range of applications. Bioconjugation to a polymer allows the protein to maintain or even enhance its activity while imparting self-assembly capabilities to the overall material, which provide control over the orientation and nanostructure of the bioconjugates, enabling the design of materials with superior transport properties and high stability. The phase behavior of globular protein-polymer bioconjugates is comparable to that of traditional synthetic polymer block copolymers and leads to the formation of many of the same nanostructures. Despite these similarities, there are also key differences between these systems. The phase behavior of protein-polymer bioconjugates is affected by coarse-grained properties of both the protein and polymer. However, a unifying theory describing the self-assembly of these materials does not yet exist.
The goal of this thesis was to understand interaction-based and structural driving forces of bioconjugate self-assembly. Partial structure factor analysis and subsequent inverse Fourier transformation showed that protein-polymer interactions could be quantified and understood in the context of physical phenomena through a real-space correlation function. The nature of these interactions can affect the propensity of a bioconjugate system to order. Polymer-water interactions were probed using small-angle neutron scattering, which showed that polymer hydration is affected by both polymer chemistry and concentration. This dependence likely underpins the significant effect that polymer chemistry has on self-assembly. On the structural side, the self-assembly of protein-rod block copolymers was investigated by imparting secondary structure to the polymer through chirality. The rigidity of the rod block was shown to drive self-assembly in inherently weakly segregated systems.
Finally, a hard sphere-soft sphere dumbbell model for protein-polymer bioconjugates was built to understand the role of coarse-grained structural properties in phase behavior. Molecular dynamics simulations reproduced the most notable features of bioconjugate self-assembly, including an asymmetrical phase diagram and a lyotropic reentrant order-disorder transition at high concentrations. The success of this coarse-grained model revealed that colloidal interactions are sufficient to effect self-assembly in the globular protein-polymer block copolymer system.
by Helen Yao.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemical Engineering

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29

Tasneem, Nuren. "Regulating self-assembly and porosity of encapsulin protein compartments." Thesis, The University of Sydney, 2022. https://hdl.handle.net/2123/29264.

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Анотація:

The work presented in this thesis explores the redesign of a class of naturally occurring protein compartment towards building nanoreactors with tuneable assembly and permeability properties. Nanoreactors with tuneable assembly properties can expand the range of catalytic reactions that can be confined within, while control over permeability properties allows creation of selective and responsive chemical systems. This thesis aims to incorporate these two properties in the encapsulin protein compartments. Chapter 1 provides an introduction to protein compartments and a general background on the encapsulin family of proteins as it existed at the start of this work. In Chapter 2, I discuss how encapsulin proteins are purified and characterised. In Chapter three, I detail the different approaches I have used in this thesis for controlling the self-assembly of encapsulins from different structural classes. As part of this, four different routes undertaken for regulating encapsulin assembly for in vitro encapsulation of synthetic molecules has been detailed. Chapter four describes our collaborative efforts in regulating encapsulin pore permeability. Here we detail how the modification of encapsulin pores affects bulk properties of encapsulin particles with the goal to broaden our current understanding of pore permeability and inform future design of tuneable nanoreactors. Chapter 5 discusses the key conclusions from the previous chapters in the context of the existing body of knowledge along with broader implications for this field at large. Together with insights into future work arising from this thesis, this Chapter aims to provide the reader with a picture of how encapsulins can be engineered to create programmable chemical systems.

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30

Branson, Thomas Reuben. "The self-assembly of nanoarchitectures via protein-ligand interactions." Thesis, University of Leeds, 2013. http://etheses.whiterose.ac.uk/5844/.

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Анотація:

Nature has evolved proteins that can spontaneously self-assemble to create complex structures such as virus particles and molecular motors. The fields of bionanoscience and synthetic biology are based on the concept that by combining biological building blocks with synthetic molecules it will be possible to construct novel nanoarchitectures and machines that can do useful work. This thesis describes strategies that have been developed using protein-ligand interactions to construct nanoscale assemblies. The B-subunit of cholera toxin (CTB) has been site-specifically modified at the N-teminus, by oxime ligation, with carbohydrate ligands and the assembly of these modified proteins was observed. A W88E non-binding mutant of CTB was made and modified with GM1os ligands. The interaction of this pentavalent protein-based ligand with wild-type CTB has been investigated and showed the formation of a protein heterodimer. The CTA2-subunit of the cholera toxin AB5 complex was also modified at the N-terminus enzymatically with depsipeptides via sortase ligation and by oxime ligation. Biotin ligands have been covalently attached and the formation of a 2:1 complex with streptavidin was observed. The structures and assemblies demonstrated herein have been analysed and characterised by a range of analytical and biophysical techniques including dynamic light scattering, analytical ultracentrifugation and isothermal titration calorimetry.

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31

Littlejohn, Jacob James. "Peroxiredoxins : a model for a self-assembling nanoscale system." Thesis, University of Canterbury. School of Biological Sciences, 2012. http://hdl.handle.net/10092/10731.

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The formation of large, complex structures from small building blocks through self-assembly is widely seen in proteins and provides a tool for the creation of functional nanoscale devices. However, the factors controlling protein self-assembly are complex and often poorly understood. Peroxiredoxins are a large family of proteins, many of which are able to form a variety of large structures from a small, basic unit. This assembly has been shown to be strongly influenced by the redox state of the enzyme, which functions as a switch, controlling self-assembly. This thesis uses a protein from this family, human peroxiredoxin 3 (hPrx3) as a model system to investigate whether the self-assembly properties of hPrx3 can be influenced by rational protein engineering. Three forms of hPrx3 were purified and examined. These were the wild type and two variants: a mutant (S78A) and a His-tagged form. Size exclusion chromatography showed that each form showed a different ratio of dimers and larger species. Both variants showed preference for larger species, especially in the His-tagged form. This was shown to be partially dependent on metal binding in the His-tagged form. Larger species formed from multiple rings were also identified. SAXS measurement indicated that in the wild type enzyme, higher order species were dodecameric rings. For the His-tagged variant, SAXS measurement showed that the species observed had a different structure than that of the wild type. Electron microscopy showed that higher order structures seen in both wild type hPrx3 and His-tagged hPrx3 were ring shaped, with dimensions consistent with dodecamers. A competitive assay showed that the wild type, with kcat/km values near 2 x 10⁷, consistent with published results. Both variant forms showed evidence of slightly higher activity than the wild type, indicating a link between activity and assembly. A peroxiredoxin from the thermophilic bacteria Thermus aquaticus, TaqPrx was also examined, in an attempt to investigate a peroxiredoxin capable of self-assembly at high temperatures, which would be very useful for a nanoscale device. TaqPrx was cloned, purified and examined, however, no evidence of self-assembly was observed. Protein modelling and dynamic light scattering measurement indicated that the protein purified was monomeric and had a structure. Sparse matrix crystal screening identified conditions that allowed crystal formation, although strongly diffracting crystals were not produced. A novel assay for peroxiredoxin activity was developed, and suggested that TaqPrx shows peroxiredoxin activity. This thesis shows that peroxiredoxins are a useful model system for the investigation of how protein self-assembly is controlled, and how it can be influenced by protein engineering.

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32

Carter, Nathan Andrew. "Design Strategies for Dynamic Self-assembled Protein Materials." Diss., Virginia Tech, 2018. http://hdl.handle.net/10919/93207.

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Structures in nature exhibit unique and complex architectures whose order propagates from nano- (10-9 m) to macro-scales (mm to m). These structures give rise to a rich diversity of adaptive function that allows for life in all environments on Earth. This complex functionality has driven research into bio-inspired materials where scientists investigate the complex relationship between sequence, structure and function of these materials. A good illustrative example of the effect that hierarchical structure can have is a brick wall. Bricks are laid so that the layer on top is shifted in either direction by half of a brick. This alternating pattern is what gives the wall its strength. If a crack occurs in the mortar, it will only propagate until it hits a boundary (a neighboring brick). Designing nanostructures can have similar effects on materials we use every day. Some of the most prevalent are adhesives that mimic the structures on gecko feet, which allow them to stick to any surface. This work presents bottom-up design strategies for self-assembling protein materials whose hierarchical structure may prove useful in a variety of applications in soft-robotics and energy storage. Proteins are a useful class of molecules, because they contain a level of structural complexity beyond that of synthetic materials. They are an inherently 'green' material feedstock; made in a lab using microbes like E. coli. Additionally, with the ease and availability of genetic engineering techniques we can easily modify the structure. This is especially true for the class of proteins, repeat proteins, which are the focus of this manuscript. Repeat proteins comprise small repeated sequences which are structurally independent from each other and can be strung together to create open, extended architectures. Here we explore the self-assembly emergent properties of the consensus tetratricopeptide repeat (CTPR18) . We show that this protein assembles into highly ordered 1D and 2D arrays that are shape tunable based the molecular environment (solvents, charge, etc). These nanomaterials may prove useful as molecular recognition scaffolds. We further explore the hierarchical self-assembled films of CTPR18. These films form highly oriented lamellar structures that seemingly propagate the entire length of the films. These lamellae directly affect the materials mechanical properties. Accordingly, by changing the film casting conditions, we can impart a structural gradient in the film, which proves useful in tuning the water-induced bending motion of these films. Herein, we show the ability to change the speed and directionality of actuation by simply changing the underlying film morphology. Lastly, we show that these films are electroresponsive as well, owing this function to ion transport through the inherently charged character of CTPR18. These dual responsive materials may prove useful in soft robotics. Additionally we are beginning investigations into the usefulness of CTPR18 films as alternate materials for ion-transport materials like those used in lithium polymer (more commonly LiPo) and sodium-ion batteries.
PHD

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33

Lessard, Ivan A. D. "Protein-protein interaction and molecular self-assembly of the pyruvate dehydrogenase multienzyme complex." Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389875.

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34

Bludin, Alexey O. "Peptide-Porphyrin Self-Assembled Materials." Bowling Green State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1308097842.

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35

Lam, Christopher N. (Christopher Nguyen). "Interactions governing the self-assembly of globular protein-polymer block copolymers." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/104208.

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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2016.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Engineering enzymes and other proteins into biocatalysts or bioelectronic devices has the potential to lead to a new generation of energy-generating and energy conversion technologies. Controlling the hierarchical structure of protein materials from the nanoscale single molecule level up to the microscale material morphology is critical to improving their function. Lithographic patterning methods such as electron beam lithography, dip-pen nanolithography, and nanograftin allow proteins to be patterned with nanoscale resolution, but parallelization to increase throughput remains a significant challenge. While templated self-assembly enables patterning in three dimensions, maximizing protein loading and controlling orientation are challenges that remain to be addressed. Self-assembly provides a low-cost method to nanopattern proteins for biofunctional devices with high operational efficiency through control over three-dimensional spatial arrangement and orientation. Complementary experimental techniques were used to investigate the phase behaviors of globular protein-polymer block copolymers and provide insight into the relevant physics and thermodynamics governing their self-assembly. In particular, methodical permutations were made to the protein block to understand the relationship between protein interactions and protein-polymer block copolymer selfassembly. Order-disorder and order-order transitions were demonstrated for the first time within a rich window of phase space of hexagonal, lamellar, perforated lamellar, and micellar phases that were dependent on coil fraction. Protein-polymer net repulsive interactions were discovered to be important for self-assembly. The type of nanostructures formed at a given coil fraction are different between globular-coil and coil-coil systems due to the anisotropy between protein and coil shape and interactions and minor differences in solvent selectivity. A set of structurally homologous proteins in which the chemical composition and surface interaction potential were varied globally throughout the entire sequence and locally through single point mutations demonstrated highly similar phase behavior, revealing that coarse-grained properties such as the protein shape, size, solubility, surface charge, and virial coefficient can capture the general shape of the phase diagram in nonselective solvents. Engineering greater changes in protein electrostatic interactions and virial coefficient demonstrated that the electrostatic environment of proteins may be designed to tune the morphologies of protein-polymer blok copolymers, both enhancing and suppressing formation of nanostructures through attractive and repulsive interactions, respectively. A combination of small-angle neutron scattering experiments, theory, and coarse-grained modeling and simulation was used to elucidate the shape of protein-polymer block copolymers in dilute solution and quantify their interactions. Modeling protein-polymer interactions using repulsive Weeks-Chandler- Andersen potentials showed that the polymer exists as a relatively unperturbed coil extended away from the protein. The coarse-grained representation additionally provides a simple way to model the conformation of protein-polymer conjugates with strong interactions that result in the polymer wrapping around the protein in a shroud-like configuration.
by Christopher N. Lam.
Ph. D.

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36

Ryan, Morris. "Exploring the mechanisms of fibrillar protein aggregation." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/8878.

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The aim of this thesis is to investigate and better understand the mechanisms of protein self-assembly. Specifically, I study three protein systems which form morphologically and structurally distinct brillar protein aggregates. The first of these studies is concerned with the self-assembly of amyloid brils formed from bovine insulin. Amyloid brils are associated with human diseases such as Alzheimers Disease and type-2 diabetes, and are also garnering interest in biomaterial applications. Fragmentation-dominated models for the self-assembly of amyloid brils have had important successes in explaining the kinetics of amyloid bril formation but predict bril length distributions that do not match experimental observations. Here I resolve this inconsistency using a combination of experimental kinetic measurements and computer simulations. I provide evidence for a structural transition demarcated by a critical bril mass concentration, or CFC, above which fragmentation of the brils is suppressed. Our simulations predict the formation of distinct bril length distributions above and below the CFC, which I confirm by electron microscopy. These results point to a new picture of amyloid bril growth in which structural transitions that occur during self-assembly have strong effects on the final population of aggregate species with small, and potentially cytotoxic, oligomers dominating for long periods of time at protein concentrations below the CFC. I further show that the CFC can be modulated by environmental conditions, pointing to possible in vivo strategies for controlling cytotoxicity. I probe the structural nature of the transition by performing small angle neutron scattering. Secondly, I study the formation of amyloid-like brils from the protein ovalbumin. I undertake kinetic experiments of self-assembly and find two key features emerge: the lack of a lag time and the existence of a slow growth regime in the long-time limit. I observe, using TEM, that these brils are worm-like in nature and form closed-loops. I find the growth kinetics are intimately connected to this particular morphology. I present a simple kinetic model which captures the features of the kinetics found in experiments by incorporating end-to-end association of brils. I comment on the ramifications this type of amyloid bril assembly may have on oligomeric toxicity. Thirdly, the DNA-mimic protein ocr is highly charged (-56e at pH 8) and forms non-amyloid brillar assemblies at very high ammonium sulphate concentrations (3.2M). The fact that ocr forms translucent brillar gels at such high salt concentrations is extremely unique. Typically under such high salt conditions, non-specific amorphous aggregates are formed. In order to better understand the mechanism of why ocr forms specific bril aggregates, I used variants of the wile-type protein in which extensive regions of surface have been removed or modified. The structural characteristics of gels formed from the variants were probed using microrheological techniques. I find that non-specific electrostatic charge screening plays an important role in ocr aggregation. However, I also locate a potentially important α-helical region which may play a part in establishing specific interactions so that ocr may form ordered brillar assemblies.

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Varga, Melinda. "Self-assembly of the S-layer protein of Sporosarcina ureae ATCC 13881." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2011. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-65141.

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Increasing the integration density of electron device components will necessitate the use of new nanofabrication paradigms that complement and extend existing technologies. One potential approach to overcome the current limitations of electron-beam lithography may involve the use of hybrid systems, in which existing lithographic techniques are coupled with “bottom up” approaches such as supramolecular self-assembly. In this respect, biological systems offer some unique possibilities as they combine both self-organization and spatial patterning at the nanometer length scale. In particular, Surface Layer Proteins (S-layers) can facilitate high order organization and specific orientation of inorganic structures as they are two-dimensional porous crystalline membranes with regular structure at the nanometer scale. In this framework, the aim of the present work was the characterization of the S-layer of Sporosarcina ureae ATCC 13881 (SslA) with respect to its self-assembling properties and modification that would allow it to be employed as a patterning element and a new building block for nanobiotechnology. In vitro recrystallization experiments have shown that wild type SslA self-assembles into monolayers, multilayers or tubes. Factors such as initial monomer concentration, Ca2+ ions, pH of the recrystallization buffer and the presence of a silicon substrate have a strong influence on the recrystallization process. SslA monolayers proved to be an excellent biotemplate for ordered assembly of gold nanoparticle arrays. The recombinant SslA after expression and purification formed micrometer sized, crystalline monolayers exhibiting the same lattice structure as the wild type protein (p4 symmetry). This remarkable property of self-assembling has been preserved even when SslA was truncated. The deletion of both, N- and C-terminal SslA domains does not hinder self-assembly; the resulting protein is able to form extended monolayers that exhibit the p4 lattice symmetry. The central SslA-domain is self sufficient for the self-assembly. The possibility to change the natural properties of S-layers by genetic engineering techniques opens a new horizon for the tuning of their structural and functional features. The SslA-streptavidin fusion protein combines the remarkable property of self-assembling with the ligand i.e. biotin binding function. On silicon wafers, this chimeric protein recrystallized into coherent protein layers and exposes streptavidin, fact demonstrated by binding studies using biotinylated quantum dots. In this way, it can serve as a functional surface for controlled immobilization of biologically active molecules but also as a platform for the synthesis of planar arrays of quantum dots. Furthermore, the results open up exciting possibilities for construction of hybrid S-layers, structures that may ultimately promote the fabrication of miniaturized, nanosized electronic devices.

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Liu, Qiao Liu. "THE INVESTIGATION ON THE SELF-ASSEMBLY DRIVING FORCE OF HBV CAPSID PROTEIN." University of Akron / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=akron152233306275171.

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39

Chen, Chao. "Self-assembly studies of hybrid nanoparticle-protein cage systems and icosahedral viruses." [Bloomington, Ind.] : Indiana University, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3331353.

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Thesis (Ph.D.)--Indiana University, Dept. of Chemistry, 2008.
Title from PDF t.p. (viewed on Jul 27, 2009). Source: Dissertation Abstracts International, Volume: 69-11, Section: B, page: 6818. Adviser: Bogdan Dragnea.

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40

Nilsson, Josefina. "Protein adaptability involved in self-assembled icosahedral capsids /." Stockholm : Department of biosciences and nutrition, Center for biotechnology, Karolinska institutet, 2006. http://diss.kib.ki.se/2006/91-7140-717-0/.

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41

Rodriguez, de Francisco Borja. "Self-assembly into functional amyloids of Aspergillus fumigatus hydrophobins." Electronic Thesis or Diss., Sorbonne université, 2019. http://www.theses.fr/2019SORUS332.

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Les hydrophobines sont des protéines fongiques qui s’assemblent aux interfaces hydrophobes/hydrophiles ou air-eau (IHH) pour former des fibres amyloïdes fonctionnelles qui s’organisent en couches dont le caractère amphiphile détermine leurs fonctions. Les spores du champignon pathogène opportuniste Aspergillus fumigatus sont couvertes d’une couche de bâtonnets constitués de fibres amyloïdes formés par l’hydrophobine RodA. Ce revêtement hydrophobe facilite la dispersion des spores et les rend inertes vis à vis du système immunitaire humain. Deux proches homologues de RodA, RodB et RodC, sont aussi présents dans la paroi cellulaire des spores. Nous avons réalisé une étude comparative de l’auto-assemblage de ces trois protéines avec un accent particulier sur RodC. Nous avons montré que RodA-C nécessitent d’une interface IHH pour s’assembler en fibres et mis en évidence l’importance de la nature de l’interface dans la morphologie de leurs assemblages. Nous avons observé une auto-inhibition de la fibrillation avec la concentration et montré que celle-ci est due à la saturation de l’interface air-eau. En étudiant l’effet de mutations ponctuelles sur les cinétiques de fibrillation de RodC, nous avons établi des similitudes et des différences par rapport aux régions importantes pour la formation de fibres pour RodC et RodA, étudiée au préalable. La transition du monomère à l’état amyloïde est accompagnée d’une perte de régions désordonnées et un gain de feuillets β intermoléculaires en accord avec les analyses par mutagénèse dirigée, qui indiquent que des résidus hydrophobes dans des régions flexibles du monomère sont impliqués dans le squelette β-croisé des fibres
Hydrophobins are fungal proteins that spontaneously self-assemble at hydrophobic/hydrophilic or air/water interfaces to form functional amyloids that associate laterally into layers. The amphiphilic character of these layers is at the origin of the hydrophobin biological roles. The spores of the airborne fungal pathogen Aspergillus fumigatus are covered by an amyloid layer with rodlet morphology made up by the RodA hydrophobin. This hydrophobic coat facilitates air-dispersal of the spores and renders these inert relative to the human immune system. Two close homologs of RodA, named RodB and RodC, are also present in the spore cell wall. We have performed a comparative study on the self-assembly of the three proteins with particular emphasis on RodC. We have shown that RodA-C require an interface to form amyloids and revealed the importance of the nature of the interface in determining the morphology of hydrophobin assemblies. We have observed that the fibrillation of RodA-C is auto-inhibited (slower at higher concentrations) and shown that this phenomenon can be explained by saturation of the air-water interface. The analysis of the effect of single point mutations on the fibrillation kinetics of RodC revealed the regions that are important for fiber formation, which showed differences and similarities relative to the previously studied RodA. The transition from monomers to amyloids is accompanied by a loss of unordered regions and a gain in intermolecular β-sheets, in agreement with the mutational analyses of RodA and RodC that indicated that hydrophobic residues in flexible loops are involved in the cross-β core of the fibers

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42

Bastardo, Zambrano Luis Alejandro. "Self assembly of surfactants and polyelectrolytes in solution and at interfaces." Doctoral thesis, KTH, Ytkemi, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-425.

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This thesis focuses on the study of the interactions between polyelectrolytes and surfactants in aqueous solutions and at interfaces, as well as on the structural changes these molecules undergo due to that interaction. Small–angle neutron scattering, dynamic, and static light scattering were the main techniques used to investigate the interactions in bulk. The first type of polymer studied was a negatively charge glycoprotein (mucin); its interactions with ionic sodium alkyl sulfate surfactants and nonionic surfactants were determined. This system is of great relevance for several applications such as oral care and pharmaceutical products, since mucin is the main component of the mucus layer that protects the epithelial surfaces (e.g. oral tissues). Sodium dodecyl sulfate (SDS) on the other hand, has been used as foaming agent in tooth pastes for a very long time. In this work it is seen how SDS is very effective in dissolving the large aggregates mucin forms in solution, as well as in removing preadsorbed mucin layers from different surfaces. On the other hand, the nonionic surfactant n-dodecyl β-D-maltopyranoside (C12-mal), does not affect significantly the mucin aggregates in solution, neither does it remove mucin effectively from a negatively charge hydrophilic surface (silica). It can be suggested that nonionic surfactants (like the sugar–based C12-mal) could be used to obtain milder oral care products. The second type of systems consisted of positively charged polyelectrolytes and a negatively charged surfactant (SDS). These systems are relevant to a wide variety of applications ranging from mining and cleaning to gene delivery therapy. It was found that the interactions of these polyelectrolytes with SDS depend strongly on the polyelectrolyte structure, charge density and the solvent composition (pH, ionic strength, and so on). Large solvent isotopic effects were found in the interaction of polyethylene imine (PEI) and SDS, as well as on the interactions of this anionic surfactant and the sugar–based n-decyl β-D-glucopyranoside (C10G1). These surfactants mixtures formed similar structures in solutions to the ones formed by some of the polyelectrolytes studied, i.e. ellipsoidal micelles at low electrolyte concentration and stiff rods, at high electrolyte and SDS concentrations.
QC 20100901

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43

Broncel, Malgorzata [Verfasser]. "Synthetic phosphopeptides and phosphoproteins as tools for studying peptide and protein self-assembly / Malgorzata Broncel." Berlin : Freie Universität Berlin, 2011. http://d-nb.info/1025552814/34.

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Weber, Jeffrey. "Coarse Grained Monte Carlo Simulation of the Self-Assembly of the HIV-1 Capsid Protein." Honors in the Major Thesis, University of Central Florida, 2014. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/1654.

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In this study, a Monte Carlo simulation was designed to observe the self-assembly of the HIV-1 capsid protein. The simulation allowed a coarse grained model of the capsid protein with defined interaction sites to move freely in three dimensions using the Metropolis criterion. Observations were made as to which parameters affected the assembly the process. The ways in which the assembly were affected were also noted. It was found that proper dimerization of the capsid protein was necessary in order for the lattice to form properly. It was also found that a strong trimeric interface could be responsible for double-layered assemblies. Further studies may be conducted by further varying of parameters or reworking the dynamics of the simulation. The possible causes of curvature within the assembly still need to be researched further.
B.S.
Bachelors
Physics
Sciences

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45

Dahal, Yuba Raj. "Equilibrium and kinetic factors in protein crystal growth." Diss., Kansas State University, 2017. http://hdl.handle.net/2097/36220.

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Doctor of Philosophy
Department of Physics
Jeremy D. Schmit
Diseases such as Alzheimer’s, Parkinson’s, eye lens cataracts, and Type 2 diabetes are the results of protein aggregation. Protein aggregation is also a problem in pharmaceutical industry for designing protein based drugs for long term stability. Disordered states such as precipitates and gels and ordered states such as crystals, micro tubules and capsids are both possible outcomes of protein–protein interaction. To understand the outcomes of protein–protein interaction and to find the ways to control forces, it is required to study both kinetic and equilibrium factors in protein–protein interactions. Salting in/salting out and Hofmeister effects are familiar terminologies used in protein science field from more than a century to represent the effects of salt on protein solubility, but they are yet to be understood theoretically. Here, we build a theory accounting both attractive and repulsive electrostatic interactions via the Poisson Boltzmann equation, ion–protein binding via grand cannonical partition function and implicit ion–water interaction using hydrated ion size, for describing salting in/salting out phenomena and Hofmeister and/or salt specific effect. Our model free energy includes Coulomb energy, salt entropy and ion–protein binding free energy. We find that the salting in behavior seen at low salt concentration near the isoelectric point of the protein is the output of Coulomb energy such that the addition of salt not only screens dipole attraction but also it enhances the monopole repulsion due to anion binding. The salting out behavior appearing after salting in at high salt concentration is due to a salt mediated depletion interaction. We also find that the salting out seen far from the isoelectric point of the protein is dominated by the salt entropy term. At low salt, the dominant effect comes from the entropic cost of confining ions within the aggregates and at high salt, the dominant effect comes from the entropy gain by ions in solution by enhancing the depletion attraction. The ion size has significant effects on the entropic term which leads to the salt specificity in the protein solubility. Crystal growth of anisotropic and fragile molecules such as proteins is a challenging task because kinetics search for a molecule having the correct binding state from a large ensemble of molecules. In the search process, crystal growth might suffer from a kinetic trap called self–poisoning. Here, we use Monte Carlo simulation to show why protein crystallization is vulnerable to the poisoning and how one can avoid such trap or recover crystal growth from such trap during crystallization. We show that self–poisoning requires only three minimal ingredients and these are related to the binding affinity of a protein molecule and its probability of occurrence. If a molecule attaches to the crystal in the crystallographic state then its binding energy will be high but in protein system this happens with very low probability (≈ 10−5). On the other hand, non–crystallographic binding is energetically weak, but it is highly probable to happen. If these things are realized, then it will not be surprising to encounter with self–poisoning during protein crystallization. The only way to recover or avoid poisoning is to alter the solution condition slightly such as by changing temperature or salt concentration or protein concentration etc.

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46

Dickerson, Matthew B. "The protein and peptide mediated syntheses of non-biologically-produced oxide materials." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2007. http://hdl.handle.net/1853/24704.

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Thesis (Ph. D.)--Materials Science and Engineering, Georgia Institute of Technology, 2008.
Committee Chair: Sandhage, Kenneth; Committee Co-Chair: Kröger, Nils; Committee Co-Chair: Naik, Rajesh; Committee Member: Hud, Nicholas; Committee Member: Marder, Seth.

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47

Powell, Tremaine Bennett. "The Use of Nanoparticles on Nanometer Patterns for Protein Identification." Diss., The University of Arizona, 2008. http://hdl.handle.net/10150/194368.

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This dissertation describes the development of a new method for increasing the resolution of the current protein microarray technology, down to the single molecule detection level. By using a technique called size-dependent self-assembly, different proteins can be bound to different sized fluorescent nanostructures, and then located on a patterned silicon substrate based on the sized pattern which is closest to the size of the bead diameter.The protein nanoarray was used to detect antibody-antigen binding, specifically anti-mouse IgG binding to mouse IgG. The protein nanoarray is designed with the goal of analyzing rare proteins. However, common proteins, such as IgG, are used in the initial testing of the array functionality. Mouse IgG, representing rare proteins, is conjugated to fluorescent beads and the beads are immobilized on a patterned silicon surface. Then anti-mouse IgG binds to the mouse IgG on the immobilized beads. The binding of the antibody, anti-mouse IgG, to the antigen, mouse IgG is determined by fluorescent signal attenuation.The first objective was to bind charged nanoparticles, conjugated with proteins, to an oppositely charged silicon substrate. Binding of negatively charged gold nanoparticles (AuNP), conjugated with mouse IgG, to a positively charged silicon surface was successful.The second objective was to demonstrate the method of size-dependent self-assembly at the nanometer scale (<100 >nm). Different-sized, carboxylated, fluorescent beads and AuNP, which were conjugated with proteins, were serially added to a patterned polymethyl methacrylate (PMMA) coated silicon surface. Size-dependent self-assembly was successfully demonstrated, down to the nanometer scale.The final objective was to obtain a signal from antibody-antigen binding within the protein array. Conjugated fluorescent beads were bound to e-beam patterns and signal attenuation was measured when the antibodies bound to the conjugated beads. The size-dependent self-assembly is a valuable new method that can be used for the detection and quantification of proteins.

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48

Jullian, Christelle Francoise. "Self-Assembly of Matching Molecular Weight Linear and Star-Shaped Polyethylene glycol Molecules for Protein Adsorption Resistance." Diss., Virginia Tech, 2007. http://hdl.handle.net/10919/29581.

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Fouling properties of materials such as polyethylene glycol (PEG) have been extensively studied over the past decades. Traditionally, the factors believed to result in protein adsorption resistance have included i) steric exclusion arising from the compression of longer chains and ii) grafting density contribution which may provide shielding from the underlying material. Recent studies have suggested that PEG interaction with water may also play a role in its ability to resist protein adsorption suggesting that steric exclusion may not be the only mechanism occurring during PEG/protein interactions. Star-shaped PEG polymers have been utilized in protein adsorption studies due to their high PEG segment concentration, which allows to increase the PEG chain grafting density compared to that achieved with linear PEG chains. Most studies that have investigated the interactions of tethered linear and star-shaped PEG layers with proteins have considered linear PEG molecules with molecular weights several orders of magnitude smaller than those considered for star-shaped PEG molecules (i.e. 10 000 g/mol vs. 200 000 g/mol, respectively). Additionally, the star-shaped PEG molecules which have been considered in the literature had up to ~70 arms and were therefore modeled by hard-sphere like structures and low chain densities near the surface due to steric hindrance. This resulted in some difficulties to achieve grafted PEG chain overlap for star molecules. Here, triethoxysilane end-functionalized linear PEG molecules have been synthesized and utilized to form star-shaped PEG derivatives based on ethoxy hydrolysis and condensation reactions. This resulted in PEG stars with up to ~4 arms, which were found to result in grafted star-shaped PEG chains with significant chain overlap. Linear PEG derivatives were synthesized so that their molecular weight would match the overall molecular weight of the star-shaped PEG molecules. These 2 PEG molecular architectures were covalently self-assembled to hydroxylated silicon wafers and the thickness, grafting density, and conformation of these films were studied. The adsorption of human albumin (serum protein) on linear and star-shaped PEG films was compared to that obtained on control samples, i.e. uncoated silicon wafers. Both film architectures were found to significantly lower albumin adsorption.
Ph. D.

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49

Phan-Xuan, Minh-Tuan. "Elaboration of microgel protein particles by controlled selfassembling of heat‐denatured beta‐lactoglobulin." Phd thesis, Université du Maine, 2012. http://tel.archives-ouvertes.fr/tel-00770331.

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Beta lactoglobulin (βlg) is a major whey protein in the bovine milk. Upon heating above its denaturation temperature (which is pH-dependent), this globular protein undergoes molecular changes leading to the irreversible aggregation. Depending on the pH and ionic strength, either protein aggregates or gels exhibiting various structures and morphologies have been described. Very recently, it was found that in a narrow range of the pH close to iso-electric point, stable suspensions of rather monodisperse spherical particles with a radius of about a hundred nanometers were formed. These spherical particles which were called microgels might be of special interest for the production of liquid dispersions of β-lactoglobulin aggregates exhibiting various functionalities for food applications. The project on which I report here was a collaboration with the Nestlé Reseach Center (Lausanne, Switzerland) and its objective was to study the formation and structural properties of the microgels in different environmental conditions. The first part of the project is to study the influence of the pH on the formation of microgels. Stable suspensions of protein microgels are formed by heating salt free βlg solutions at concentrations up to about C = 50 g.L-1 if the pH is set within a narrow range between 5.75 and 6.1. The internal protein concentration of these spherical particles is about 150 g.L-1 and the average hydrodynamic radius decreases with increasing pH from 200 nm to 75 nm. The formation of the microgels leads to an increase of the pH, which is a necessary condition to obtain stable suspensions. The spontaneous increase of the pH during microgel formation leads to an increase of their surface charge density and inhibits secondary aggregation. This self-stabilization mechanism is not sufficient if the initial pH is below 5.75 in which case secondary aggregation leads to precipitation. Microgels are no longer formed above a critical initial pH, but instead short curved protein strands are obtained with a hydrodynamic radius of about 15-20 nm. The second part of the work is about the formation of microgels driven by the addition of calcium ions. We found that stable suspensions of spherical protein particles (microgels) can be formed by heating βlg solutions in the presence of calcium ions. The conditions for the calcium induced microgel formation were studied at different pH between 5.8 and 7.5 and different protein concentrations between 5 - 100 g.L-1. The results showed that a critical molar ratio of calcium to proteins (R) is needed to form microgels independent of the protein concentration. R decreases with decreasing pH. The microgels have a hydrodynamic radius ranging from 100 to 300 nm and their internal protein concentration ranges from 0.2 to 0.45 g.mL-1. The determination of calcium bound to the microgels suggests that the crucial parameter for microgel formation is the net charge density of the native proteins. The microgel suspensions are stable in a narrow range of R but aggregate at higher Ca2+ concentrations. In the third part, we continued to investigate the formation of microgels at initial step and how it is growing in the presence of calcium ions. We have proposed a mechanism of formation of blg microgels which follows a nucleation and growing process. The nucleus with define size are formed at the initial state and that is growing in size to reach final size of aggregates. At low calcium concentration it stabilizes and then we obtain a stable suspension of microgels. But at high concentrations, the microgels here can jump to form big aggregates and finally a gel. The structure of gel from microgels is heterogenous at the scale of confocal microscopy and similar to those formed in the presence of NaCl 0.3 M. However the process of formation of these gels is not the same...

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Gordo, Villoslada Susana. "Use of calix[4]arenes to recover the self-assembly ability of mutated p53 tetramerization domains." Doctoral thesis, Universitat de Barcelona, 2008. http://hdl.handle.net/10803/2819.

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Protein-protein interactions are essential in biological processes and thus, they have become very promising therapeutic targets. Nevertheless, the artificial modulation of protein complexes remains a challenge. Since most work to date has been focused on the inhibition of protein-protein interactions, there is little precedent on the design of molecules which can induce, stabilize or recover the oligomerization state of proteins. In this context, the system comprised of the tetramerization domain of protein p53 (p53TD) and its oncogenic mutants with defective oligomerization properties is an outstanding case of study for the design and evaluation of molecules which can recover the tetrameric structure.
Through a collaboration with Prof. Javier de Mendoza, a family of para-guanidinomethyl-calix[4]arenes able to interact simultaneously with the four monomers of the p53 tetramerization domain was rationally designed; hence, the interaction with these compounds would stabilize the whole tetrameric assembly.
In order to evaluate experimentally said calix[4]arenes, three natural oncogenic mutants of the p53TD with defective assembly abilities were biosynthesized. Namely, they are: G334V, R337H and L344P.
Once synthesized and purified the calix[4]arenes compounds, their molecular recognition properties were tested through a battery of biophysical techniques, including nuclear magnetic resonances (on both the protein and the ligand), circular dichroism, differential scanning calorimetry, crystallography, mass spectrometry and chemical cross-linking. The results clearly show that these calix[4]arenes interact with the proteins as intended and, the most important, they can thermally and kinetically stabilize the tetrameric state.
These results are the perfect evidence of the proof-of-concept initially sought: a little synthetic ligand can stabilize the oligomeric state of proteins which are structurally defective. In addition, the study of several ligands with different functionalizations provides further understanding about the basis of molecular recognition events. On the one hand, the guanidinium group has a vital role for high affinity interactions. On the other hand, structural flexibility, in both the protein and the ligand, enables the molecules to adopt the optimal conformation for the tightest interaction, thereby underscoring the ambiguous and unpredictable role of the entropy in interaction processes.
Las interacciones proteína-proteína son esenciales en muchos procesos biológicos y por ello resultan dianas terapéuticas muy prometedoras. Sin embargo, modular artificialmente complejos proteicos resulta todavía un gran reto. Hasta la fecha, los esfuerzos se han dirigido básicamente hacia la inhibición de interacciones proteína-proteína; pocos precedentes describen el diseño de moléculas que puedan inducir, estabilizar o recuperar el estado oligomérico de proteínas. En relación a lo último, el sistema formado por el dominio de tetramerización de la proteína p53 (p53TD) y sus mutantes oncogénicos con oligomerización defectuosa representa un excelente modelo para el diseño y la evaluación de moléculas que puedan recuperar el estado tetramérico nativo.
En colaboración con el Prof. Javier de Mendoza, se diseñaron racionalmente compuestos para-guanidinometil-calix[4]arenos capaces de interaccionar con simultáneamente con las cuatros unidades que estructuran el dominio de tetramerización de p53, de tal modo que podrían estabilizar el estado oligomérico de la proteína.
Para la evaluación experimental de dichos ligandos calix[4]arenos, se biosintetizaron tres mutantes naturales de p53TD con tetramerización defectuosa: G334V, encontrado en cánceres de pulmón; R337H, asociado al carcinoma adrenocortical infantil; y L344P, asociado al síndrome Li-Fraumeni.
Tras la síntesis y purificación de los compuestos guanidinometil-calix[4]arenos, sus capacidades de interacción con las proteínas se estudiaron por técnicas biofísicas, que incluyen resonancia magnética nuclear (sobre la proteína y sobre el ligando), dicroismo circular, calorimetría diferencial de barrido, calorimetría isotérmica de titulación, cristalografía, espectrometría de masas y entrecruzamiento químico. Los resultados muestran claramente que los calix[4]arenos pueden interaccionar con las proteínas tal y como se habían diseñado; en consecuencia, estos ligandos son capaces de estabilizar térmica y cinéticamente las proteínas mutantes, recuperando así su estado tetramérico.
Estos resultados son la perfecta prueba del concepto inicialmente planteado: un pequeño ligando sintético diseñado puede estabilizar el estado oligomérico de proteínas estructuralmente defectuosas. El estudio de varios ligandos con diferentes grupos funcionales también pone de manifiesto otros fenómenos de particular relevancia en el campo del reconocimiento de superficies proteicas. Por una parte, el grupo guanidinio tiene un papel clave para la afinidad de la interacción. Por otra parte, la flexibilidad estructural de ambos componentes: la proteína y el ligando, permite que se establezcan interacciones más estrechas y fuertes, lo que refleja el papel tan ambiguo e impredecible de la entropía en procesos de interacción.

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