Dissertations / Theses on the topic 'Protein surfaces'

This thesis is concerned with the fundamental principles affecting protein adsorption. The effects of surface chemistry and topography on protein adsorption characteristics have been identified and quantified. Particular attention has been made to understand how the conformation of surface-bound proteins was affected by the surface onto which they adsorbed. Quartz crystal microbalance (QCM), UV-Vis spectroscopy and fluorometry were used to assess protein-surface affinity and amounts of protein adsorbed at surface saturation levels. Infrared spectroscopy was used to quantify protein conformational changes incurred upon adsorption. A fluorescent assay protocol was developed for use as an external calibration method for the quantification of adsorbed protein an d the results obtained were compared with QCM and an amido black protein assay of the same systems. Model experiments were performed using bovine fibrinogen (an elongated molecule) and albumin (a globular molecule) adsorbing onto flat hydrophilic (OH terminated) and hydrophobic (CH3 terminated) surfaces in the first instance, but later superhydrophilic and superhydrophobic surfaces were also studied. Surface curvature on the nano-scale was used to model topography, wherein protein molecules adsorbed onto spherical substrates (15-165 nm diameter) having chemically defined surfaces. Results obtained indicate that both proteins exhibit a less organised secondary structure upon adsorption onto hydrophobic compared to hydrophilic surfaces, with this effect being greatest for albumin. Adsorption rates and binding affinities were found to be higher on hydrophobic surfaces although the amounts adsorbed at saturation were lower. Supporting spectroscopic data suggests that proteins undergo surface induced deformation upon adsorption. Topography was shown to compound the effects of surface chemistry, with fibrinogen being more denatured on surfaces presenting high surface curvature whereas albumin was more denatured on larger substrates. These effects are most probably due to the differing size and shape of the proteins investigated. This study highlights the possibility of using tailor-made surfaces to influence binding rates and the conformation of bound proteins through protein-surface interactions. The data presented in this thesis demonstrates our ability to control protein adsorption characteristics through careful consideration of the underlying surface, which may facilitate the development and fabrication of materials / surface coatings with tailored bioactivity.

Is it possible to find an easy, generic method for protein refolding? The preparation of functionally active protein molecules from the unfolded state can be a difficult task. Although there are many well-established techniques for protein refolding, such as dilution, dialysis, chromatography and others, in many instances these methods can be time consuming and inefficient. A rapid, inexpensive and simple method for protein folding is a much sought after technique. Proteins in the unfolded state (either inclusion bodies or unfolded by chemical or physical means) are generally solubilised in solutions containing urea or guanidine hydrochloride. The removal of these molecules from the protein environment is commonly utilised as a method for triggering refolding. A new method for the refolding of biomolecular species has been developed via the formation of Protein Coated Micro-crystals (PCMC). The formation of PCMC is a recently developed method for the immobilisation protein upon the surface of a watersoluble excipient (salt, amino acid or sugar) via a co-precipitation reaction in a water miscible organic solvent. These proteins can then be used as immobilised biocatalysts in both the aqueous and organic phase. In the immobilisation of unfolded, solubilised protein, the solubilising agents (e.g. urea or guanidine hydrochloride) are removed from the protein environment as they are soluble in the organic phase. The removal of these molecules initiates protein folding during the coprecipitation process. In the course of this project, a number of proteins were studied in order to observe their behaviour in this immobilisation and simultaneous folding process. Lysozyme was utilised as it is an enzyme which is relatively simple to refold from the chemically unfolded state by conventional methods such as dilution. Upon immobilisation of lysozyme from the chemically unfolded state, up to 92% of the activity of the native protein was regained. The enzyme lipase, which is notoriously difficult to fold, was also used to determine the efficiency of this method under more challenging conditions. Lipase immobilised from the chemically unfolded state was seen to regain up to 36 % of the activity of the native protein.

Thesis (M. S.)--Materials Science and Engineering, Georgia Institute of Technology, 2008.
Committee Chair: Tsukruk, Vladimir; Committee Member: Kalaitzidou, Kyriaki; Committee Member: Valeria Milam.

The aim of the project was to modify polydimethylsiloxane (PDMS) surfaces in order to minimize adsorption of proteins. PDMS is used in micro-fluidic devices that control the delivery of samples to a sensor chip in Biacore instrumentation. These instruments are used to characterize interactions between biomolecules with a detection principle based on surface plasmon resonance (SPR). To minimize adsorption of proteins poly-ethylene-oxide (PEO) based surfactants, were added to the buffer. The added PEO surfactants were P20, Pluronic F-127 and Brij 35. Interaction of these surfactants with the sensor chip in Biacore instruments was also examined. Creating a more hydrophilic surface layer on PDMS by oxidation was also examined.

When surfactants were continuously added to protein samples, as in dynamically coating of PDMS surfaces, Brij 35 resulted in the strongest reduction in protein adsorption. Brij 35 was also the surfactant that was easiest to remove from both PDMS and the sensor surfaces. Pluronic bound strongest to surfaces, and is most suitable when only adding surfactant to the buffer in a pre-coating step. All surfactants did reduce protein adsorption considerably (99% or more) and addition is necessary when working with protein solutions and hydrophobic surfaces as PDMS. Another alternative is oxidation of PDMS surface, which is an easy procedure that decreased the protein adsorption to about 10% compared to adsorption to untreated surface.

The objective of this thesis was to investigate and characterize the interaction between blood proteins and different surfaces with emphasis on protein adsorption to bioceramics and model surfaces. Special effort was made to monitor the spontaneous and selective adsorption of proteins from human plasma and to examine the orientation, conformation and functional behavior of single proteins after adsorption.

Five different ceramic biomaterials: alumina (Al₂O₃), zirconia (ZrO₂), hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) and two glass-ceramics, AP40 (SiO₂-CaO-Na₂O-P₂O₅-MgO-K₂O-CaF₂) and RKKP (AP40 with Ta₂O₃-La₂O₃), were exposed to human plasma and their protein binding capacities and affinities for specific proteins were studied by chromatography, protein assays, two-dimensional gel electrophoresis and Western blotting. The studies showed that all materials adsorbed approximately the same high amount of plasma proteins and that they therefore should be fully covered by proteins in an in vivo setting. The adsorbed proteins were different for most materials which could explain their previously observed different levels of tissue integration in vivo.

Four of the proteins that behaved differently, ceruloplasmin, prothrombin, α₂-HS-glycoprotein and α₁-antichymotrypsin, were selected for characterization with atomic force microscopy and ellipsometry. The studies, which were performed on ultraflat silicon wafers (silica), showed that the proteins oriented themselves with their long axis parallel to the surface or as in case of ceruloplasmin with one of its larger sides towards the surface. All of them had globular shapes but other conformational details were not resolved. Furthermore, prothrombin (none of the others) formed multilayers at high proteins concentrations.

The functional behaviour of the adsorbed proteins, referring to their cell binding and cell spreading capacity on silica and a positive cell adhesion reference surface (Thermanox®), was affected by the underlying substrate. Ceruloplasmin, α₂-HS-glycoprotein and α₁-antichymotrypsin stimulated cell attachment to silica, but suppressed attachment to Thermanox®. Prothrombin stimulated cell attachment to both surfaces. The attachment was in most cases mediated both by cell membrane-receptors (integrins) and by non-specific interactions between the cell and the material.

This thesis showed that the compositional mixture, orientation, conformation and functional behavior of the adsorbed proteins are determined by the properties of the underlying surface and if these parameters are controlled very different cellular responses can be induced.

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.

The work described in this thesis is concerned with the development of new applications of the photo-CIDNP (photochemically induced dynamic nuclear polarization) technique to aspects of protein structure and folding. Chapters 1 and 2 are introductory chapters; Chapter 1 describes the theoretical basis of the CIDNP phenomenon in terms of the underlying spin chemistry of the radical pair mechanism, while Chapter 2 presents the apparatus, photosensitizer and pulse sequences used, along with some important experimental considerations. Chapter 3 describes how ¹⁵N CIDNP can be used to probe the accessibility of tryptophan side-chains in both native and denatured states of proteins. The polarization of indole nitrogens in uniformly ¹⁵N labeled protein is detected in a two-dimensional ¹⁵N-¹ H NMR heteronuclear correlation experiment. Chapter 4 describes two new techniques offering considerable improvements in the quality of photo-CIDNP spectra of proteins. Both focus on the problem of progressive photo-degradation of the flavin dye and in both cases a larger number of scans can be accumulated before the flavin is exhausted than would otherwise be possible. In Chapter 5, the potential of stopped-flow photo-CIDNP spectroscopy for the study of protein folding is explored. Rapid dilution of denatured protein into a buffer solution is used to initiate a refolding process which is followed using short laser pulses to generate ¹H CIDNP in the side-chains of exposed aromatic residues. In Chapter 6, the field dependence of amino acid photo-CIDNP intensities is investigated using a stopped-flow CIDNP device that allows sample irradiation over a range of magnetic fields (0.1-7 T) within the bore of a 9.4 T NMR magnet and rapid transfer into the NMR tube for detection. Finally, in Chapter 7 two photo-CIDNP techniques that probe the exposure of aromatic residues in partially folded states are described. Both involve transfer of polarization to the native state for detection. One approach achieves this kinetically by rapid refolding, and the other involves monitoring exchange cross peaks in a two-dimensional CIDNP spectrum under conditions where the two states are interconverting.

Cullin RING E3 ubiquitin ligases (CRLs) function in the ubiquitin proteasome system by catalysing the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins. CRLs are large dynamic multi-subunit complexes that control the fate of many proteins in cells and, therefore, constitute attractive drug targets for the development of small-molecule tools and potential drug leads, such as inhibitors and chemical inducers of protein degradation. This work presents the first crystal structure of the pentameric human CRL2VHL complex, composed of Cul2, Rbx1, Elongin B (EloB), Elongin C (EloC) and pVHL. The structure presents a closed state of full-length Cul2 and a new conformation of Rbx1, thought to be in a trajectory from inactive to active state. The thermodynamic signature of the interaction between Cul2 and pVHL-EloBC (VBC) was determined as well as mutations that contribute toward a selectivity switch for Cul2 versus Cul5 recognition. In addition, this work focused on an extensive approach to probe the VBC surface with peptides. A first methodology involved the structure-based design aimed at targeting the Cul2 VBC interaction. Three peptides have been shown to bind at the Cul2 binding site on EloC, however, with very weak affinities that could not be optimised, suggesting its poor ligandability. The second methodology included an unbiased screening using phage display libraries of bicyclic peptides. The screening campaign yielded peptide hits but their physicochemical properties, especially poor solubility, constituted an obstacle to the progress toward a biophysical characterisation of their binding to VBC and the development into high-affinity probes. The findings of this work provide structural and biophysical contributions into the whole CRL2VHL complex assembly and functioning and provide insights and tools that could aid future targeting of this CRL.

In order to measure the thickness of a protein layer on a structured surface of silicon rubber, we have used ellipsometry and Fourier transform infrared (FTIR)-spectroscopy. The aim was to determine whether this type of measurement method can be used on protein layers or not. By hot-embossing a specific pattern of micrometre-sized pillars was created on the surface of the silicon rubber, which then was exposed to a phosphate buffer solution (PBS) containing human serum albumin (HSA) protein. FTIR measurements confirmed that proteins had attached to the surface. Ellipsometric studies were made and even though the protein layer was too thin to be measured, a simulation was made that revealed that a protein layer needs to be at least 1,5 nm to be measured properly with this method. We can also see that the protein molecules can get out of the solution, to find their way into the small pits of the samples.

The engineering of bacterial surfaces has in recent yearsattracted a lot of attention with applications in manydifferent areas of bioscience. Here we describe the use of twodifferent surface display systems for the gram-positivebacteria Staphylococcus carnosus and Staphylococcus xylosus invarious biotechnological applications.

Environmental microbiology currently attracts a lot ofattention since genetically engineered plants and bacteriamight be used as bioadsorbents for sequestration of toxicmetals. Bacterial surface display of metal-binding peptidesmight enable recycling of the biomass by desorption ofaccumulated heavymetals. In an attempt to recruitstaphylococcal display systems for bioremediation purposes,polyhistidyl peptides were successfullly displayed on thesurface of recombinant S. carnosus and S. xylosus cells.Whole-cell Ni2+-binding assays demonstrated that therecombinant cells had gained metal-binding capacity compared towild-type cells.

Tailor-made, metal-binding staphylococci was created using apreviously constructed phage-display combinatorial proteinlibrary based on a fungal cellulose-binding domain (CBD)derived from the cellobiohydrolase Cel7A of Trichoderma reseii.Novel metal-binding CBDs were generated through a phagemediated selection procedure. Selected CBD variants, now devoidof cellulose binding, were randomly selected and sequenceanalysis of selected variants revealed a marked preference forhistidine residues at the randomized positions. Surface displayof these novel CBD variants resulted in recombinantstaphylococci with increased metal-binding capacity compared tocontrol strains, indicating that this could become a generalstrategy to engineer bacteria for improved binding to specificmetal ions.

Directed immobilization of cells with surface displayedheterologous proteins have widespread use in modernbiotechnology. Among other things they could provide aconvenient way of generating biofilters, biocatalysts orwhole-cell diagnostic devices. It was therefore investigatedwhether directed immobilization of recombinant staphylococci oncotton fibers could be achieved by functional display of afungal cellulose-binding domain (CBD). Recombinant S. carnosuscells with surface anchored CBDs from Trichoderma reseii Cel6Awere found to efficiently bind to cotton fibers creating almosta monolayer on the fibrous support. The co-expression of thisCBD together with previously described metal-binding proteinson the surface of our staphylococci would create means fordeveloping effective bioadsorbents for remediationpurposes.

The original plasmid vector, designed for heterologoussurface display on recombinant S. carnosus cells has exhibitedproblems related to structural instability, possibly due to thepresence of a phage f1 origin of replication in the vectorsequence. This would be a problem if using the vector systemfor library display applications. Therefore, novel surfacedisplay vectors, lacking the phage ori were constructed andevaluated by enzymatic and flow cytometric whole-cell assays.One such novel vector, pSCXm, exhibited dramatically increasedplasmid stability with the retained high surface density ofexpressed heterologous proteins characteristic for the originalS. carnosus display vector, thus making it potentially moresuitable for library display applications.

The successful engineering of our staphylococcal displaysystem encouraged us to further evaluate the potential to usethe staphylococcal system for display of combinatorial proteinlibraries and subsequent affinity based selections using flowcytometric cell sorting. A model system of recombinant S.carnosus cells with surface displayed engineered protein Adomains was constructed. It was demonstrated that target cellscould be sorted essentially quantitatively from a moderateexcess of background cells in a single sorting-step.Furthermore, the possibility of using staphylococcal surfacedisplay and flow cytometric cell sorting also for specificenrichment of very rare target cells by multiple rounds ofcell-sorting and in between amplification was demonstrated.

Key words:affibody, albumin binding protein, bacterialsurface display, cell immobilization, bioremediation,combinatorial protein engineering, flow cytometry,Gram-positive, metal binding, staphylococcal protein A,Staphylococcus carnosus, Staphylococcus xylosus, whole-celldevices

The present thesis is based on the development and optimization of a solid-phase strategy to prepare biaryl bicyclic pentapeptides. Suzuki-Miyaura cross-coupling reactions were used to obtain the biaryl bridge. The metholodogy was compatible with the introduction of amino acids with functional groups in their side-chains (lysine, arginine and serine). The bicyclic pentapeptides were considered privileged structures regarding the evaluated properties. The introduction of the biaryl stapled enhanced passive diffusion permeability across the blood-brain barrier (BBB). Furthermore, protease resistance in human serum was also improved due to the presence of the biaryl. Moreover, no cytotoxicity was observed in HeLa cells at 500µM concentration. The developed methodology was expanded to a new kind of compounds. While the compounds synthesized in chapter 1 displayed a phe-phe stapled, the peptides prepared in chapter 3, presented the stapling between two tryptophans (trp-trp). Permeability studies demonstrated that the nature of the stapled was critical to improve the passive transport across the BBB. Cyclic peptides with bromine at different positions (5, 6 or 7) of the indole group of tryptophan were also synthesized. These compounds showed high passive permeability across the BBB. High cell survival rates in HeLa cells were observed for these peptides, except for the analog with bromine at position 5 of the indole. We aimed to evaluate the recovery of the self-assembly of a mutated p53 protein with some of the previously described peptides. Protein p53 is known as the “genome guardian”. The alteration of the balance of p53 pathway is mainly related with cancer. We were interested in the region that controls the proper folding of the protein, named the tetramerization domain. This domain is a dimer of dimer that contains 37 residues. The most relevant point mutation in this region is R337H. This mutant is mainly associated with the atypically frequent cases of pediatric adrenocortical carcinoma (ACC) in southern Brazil. Moreover, it is also associated with high prevalence of breast cancer in women from this region. The native p53TD protein, as well, as the R337H mutant were synthesized and characterized by circular dichroism (CD). The tetramer formed by R337H mutant was unstable, especially at higher pH. In order to stabilize the folding of this mutant, we used two different peptides prepared during the synthesis as ligands for this protein. Previous studies in our group demonstrated that calixarenes displaying guanidinium groups were able to recover the self-assembly of R337H mutant. Therefore, the selected peptides were from the arginine family. The bicyclic peptide, cyclo(Arg- (4&)Phe-Arg-(4&)Phe-D-Pro), was used as ligand of the protein R337H mutant. Unfolding experiments by CD did not show a relevant stabilization of the tetramer formed by the mutant in the presence of this ligand. Nevertheless, a more promising result was obtained for the linear stapled peptide, H-Arg-(4&)Phe-Arg-(4&)Phe-D-Pro- OH. The thermal stability of the protein R337H mutant in the presence of the linear stapled ligand was increased, transition temperature was 7ºC higher. Therefore, we could find an interesting application of one of these peptides synthesized by our previously optimized methodology.
La present tesis es centra en el desenvolupament i optimització d’una metodologia per a la obtenció de pèptids biaril bicíclic mitjançant una estratègia de fase sòlida. Per tal d’obtenir l’anell biaril es van emprar les reaccions de Suzuki-Miyaura. La correcta selecció del grup protectors va permetre expandir la metodologia incorporant aminoàcids tals com lisina, arginina i serina. Es va decidir avaluar algunes propietats farmacològiques dels compostos per tal de conèixer l’efecte d’introduir l’anell biaril. Pel que fa a la permeabilitat per difusió passiva a través de la barrera hematoencefàlica (BHE), va resultar beneficiós incorporar la grapa biaril a l’estructura peptídica. Atenent a la resistència del pèptids a les proteases presents en el sèrum humà, els pèptids que incorporen l’anell biaril van mostrar major resistència. En els assajos de permeabilitat cel·lular duts a terme amb cèl·lules HeLa no es va observar una mortalitat rellevant a una concentració de 500µM. Emprant triptòfans halogenats a diferents posicions del grup indole, es va dur a terme la síntesis de pèptids bicíclic amb una unió carboni-carboni entre les diferents posicions dels triptòfans, trp-trp. Aquests compostos van mostrar valors rellevants de permeabilitat per difusió passiva a través de la BHE. Tanmateix, els valor obtinguts pels pèptids units entre fenilalanines van ser majors, denotant la importància del aminoàcids involucrats en l’enllaç carboni-carboni d’aquests pèptids. Estudis previs del nostre grup van demostrar que l’ús de calixarens amb guanidinis permetia recuperar l’autoensamblatge d’un mutant de la proteïna p53. La proteïna p53 està altament relacionada amb el càncer. El mutant R337H és el més freqüent en el domini de tetramerització i tot i ser capaç de formar un tetràmer, aquest no és estable. Es van seleccionar dos pèptids de la família d’arginines, d’entre ells, el pèptid grapa H-Arg-(4&)Phe-Arg-(4&)Phe-D-Pro, va donar lloc a un resultat interessant. Per dicroisme circular, es va observar una major estabilització del tetràmer format pel mutant en presència del pèptid grapa. De manera que es va obtenir un resultat prometedor amb un compost sintetitzat amb la metodologia prèviament descrita.

Addressing the problem of protein crystallization bottlenecks is a broadly regarded, but complex, research topic. High-throughput screening techniques have been developed in the recent decades to proceed with the investigations in this field. Some of these techniques are regularly used to detect the best crystallizing formulations and form crystals that are suitable for characterization. However, although slow crystallization processes can produce regular (or large) crystals, a faster growth rate is aimed for. Videlicet, gaining a certain control over the nucleation (or crystal growth) rate of proteins would be a breakthrough in modern science. In this work, we focus on developing a low-concentration crystallizing solution and on the design of a novel device to crystallize lysozyme from the above solution within an airdepleted micro-batch environment. It was, de facto, observed, during this project, that the heterogeneous crystallization of lysozyme in standard laboratories and from its conventionally formulated solutions, hardly, would lead to a real understanding of this process. Videlicet, the effect of the solid substrate topographies (or chemistries) on the lysozyme heterogeneous nucleation rate might be altered also by interfering solution factors. Hence, the introduction of a controlled crystallization environment, including surfaces bearing highly controlled features, and the formulation of a low-salt precipitating solution were necessary. This would also mean that, although, so far, a variety of surface effects on the crystallization of conventionally formulated protein solutions have been observed, a far better characterization of protein heterogeneous nucleation, and crystal growth, could be attained. Videlicet, reducing the local effect of interfering, or uncontrollable, factors, almost certainly, would lead to a better definition, and control, of the protein nucleation dynamics. Also, for the same reason, it is pivotal that substrates with strictly controlled chemistries (or topographies) are employed. Indeed, gaining a real understanding of the criteria that govern the protein heterogeneous nucleation would be the ultimate scope of any work in this research field.

The main focus of this thesis has been directed towards developing novel surface-confined transition metal complexes for applications in heterogeneous catalysis and for the preparation of anisotropic particle surfaces. The first part describes the heterogenization of a homogeneous transition metal-based catalyst tetraphenyl cobalt porphyrin (CoTPP) on silicon wafers and on silica particles. The activity in hydroquinone oxidation for the silica particle-immobilized CoTPPs was found to be increased 100-fold compared to its homogeneous congener whereas the silicon wafer-immobilized CoTPPs achieved lower activity due to the formation of clusters of catalyst molecules on the support surface as detected with atomic force microscopy (AFM). The second part of this thesis describes the development and characterization of anisotropic particle-surfaces by electrochemical site-specific oxidation of surface-confined thiols. Reactive patches or gold gradients could be obtained on the particle surfaces depending on the type of working electrode used and on the electrolyte composition. The particle surface functionalities were characterized with X-ray photoelectron spectroscopy (XPS) and the particle-surface-confined patches and gradients were conjugated with proteins to obtain fluorescence for investigation using fluorescence microscopy. Gold-functionalized siliceous mesocellular foams were further demonstrated to be highly efficient and selective catalysts in the cycloisomerization of 4-alkynoic acids to lactones. The final part of this thesis describes the preparation and characterization of palladium nanoparticles heterogenized in the pores of siliceous mesocellular foam. The nanoparticles were analyzed with transmission electron microscopy (TEM) and found to have a size of 1-2 nm. Primary- and secondary benzylic- and allylic alcohols were oxidized by the heterogeneous palladium nanoparticles in high to excellent yields using air atmosphere as the oxygen source. The nanopalladium catalyst was used up to five times without any decrease in activity and the size of the nanoparticles was retained according to TEM.

At the time of doctoral defence the following paper were unpublished and had a status as follows: Paper1: Manuscript; Paper 4: Manuscript

Ces travaux de recherche s'inscrivent dans le cadre du développement de nouvelles surfaces biocompatibles capables de contrôler l'adhésion d'agents pathogènes responsables de maladies neurodégénératives telles que les maladies de Creutzfeld Jacob, Alzheimer, Parkinson et Lewis. Deux axes de recherche ont été privilégiés. Notre approche se focalise en amont des dosages sur l'amélioration des procédures de stockage des prélèvements biologiques réalisés dans des tubes de type Eppendorf. Ces tubes en polypropylène induisent une perte du matériel génétique de plus de 70% accentuant la faible concentration en agent pathogène pour la détection immunoenzymatique. Dans le but de réduire les phénomènes indésirables d'adhésion des agents pathogènes à la surface des supports de stockage, deux voies de traitement ont été envisagées dans ce travail de thèse. La première consiste à modifier la surface du tube Eppendorf en une étape par décharge plasma fluoré, la seconde à créer de nouvelles surfaces hydrophiles en deux étapes couplant la technique des plasmas froids au greffage de polymères, les agents pathogènes pouvant être hydrophiles ou hydrophobes. Avec cette dernière technique, une voie originale a été abordée de part l'utilisation de solutions de greffage complexes composées à la fois de polymères et de molécules tensioactives. Les surfaces ainsi obtenues présentent une nano-structuration. Toutes les étapes de modification de la surface interne des tubes de stockage ont été caractérisées. Ces surfaces sont alors décrites selon leur caractère hydrophile ou hydrophobe grâce à la détermination des énergies de surface polaire et apolaire, selon leur charge de surface obtenue par mesure du potentiel d'écoulement, selon leur composition chimique déterminée par spectroscopie à photoélectrons X (XPS) et enfin selon leur topographie et leur rugosité relevées par microscopie à force atomique (AFM). Les interactions entre les groupements fonctionnels ainsi obtenus à la surface des tubes de stockage après les divers traitements et les protéines antigéniques considérées ont été interprétées en se référant aux différents modèles de l'adhésion pour des gammes de pH proches des protocoles biologiques usuels. Afin de s'assurer que ces nouvelles surfaces permettent bien une diminution de l'adhésion des agents infectieux sur la paroi interne des tubes de polypropylène, des analyses immunoenzymatiques ont été réalisées au sein des centres hospitaliers participant au projet STREP NEUROSCREEN n° LSHB-CT 2006-03 7719 (CRPP de Liège et CHU de Lyon). Ces analyses ont permis de montrer que la modification des surfaces entraîne une diminution de l'absorption des agents pathogènes jusqu'à 100% permettant ainsi une meilleure détection.

Mucous membranes are covered with mucus, a viscoelastic hydrogel that plays an essential role in their protection from shear and pathogens. The viscoelasticity of mucus is owing to mucins, a group of densely glycosylated proteins. Mucins can interact with a wide range of surfaces; thus, there is big interest in exploring and manipulating such interactions for biomedical applications. This thesis presents investigations of mucin interactions with hydrophobic surfaces in order to identify the key features of mucin lubricity, as well as describes the development of materials that are optimized to interact with mucins.   In Paper I we investigated the domains which make mucins outstanding boundary lubricants. The results showed that the hydrophobic terminal domains of mucins play a crucial role in the adsorption and lubrication on hydrophobic surfaces. Specifically, protease digestion of porcine gastric mucins and salivary mucins resulted in the cleavage of these domains and the loss of lubricity and surface adsorption. However, a “rescue” strategy was successfully carried out by grafting hydrophobic phenyl groups to the digested mucins and enhancing their lubricity. This strategy also enhanced the lubricity of polymers which are otherwise bad lubricants.   In Paper II we developed mucoadhesive materials based on genetically engineered partial spider silk proteins. The partial spider silk protein 4RepCT was successfully functionalized with six lysines (pLys-4RepCT), or the Human Galectin-3 Carbohydrate Recognition Domain (hGal3-4RepCT). These strategies were aiming to either non-specific electrostatic interactions between the positive lysines and the negative mucins, or specific binding between the hGal3 and the mucin glycans. Coatings, fibers, meshes and foams were prepared from the new silk proteins, and the adsorption of porcine gastric mucins and bovine submaxillary mucins was measured, demonstrating enhanced adsorption.   The work presented demonstrates how mucin-material interactions can provide us with valuable information for the development of new biomaterials. Specifically, mucin-based and mucin-inspired lubricants could provide desired lubrication to a wide range of surfaces, while our new silk based materials could be valuable tools for the development of mucosal dressings.
Slemhinnor täckts av slem, en viskoelastisk hydrogel som spelar en viktig roll för att skydda mot mekanisk nötning och patogener. Muciner, en grupp av tätt glykosylerade proteiner, spelar en viktig roll i viskoelasticiteten av slem. Eftersom muciner kan interagera med diverse ytor är det av stort intresse att utforska och manipulera sådana interaktioner för biomedicinska tillämpningar. Denna avhandling presenterar undersökningar av mucininteraktioner med hydrofoba ytor för att identifiera de viktigaste egenskaperna hos mucinsmörjning, samt beskriver utveckling av material som optimerades för att interagera med muciner.   I Artikel I undersökte vi de domäner som bidrar till  mucinernas enastående kapacitet som smörjmedel. Resultaten visade att mucinernas hydrofoba terminaldomäner spelar en avgörande roll vid adsorption och smörjning på hydrofoba ytor. Mer specifikt, proteasklyvning av svinmagemuciner och salivmuciner resulterade i klyvningen av dessa domäner och förlust av smörjning och ytadsorption. Genom att länka hydrofobiska fenylgrupper till de uppbrutna mucinerna, lyckades deras smörjningsegenskaper förbättras. Denna strategi förbättrade också smörjningsegenskaper hos andra polymerer som annars har  dåliga smörjningsegenskaper.   I Artikel II utvecklade vi mukoadhesiva material baserade på genetiskt modifierade partiella spindelsilkeproteiner. Spindelsilkeproteinet 4RepCT funktionaliserades framgångsrikt med tillsats av sex lysiner (pLys-4RepCT), eller den mänskliga Galectin-3 karbohydrat igenkänningsdomänen (hGal3-4RepCT). Syftet med dessa strategier var antingen att öka ospecifika elektrostatiska interaktioner mellan de positiva lysinerna och de negativa mucinerna, eller den specifika bindningen mellan hGal3 och mucin-glykanerna. Beläggningar, fibrer, nät och skum framställdes från de nya silkeproteinerna. Efter att adsorption av svinmagsmuciner och bovina submaxillära muciner uppmätts, visade de nya silkeproteinerna förbättrad mucin adsorption.   Detta arbete visar hur interaktioner mellan mucin-material kan bidra med värdefull information för utvecklingen av nya biomaterial. Mucinbaserade och mucininspirerade smörjmedel kan ge önskad smörjning till ett brett spektrum av ytor, medan vår nya silkesbaserad material kan vara ett värdefullt verktyg för utvecklingen av slemhinneförband.

QC 20190412

In this work we studied protein adsorption on chemically well-controlled surfaces. The focus is put on linking physico-chemical properties of surfaces (hydrophobicity/charge) to the structural properties of the adsorbed proteins. To this end, alkyl thiols differing by their end group were used to build self-assembled monolayers on gold substrates (SAM) that serve as templates for protein adsorption or covalent grafting. SAM surfaces before and after protein adsorption were characterized with a combination of techniques. Ex situ analysis were carried out, in air with polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), or in vacuum using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). ToF-SIMS results were analyzed statistically in principal component analysis (PCA) to reveal preferential orientations based on amino acids fragments distributions. Protein adsorption was also followed directly in situ (i.e. in the liquid phase) with quartz crystal microbalance with dissipation monitoring (QCM-D). Two model proteins - β-Lactoglobulin (βLG) and bovine serum albumin (BSA) - were first studied. They are both model globular proteins with different structural properties (βLG is hard while BSA is soft). Different orientations were proposed for both proteins on each SAM surface. A more complex case was then studied with the adsorption and grafting of a monoclonal antibody on the SAM. Again differences in orientations were determined and correlated to biorecognition measurements. In conclusion, this thesis establishes a methodology for the direct label free determination of protein orientation on surfaces.

Les interactions protéine-protéine jouent un rôle primordial dans de nombreux processus biologiques. L’importance de ces interactions a suscité le développement de nouvelles approches thérapeutiques qui ciblent ces complexes protéiques. Nous nous proposons d’inhiber ces interactions en élaborant une stratégie de reconnaissance de surfaces de protéines par des molécules synthétiques de taille intermédiaire, les foldamères d’oligoquinoline. Ces composés se replient en des structures hélicoïdales stables dont chaque élément constitutif peut être fonctionnalisé pour permettre des propriétés de reconnaissance de surface de protéine.Afin de valider ce concept, l’interaction entre l’anhydrase carbonique humaine de type II (HCAII) et son inhibiteur N-benzyl-4-sulphamoylbenzamide (SBB) a été sélectionnée comme système modèle. Plusieurs étapes de synthèse ont permis de concevoir de nouveaux foldamères capables de former un complexe avec l’enzyme par l’intermédiaire de l’inhibiteur SBB et d’un espaceur approprié. Chaque complexe protéine-foldamère a été co-cristallisé et l’affinité des interactions a été caractérisée par dichroïsme circulaire induit et par résonance plasmonique de surface. Ce concept a ensuite été appliqué à une interaction protéine-protéine d’intérêt thérapeutique, le complexe IL-4/IL-4R, dans le cadre du programme européenFOLDAPPI (FP7-PEOPLE-IAPP-2008)
Protein-protein interactions play key roles in many biological processes as well as in many diseases. The importance of these interactions has led to the development of new therapeutic approaches that target protein interfaces. We have developed a protein surface recognition strategy to inhibit protein-protein interactions by using intermediate size organicmolecules called oligoquino line foldamers, that result in very stable and well defined helical structures. These helical backbones are used as templates within each building block can be modulated to allow protein surface recognition.In order to validate this concept, the well-characterized interaction between the enzyme human carbonic anhydrase II (HCAII) and its N-benzyl-4-sulphamoylbenzamide (SBB) inhibitor was selected as a model system. Multi-steps synthesis allowed functionalization of new foldamers able to bind to the enzyme through the SBB inhibitor attached by a spacer.Each foldamer–protein complex was cocrystallized and the affinity of the interactions was assayed using both induced circular dichroïsm and surface plasmon resonance. The concept of using a foldamer against protein-protein interaction was then applied to a protein complex of therapeutic interest, IL-4/IL-4R, within the European FOLDAPPI program (FP7-PEOPLEIAPP- 2008)

In this thesis, different aspects within the research field of protein spectro- and electro-chemistry on nanostructured materials are addressed. On the one hand, this work is related to the investigation of nanostructured transparent and conductive metal oxides as platform for the immobilization of electroactive enzymes. On the other hand the second part of this work is related to the immobilization of sulfite oxidase on gold nanoparticles modified electrode. Finally direct and mediated spectroelectrochemistry protein with high structure complexity such as the xanthine dehydrogenase from Rhodobacter capsulatus and its high homologues the mouse aldehyde oxidase homolog 1. Stable immobilization and reversible electrochemistry of cytochrome c in a transparent and conductive tin-doped and tin-rich indium oxide film with a well-defined mesoporosity is reported. The transparency and good conductivity, in combination with the large surface area of these materials, allow the incorporation of a high amount of electroactive biomolecules (between 250 and 2500 pmol cm-2) and their electrochemical and spectroscopic investigation. Both, the electrochemical behavior and the immobilization of proteins are influenced by the geometric parameters of the porous material, such as the structure and pore shape, the surface chemistry, as well as the protein size and charge. UV-Vis and resonance Raman spectroscopy, in combination with direct protein voltammetry, are employed for the characterization of cytochrome c immobilized in the mesoporous indium tin oxide and reveal no perturbation of the structural integrity of the redox protein. A long term protein immobilization is reached using these unmodified mesoporous indium oxide based materials, i.e. more than two weeks even at high ionic strength. The potential of this modified material as an amperometric biosensor for the detection of superoxide anions is demonstrated. A sensitivity of about 100 A M-1 m-2, in a linear measuring range of the superoxide concentration between 0.13 and 0.67 μM, is estimated. In addition an electrochemical switchable protein-based optical device is designed with the core part composed of cytochrome c immobilized on a mesoporous indium tin oxide film. A color developing redox sensitive dye is used as switchable component of the system. The cytochrome c-catalyzed oxidation of the dye by hydrogen peroxide is spectroscopically investigated. When the dye is co-immobilized with the protein, its redox state is easily controlled by application of an electrical potential at the supporting material. This enables to electrochemical reset the system to the initial state and repetitive signal generation. The case of negative charged proteins, which does not have a good interaction with the negative charged indium oxide based films, is also explored. The modification of an indium tin oxide film with a positive charged polymer and the employment of a antimony doped tin oxide film were investigated in this work in order to overcome the repulsion induced by similar charges of the protein and electrode. Human sulfite oxidase and its separated heme-containing domain are able to direct exchange electrons with the supporting material. A study of a new approach for sulfite biosensing, based on enhanced direct electron transfer of a human sulfite oxidase immobilized on a gold nanoparticles modified electrode is reported. The spherical gold nanoparticles were prepared via a novel method by reduction of HAuCl4 with branched poly(ethyleneimine) in an ionic liquid resulting in particles of about 10 nm in hydrodynamic diameter. These nanoparticles were covalently attached to a mercaptoundecanoic acid modified Au-electrode and act as platform where human sulfite oxidase is adsorbed. An enhanced interfacial electron transfer and electrocatalysis is therefore achieved. UV-Vis and resonance Raman spectroscopy, in combination with direct protein voltammetry, were employed for the characterization of the system and reveal no perturbation of the structural integrity of the redox protein. The proposed biosensor exhibited a quick steady-state current response, within 2 s and a linear detection range between 0.5 and 5.4 μM with high sensitivity (1.85 nA μM-1). The investigated system provides remarkable advantages, since it works at low applied potential and at very high ionic strength. Therefore these properties could make the proposed system useful in the development of bioelectronic devices and its application in real samples. Finally protein with high structure complexity such as the xanthine dehydrogenase from Rhodobacter capsulatus and the mouse aldehyde oxidase homolog 1 were spectroelectrochemically studied. It could be demonstrated that different cofactors present in the protein structure, like the FAD and the molybdenum cofactor, are able to directly exchange electrons with an electrode and are displayed as a single peak in a square wave voltammogram. Protein mutants bearing a serine substituted to the cysteines, bounding to the most exposed iron sulfur cluster additionally showed direct electron transfer which can be attributable to this cluster. On the other hand a mediated spectroelectrochemical titration of the protein bound FAD cofactor was performed in presence of transparent iron and cobalt complex mediators. The results showed the formation of the stable semiquinone and the fully reduced flavin. Two formal potentials for each single electron exchange step were then determined.
In dieser Arbeit werden verschiedenen Aspekte im Forschungsfeld der Protein-Spekro- und Elektro-Chemie an nanostrukturierte Materialien behandelt. Zum einen werden in dieser Arbeit nanostrukturierte, transparente und leitfähige Metalloxide als Basis für die Immobilisierung von elektroaktiven Enzym untersucht. Des Weiteren behandelt diese Arbeit die Immobilisierung von humaner Sulfitoxidase auf einer Gold-Nanopartikel-modifizierten Elektrode. Schließlich wird die direkte und die vermittelte Elektrochemie von Xanthindehydrogenase aus Rhodobacter capsulatus und Aldehydoxidase Homolog 1, aus Mause, vorgestellt. Im ersten Teil der Arbeit wird über die stabile Immobilisierung und reversible Elektrochemie von Cytochrom c in einem transparenten und leitfähigen Zinn-dotierten und Zinn-reichen Indiumoxid Film mit einer gut definierten Mesoporosität berichtet. Die Transparenz und gute Leitfähigkeit in Kombination mit der großen Oberfläche dieser Materialien erlauben die Inkorporation einer große Menge elektroaktiver Biomoleküle (zwischen 250 und 2500 pmol cm-2) und deren elektrochemische und spektroskopische Untersuchung. Das elektrochemische Verhalten und die Proteinimmobilisierung sind durch die geometrischen Parameter des porösen Materials, wie die Struktur und Porenform, die Oberflächenchemie, sowie die Größe und Ladung des Proteins beeinflusst. UV-Vis und Resonanz-Raman-Spektroskopie in Kombination mit direkter Protein-Voltammetrie werden für die Charakterisierung von Cytochrom c eingesetzt und zeigen keine Störung der strukturellen Integrität des Redox-Proteins durch die Immobilisierung. Eine langfristige Immobilisierung des Proteins von mehr als zwei Wochen auch bei hoher Ionenstärke wurde unter Verwendung dieser unmodifizierten mesoporösen Indiumoxid-basierten Materialien erreicht. Das Potential dieses modifizierten Materials für die Verwendung in einem amperometrischen Biosensor zum Nachweis von Superoxid-Anionen wurde aufgezeigt. Es wurde eine Empfindlichkeit von etwa 100 A M-1 m-2, in einem linearen Messbereich der Superoxidkonzentration zwischen 0,13 und 0,67 µM, erreicht. Außerdem wurde ein elektrochemisch umschaltbares Protein-basiertes optisches Gerät konzipiert mit Cytochrom c und der mesoporösen Indiumzinnoxidschicht. Ein redox-sensitiver Farbstoff wurde als schaltbare Komponente des Systems verwendet. Die Cytochrom c Oxidation des Farbstoffs durch Wasserstoffperoxid wurde spektroskopisch untersucht. Der Redox-Zustand des Farbstoffs, co-immobilisiert mit dem Protein, ist leicht durch das Anlegen eines elektrischen Potentials an das Trägermaterial kontrollierbar. Dadurch wird die elektrochemische Zurücksetzung des Systems auf den Anfangszustand und eine repetitive Signalerzeugung ermöglicht. Für negativ geladene Proteine, die keine gute Interaktion mit dem negativ geladenen Indiumoxid-basierten Film zeigen wurden die Modifikation der Indiumzinnoxidschicht mit einem positiv geladenen Polymer sowie die Verwendung eines Antimon-dotierten Zinnoxid Films vorgeschlagen. Dadurch konnte die Abstoßung induziert durch die ähnliche Ladung des Proteins und der Elektrode überwunden werden. Es gelang für die humane Sulfit-Oxidase und die separate Häm-haltige Domäne der Austausch von Elektronen mit dem Trägermaterial. Im zweiten Teil der Arbeit wird über eine neue Methode für die Biosensorik von Sulfit berichtet, bei der direkte Elektronentransfer von humaner Sulfitoxidase immobilisierten auf einer mit Gold-Nanopartikeln modifizierten Elektrode verstärkt wurde. Die sphärischen Gold-Nanopartikeln, von etwa 10 nm im Durchmesser, wurden über eine neue Methode durch Reduktion von HAuCl4 mit verzweigtem Polyethylenimin in einer ionischen Flüssigkeit synthetisiert. Diese Nanopartikel wurden kovalent an eine mit Mercaptoundecansäure modifizierten Gold-Elektrode immobilisiert und dienen als Basis für die Adsorption von Sulfitoxidase adsorbiert wurde. Dadurch wurde ein schneller heterogener Elektronen-Transfer und verbesserte Elektrokatalyse erreicht. Für die Charakterisierung des verwendeten Systems eingesetzt wurden UV-Vis und Resonanz-Raman-Spektroskopie in Kombination mit direkter Protein-Voltammetrie. Es wurde keine Störung der strukturellen Integrität des Redox-Proteins beobachtet. Der vorgeschlagene Biosensor zeigte eine schnelle steady-state Stromantwort innerhalb von 2 s, eine lineare Detektion im Bereich zwischen 0,5 und 5,4 µM Sulfit mit einer hohen Empfindlichkeit (1,85 nA µM-1). Das untersuchte System bietet bemerkenswerte Vorteile da es ermöglicht bei niedriger angelegter Spannung und bei sehr hoher Ionenstärke zu arbeiten. Aufgrund dieser Eigenschaften hat das vorgeschlagene System großes Potential für die Entwicklung von bioelektronischen Geräten und der Anwendung in realen Proben. Schließlich werden im letzten Teil der Arbeit die komplexeren Enzymen Xanthindehydrogenase aus Rhodobacter capsulatus und Maus Aldehydoxidase Homolog 1 spektro- und elektrochemisch untersucht. Es konnte gezeigt werden, dass verschiedene Kofaktoren in der Proteinstruktur, wie FAD und der Molybdän Kofaktor direkt Elektronen mit einer Elektrode austauschen können, was durch einzelne Peaks im Square Wave Voltammogramm angezeigt wird. Es konnte eine zusätzliche redoxaktive Gruppe mit direktem Elektronen-Transfer nach Austausch eines Cysteins durch Serin am exponierten Eisen-Schwefel-Cluster gezeigt werden. Außerdem wurde eine vermittelte spektroelektrochemische Titration des FAD-Kofaktors in Anwesenheit von Mediatoren der Klasse der Eisen und Kobalt-Komplexe durchgeführt. Die Ergebnisse zeigen, dass FAD in R. capsulatus XDH zu einem stabilen Semichinone reduziert werden kann. Es gelang die formalen Potentiale für die zwei einzigen Elektrontransferprozesse zu bestimmen.

The thesis presents an investigation of the nano-patterning of hydrogenated amorphous carbon (a-C:H) surfaces to control protein adsorption. The relevant literature is first reviewed, noting the link between protein adsorption and mis-folding and its relevance to bio-compatibility and nano-toxicity. It then identifies how nano-topography influences protein adsorption, the debates and conflicts in the literature regarding this effect and the issues associated with controlling nanotopography independently of local surface composition. The first experimental chapter deals with the preparation and analysis of a-C:H patterns made by focused ion beam (FIB) milling and atomic force microscopy (AFM) nanoindentation. These methods resulted in nano-patterns with 2 nm height amplitude and 60 nm spacing, hence of size commensurate with that of proteins. The challenges associated with the production of such patterns are discussed, particularly the analysis and simulation of the implanted gallium profile in the FIB patterns. Advanced AFM techniques were used to investigate the possible compositional nature of the patterns. The Interleave/Lift method detected an onset of long range interactions between a protein coated tip and the patterned surfaces at a 38 nm tip-surface distance for both patterning methods. A hill/valley compositional contrast was also noted, stronger in the case of the FIB pattern. Short range adhesive tip/surface forces were mapped with the digital pulse force method (DPFM). Again, this showed stronger compositional contrast for the FIB pattern. These effects were interpreted as arising from the electrostatic interactions between the negatively charged protein coated tip and the patterned surfaces. Using SRIM modelling and the measured contrast values, a negative charge per adsorbed protein of 1.3-3e was estimated. The second half of the thesis investigates protein adsorption on a-C:H for four different protein/solvent systems; bovine serum albumin (BSA) and bovine plasma fibrinogen (BPF) in de-ionized (DI) water or phosphate buffer saline (PBS) solutions. Fourier transform infrared (FTIR) analysis revealed that there is a significant change in the secondary structure of the proteins once they adsorbed onto a-C:H, corresponding to an increase of the 0 -sheet component, often associated with exposure of the buried hydrophobic groups. This is also consistent with the large surface footprint of the adsorbed proteins, measured by AFM microscopy. Adsorption on FIB-patterned surfaces reveals changed adsorption behaviours, with significant increases in adsorbed foot-prints for all systems expect for the BSA-DI system where this footprint decreases slightly. Finally, adsorption experiments were carried out on patterns made by the FIB and AFM techniques. This comparison indicates that, for the FIB pattern, BSA adsorbs preferentially in the valleys whereas, for the AFM pattern, it resides on the hills. This effect, consistent with the previous analysis, was attributed to the buried charges in the valleys of the FIB pattern. Overall, the work presented in this thesis showed that nano-patterned a-C:H model surfaces are useful to study and control protein adsorption, suggesting that, in the case studied here, nano-topography modifies qualitatively the adsorption process. In addition, the methods developed here can be extended to other patterning techniques and protein systems to study independently the influence of topography and composition on protein adsorption.

Accumulating evidence indicates that oxygen free radicals are involved in many diseases and pathological conditions, such as aging, inflammation, reperfusion damage of ischemic tissue and various cardiovascular diseases. Extracellular superoxide dismutase (ECSOD) thus plays a major role in the maintenance of cells by providing protection against these toxic substances in the extracellular space. Various animal studies have shown that ECSOD has the ability to protect against many of these disorders, and interest has therefore evolved in the potential therapeutic use of the enzyme. However, despite strenuous efforts, large-scale production of the enzyme has not been achieved. To overcome this problem, a mimic of the enzyme, PseudoECSOD, has previously been constructed. This chimera is easy to produce in large amounts and has all the structural, enzymatic and heparin-binding characteristics of ECSOD, making it a potential substitute for ECSOD in therapeutic situations. However, the copper content of PseudoECSOD has been shown to be rather low, and since the copper ion is very important for the catalytic function of the enzyme, a production system that utilizes a copper chaperone for proper insertion of copper into the active site of the enzyme was constructed. The results show that the copper content of PseudoECSOD produced by this system is close to 100 %. In order to use PseudoECSOD therapeutically, further investigations of its binding capability and protective properties are needed. Therefore, the binding of ECSOD and PseudoECSOD to heparin was investigated using isothermal titration calorimetry. The results show that although some purely ionic interactions are important for the binding between ECSOD and heparin, there is also a substantial contribution from non-ionic interactions. The investigation also showed that the C-terminal domain is the only part of ECSOD that contributes to productive binding, and that the binding of PseudoECSOD and ECSOD to heparin is similar. In addition, analysis of mutant proteins strongly indicated that the amino acids R210, K211 and R214 are important for optimal binding of ECSOD to heparin, accounting for about 30 % of the total binding energy. The structural placement of these amino acids in an α-helix also confirms the hypothesis postulated by Margalit et al., that a common structural motif for heparin-binding proteins may be two positively charged amino acids at a distance of approximately 20 Å in the 3D-structure, facing opposite directions of a α-helix. The importance of these residues was also confirmed by analysis of a phage display library of the C-terminal domain of ECSOD. The binding of PseudoECSOD to heparan sulfate on cell surfaces of two different cell types, HepG2 and endothelial cells, was also investigated. The results clearly show that PseudoECSOD binds to these cells in a very similar manner to ECSOD. To investigate the protective properties of PseudoECSOD against ischemia-reperfusion injuries, an isolated rabbit heart model was used. The results indicate that the enzyme has a protective effect. However, more experiments using the rabbit heart and other animal models are needed to identify the optimal dose for protective purposes. The protective properties of PseudoECSOD in human tissue should also be thoroughly investigated. In summary, the findings in these studies, together with earlier results showing the close resemblance of PseudoECSOD to ECSOD in structural, enzymatic and heparin-binding properties, further support the proposition that PseudoECSOD may be a good substitute for ECSOD to use in therapeutic interventions.

Cell adhesion to proteins adsorbed onto implanted surfaces is particularly important to host responses in biomedical and tissue engineering applications. Biomaterial surface properties influence the type, quantity and functional presentation (activity) of proteins adsorbed upon contact with physiological fluids, and modulate subsequent cell response. Cell adhesion to extracellular matrix proteins (e.g. fibronectin) is primarily mediated by the integrin family of cell-surface receptors. Integrins not only anchor cells, supporting cell spreading and migration, but also trigger signals that regulate survival, proliferation and differentiation. A fundamental understanding of the adhesive interactions at the biomaterial interface is critical to the rational design of biomaterial surfaces. Using model surfaces of self-assembled monolayers of alkanethiols on gold presenting well-defined surface chemistries (CH3, OH, COOH, NH2), we investigated the effects of surface chemistry on osteoblastic differentiation. We report that surface chemistry effectively modulates fibronectin adsorption, integrin binding, focal adhesion assembly and signaling to direct the osteoblast cellular functions of adhesion strength, gene expression and matrix mineralization. Specifically, surfaces presenting OH and NH2 functionalities provide enhanced functional presentation of adsorbed fibronectin, promoting specificity of integrin binding as well as elevating focal adhesion assembly and signaling. Furthermore, the OH and NH2 surfaces supported elevated levels of osteoblast differentiation as evidenced by osteoblast-specific gene expression and matrix mineralization. These results contribute to the development of design principles for the engineering of surfaces that direct cell adhesion for biomedical and tissue engineering applications. In particular, the understanding provided by this analysis may be useful in the engineering of surface properties for bone tissue repair and regeneration.

The interaction of proteins with surfaces is a major problem involved in protein microarrays. Understanding protein/surface interactions is key to improving the performance of protein microarrays, but current understanding of the behavior of proteins on surfaces is lacking. Prevailing theories on the subject, which suggest that proteins should be stabilized when tethered to surfaces, do not explain the experimentally observed fact that proteins are often denatured on surfaces. In an attempt to develop some predictive capabilities with respect to protein/surface interactions, it was asked in previous works if the stabilization/destabilization of proteins on surfaces could be correlated to secondary structure and found that no link existed. However, further investigation has revealed that proteins with similar tertiary structure show predictable stabilization patterns. In this research, it is reported how five, alpha-helical, orthogonal-bundle proteins behave on the surface compared to the bulk. By measuring stabilization using melting temperatures and the Gibbs energies of folding, it is shown that the stability of proteins tethered to surfaces can be correlated to the shape of the loop region where the tether is placed and the free rotation ability of the part of proteins near surfaces. It is also shown that any destabilization that occurs because of the surface is an enthalpic effect and that surfaces always stabilize proteins entropically. Furthermore, the entropical stabilization effect comes from unfolded states of the tethered protein, while the enthalpical destabilization effect is from the folded states of protein. A further analysis of surface induced change of folding mechanism is also studied with a multi-state protein 7LZM in this research. The result showed that by tethering a protein on a surface, the melting temperature of part of the protein changed, which leads to a miss of state.

Healthy articular joints exhibit remarkable lubrication due in large part to the complex rheological and tribological behavior of the synovial fluid (SF) that lubricates the joints. Current approaches that seek to elucidate such remarkable lubrication usually focus on the roles of high molecular weight SF components such as lubricin and hyaluronic acid but frequently overlook the role of serum albumin (SA), although it represents 90% of the protein content of SF. In this thesis, we used the Surface Forces Apparatus to investigate in detail the structural and tribological response of SA thin films when sheared between model surfaces and subjected to a large range of shearing parameters. Our data indicate that, under shear, SA films reproduce closely the shear response previously reported for SF, i.e., film thickening and formation of numerous long-lived aggregates accompanied by low friction and efficient surface protection against damage. More specifically, our detailed investigation of shear parameters reveals that (i) strong anchoring of SA to surfaces promotes the formation of large rod-like shaped aggregates that enable rolling friction and keep surfaces far apart, preventing damage, (ii) aggregation mechanism is irreversible, which makes aggregates long-lived (though mobile) in the contact, and (iii) aggregate formation only occur when SA was sheared above a ‘critical’ amplitude Ac and a critical shear velocity Vc. Collectively, our results provide experimental evidence of the role of globular proteins, such as SA, in lubrication and establish a correlation between shearing parameters, formation and stability of aggregates, low friction and wear protection. Although our findings are based on experiments involving rigid, nonporous surfaces hence can hardly be generalized to compliant and porous cartilage surfaces, they are applicable to other rigid tribosystems such as artificial joints and will certainly advance our understanding of joint implants’ lubrication in SF mediated by protein aggregation, with implications for future design of artificial joints and therapeutic interventions.

Proteins are involved in numerous functions in the human body, including chemical transport, molecular recognition, and catalysis. To perform their function most proteins must adopt a specific structure (often referred to as the folded structure). A microscopic description of folding is an important prerequisite for elucidating the underlying basis of protein misfolding and rational drug design. However, protein folding occurs on heterogeneous length and time scales, presenting a grand challenge to both experiments and simulations. In computer simulations, challenges are generally mitigated by adopting coarse-grained descriptions of the physical environment, employing enhanced sampling strategies, and improving computing code and hardware. While significant advances have been made in these areas, for numerous systems a large spatiotemporal gap between experiment and simulations still exists, due to the limited time and length scales achieved by simulation, and the inability of many experimental techniques to probe fast motions and short distances. In this thesis, kinetic transition networks (KTNs) are constructed for various protein folding systems, via approaches based on the potential energy landscape (PEL) framework. By applying geometry optimisation techniques, the PEL is discretised into stationary points (i.e.~low-energy minima and the transition states that connect them). Essentially, minima characterise the low-lying regions of the PEL (thermodynamics) and transition states encode the motion between these regions (dynamics). Principles from statistical mechanics and unimolecular rate theory may then be employed to derive free energy surfaces and folding rates, respectively, from the KTN. Furthermore, the PEL framework can take advantage of parallel and distributed computing, since stationary points from separate simulations can be easily integrated into one KTN. Moreover, the use of geometry optimisation facilitates greater conformational sampling than conventional techniques based on molecular dynamics. Accordingly, this framework presents an appealing means of probing complex processes, such as protein folding. In this dissertation, we demonstrate the application of state-of-the-art theory, combining PEL analysis and KTNs to three diverse protein systems. First, to improve the efficiency of protein folding simulations, the intrinsic rigidity of proteins is exploited by implementing a local rigid body (LRB) approach. The LRB approach effectively integrates out irrelevant degrees of freedom from the geometry optimisation procedure and further accelerates conformational sampling. The effects of this approach on the underlying PEL are analysed in a systematic fashion for a model protein (tryptophan zipper\,1). We demonstrate that conservative local rigidification can reproduce the thermodynamic and dynamic properties for the model protein. Next, the PEL framework is employed to model large-scale conformational changes in proteins, which have conventionally been difficult to probe in silico. Methods based on geometry optimisation have proved useful in overcoming the broken ergodicity issue, which is associated with proteins that switch morphology. The latest PEL-based approaches are utilised to investigate the most extreme case of fold-switching found in the literature:~the α-helical hairpin to β-barrel transition of the C-terminal domain of RfaH, a bacterial transcription factor. PEL techniques are employed to construct the free energy landscape (FEL) for the refolding process and to discover mechanistic details of the transition at an atomistic level. The final part of the thesis focuses on modelling intrinsically disordered proteins (IDPs). Due to their inherent structural plasticity, IDPs are generally difficult to characterise, both experimentally and via simulations. An approach for studying IDPs within the PEL framework is implemented and tested with various contemporary potential energy functions. The cytoplasmic tail of the human cluster of differentiation 4 (CD4), implicated in HIV-1 infection, is characterised. Metastable states identified on the FEL help to unify, and are consistent with, several earlier predictions.

Dans la cellule, les protéines évoluent dans un environnement très dense et interagissent ainsi avec un grand nombre de partenaires spécifiques et non-spécifiques qui entrent en compétition. L’objectif de ma thèse est de caractériser les propriétés physiques et évolutives des surfaces protéiques pour comprendre comment la pression de sélection s’exerce sur les protéines, façonnant leurs interactions et régulant ainsi cette sévère compétition.Pour cela, j’ai développé une méthodologie permettant de caractériser la propension des protéines à interagir avec les protéines de leur environnement, par des approches de docking. La cartographie moléculaire permettant la visualisation et la comparaison des propriétés de la surface des protéines, j’ai donc mis en place un nouveau cadre théorique basé sur une représentation des paysages énergétiques d'interaction par des cartes d'énergies. Ces cartes (en deux dimensions) reflètent de manière synthétique la propension des surfaces protéiques à engager des interactions avec d’autres protéines. Elles sont donc d’un grand intérêt pratique pour déterminer les régions des surfaces protéiques les plus enclines à engager des interactions avec d’autres molécules.Ce nouveau cadre théorique a permis de montrer que les surfaces des protéines comprennent des régions de différents niveaux d'énergies de liaison (régions chaudes, intermédiaires et froides pour les régions d'interaction favorables, intermédiaires et défavorables respectivement).Une partie importante de la thèse a consisté à caractériser les propriétés physico-chimiques et évolutives de ces différentes régions. L'autre partie a consisté à appliquer cette méthode sur plusieurs systèmes : complexes homomériques, protéines du cytosol de S. cerevisiae, familles d'interologues. Ce travail ouvre la voie à un grand nombre d'applications en bioinformatique structurale, telles que la prédiction de sites de liaison, l’annotation fonctionnelle ou encore le design de nouvelles interactions.En conclusion, la stratégie mise en place lors de ma thèse permet d’explorer la propension d’une protéine à interagir avec des centaines de partenaires d'intérêts, et donc d'investiguer le comportement d’une protéine dans un environnement cellulaire spécifique. Cela va donc au-delà de l'utilisation classique du docking "binaire" puisque notre stratégie fournit une vision systémique des interactions protéiques à l’échelle des "résidus"
In the crowded cell, proteins interact with their functional partners, but also with a large number of non-functional partners that compete with the functional ones. The goal of this thesis is to characterize the physical properties and the evolution of protein surfaces in order to understand how selection pressure exerts on proteins, shaping their interactions and regulating this severe competition.To do this I developed a framework based on docking calculations to characterize the propensity of protein surfaces to interact with other proteins. Molecular cartography enables the visualization and the comparison of surface properties of proteins. I implemented a new theoretical framework based on the representation of interaction energy landscapes by 2-D energy maps. These maps reflect in a synthetic manner the propensity of the surface of proteins to interact with other proteins. These maps are useful from a practical point view for determining the regions of protein’s surface that are more prone to interact with other proteins. Our new theoretical framework enabled to show that the surface of proteins harbor regions with different levels of propensity to interact with other proteins (hot regions, intermediate and cold regions to favorable, intermediate and unfavorable regions respectively).A large part of this thesis work consisted in characterizing the physico-chemical properties and the evolution of these regions. The other part of this thesis work consisted in applying this methodology on several study systems: homomeric complexes, cytosolic proteins from S. cerevisiae, families of interologs. This work opens the way to numerous practical applications in structural bioinformatics, such as binding site prediction, functional annotation and the design of new interactions.To conclude, the strategy implemented in this work enable the exploration of the propensity of a protein to interact with hundred of protein partners. It thus enables the investigation of the behavior of a protein in a crowded environment. This application goes beyond the classical use of protein docking as a, because our strategy provides a systemic point of view of protein interactions at an atomic resolution

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2006.
Vita.
Includes bibliographical references (leaves 128-137).
T cells are activated by recognition of foreign peptides displayed on the surface of antigen presenting cells (APCs), an event that triggers assembly of a complex microscale structure at the T cell-APC interface known as the immunological synapse (IS). It remains unresolved whether the unique physical structure of the synapse itself impacts the functional response of T cells, independent of the quantity and quality of ligands encountered by the T cell. As a first step toward addressing this question, we fabricated multicomponent protein surfaces that surrogate the role of APCs and studied T cell responses as a function of synapse structure. To pattern multiple proteins on surfaces, we synthesized and characterized a new polymer, poly(o-ntrobenzyl methacrylate-r-methyl methacrylate-poly(ethylene glycol) methacrylate (PNMP), a photoresist that can be processed under mild aqueous conditions. Based on the pH- and temperature-sensitive solubility of UV-exposed PNMP random terpolymers in aqueous buffers, two-component protein patterning was achieved under conditions that avoid exposing proteins to conditions outside the narrow range of physiological pH, ionic strength, and temperature where their stability is greatest.
(cont.) Using a photolithographic strategy we developed employing this novel PNMP photoresist polymer, we created multicomponent protein surfaces presenting micron-scale arrays of tethered T cell receptor (TCR) ligands (anti-CD3 'activation sites') surrounded by a field of tethered intercellular adhesion molecule-I (ICAM-1), as a model substrate on which T cells could be seeded to mimic T cell-APC interactions. CD4+ T cells seeded on these surfaces polarized and migrated; on contact with activation sites, T cells assembled an IS with a structure modulated by the physical pattern of ligand encountered. On surfaces patterned with focal spots of TCR ligand, T cells stably interacted with activation sites, proliferated, and secreted cytokines. In contrast, T cells interacting with activation sites patterned to preclude centralized clustering of TCR ligand failed to form stable contacts with activation sites, exhibited aberrant PKC-[Theta] clustering in a fraction of cells, and had significantly reduced production of interferon-[gamma]. These results suggest that focal clustering of TCR ligand characteristic of the 'mature' IS may be required under some conditions for full T cell activation.
by Junsang Doh.
Ph.D.

Les interactions protéine - protéine sont au centre de nombreux processus biologiques, et représentent des cibles thérapeutiques pertinentes pour le traitement de certaines maladies. La conception de molécules antagonistes visant à inhiber ces interactions requiert la reconnaissance spécifique d’une des surfaces protéiques impliquées. Les foldamères de type oligoamides de quinoline constituent de bons candidats. Leur production et leur fonctionnalisation sont relativement aisées. Ils adoptent des structures hélicoïdales semblables à celles rencontrées dans les protéines. Grâce à différentes techniques biophysiques (CD, SPR, diffraction des rayons X), nous montrons que ces molécules sont aptes à reconnaître une surface protéique. Deux protéines cibles ont été choisies : l’interleukine 4 et l’anhydrase carbonique humaine II.La stratégie retenue dans ce travail (ancrage du foldamère à la protéine via un bras espaceur) nous a permis d’obtenir des informations structurales sur les interactions foldamère – protéine avant toute optimisation de ces interactions. La première structure 3D d’un complexe foldamère de quinoline complexée à une protéine est décrite
Protein-protein interactions are a central issue in biological processes and represent relevant therapeutic targets for the treatment of diseases. The design of antagonistic molecules directed towards the disruption of these interactions requires the specific recognition of protein surfaces. Quinoline oligoamide foldamers present all the properties to reach that point. They are easily synthesized and fold into helices (similar to α helices) which can be decorated. Thanks to biophysical studies (CD, SPR, RX diffraction), we demonstrate that these molecules are able to recognize protein surfaces. Two proteins have been studied: the human interleukin 4 and the human carbonic anhydrase II.The applied strategy (keeping the foldamer close to the protein surface via a linker) allowed us to gather structural information about foldamer protein interactions before any strong binding is established. The first crystal structure between a protein and an aromatic amide foldamer is reported

A major challenge in nanotechnology is the spatially controlled transport of cargo on the nanometer scale. The use of a nanoscale transport system based on molecular motors and filaments of the cytoskeleton proved as a promising approach to this problem. Therefore, the objective of this work was to pattern planar surfaces with motor proteins in a way that allows controlled and guided movement of microtubule-shuttles. The first part of the work was in particular focused on generating nanometer–sized tracks of motor proteins on unstructured surfaces. Specifically, microtubules themselves were used as biological templates for the stamping and alignment of motor proteins. Compared to other soft lithography techniques like microcontact printing this approach circumvented protein denaturation due to drying and conformational changes caused by mechanical stress. Given the large persistence length of microtubules their encounters with the boundaries of the nanotracks are limited to shallow approach angles. This way, the generated structures proved very efficient for the guiding of microtubules without topographical barriers. Topography-free guiding, as demonstrated in this work, is expected to significantly ease the design and fabrication of microtubule-transport systems and opens up the possibility to transport cargo of unlimited size, i.e. without any constraints by the dimensions of topographic guiding channels. Moreover, the biotemplated patterning is a promising tool for in vitro studies on the individual and cooperative action of motor proteins. In particular it might be helpful for the reconstitution of complex subcellular machineries in synthetic environments. As an example, microtubule-microtubule sliding by the biomolecular motor ncd has been shown to induce directional sliding between antiparallel microtubules and static cross-linking between parallel ones. The second part of the work explored an in-situ patterning technique for motor proteins to enable user-defined pattern designs, and investigated the achievable resolution. Photothermal patterning, based on localized light-to-heat conversion combined with a thermoresponsive polymer layer, was presented as a novel method. Specifically, the conformation of poly(N-isopropylacrylamide) (PNIPAM) molecules in aqueous solution was switched between the swollen state at T < 30°C (protein-repelling conformation) to the collapsed state at T > 33°C (protein-binding conformation) by optical signals of visible light to generate heat in a highly-localized manner. Upon heating of a light-absorbing layer on the substrate, the surface-grafted PNIPAM molecules collapsed locally and allowed motor proteins in solution to bind in the illuminated areas. To confirm the successful patterning of kinesin-1 molecules and their functionality microtubule-based gliding motility assays were performed. It was shown that the microtubules bind to the patterned kinesin-1 molecules and are transported exclusively in the patterned areas. While the achieved pattern sizes were currently in the range of ten micrometers, finite element modeling (implemented in COMSOL) showed that increased optical intensities possibly combined with cooling of the sample allow to significantly scale down the pattern dimensions. The produced patterns can be reversibly activated and deactivated at high and low temperature, respectively. Moreover, sequential patterning of multiple kinds of proteins on the same surface will be possible in a similar way without the need for specific linker molecules or elaborate surface preparation. Another advantage of the method is the use of visible light, which is versatile as any wavelength can be applied. In addition visible light is in comparison to other UV-based photopatterning techniques non-damaging to proteins
Der räumlich kontrollierte Transport von nanoskaligen Objekten ist eine große Herausforderung auf dem Gebiet der Nanotechnologie. Ein auf molekularen Motoren und Filamenten des Zellskeletts basierendes Nanotransportsystem hat sich dabei als ein viel versprechender Ansatz erwiesen. Das Ziel der vorgelegten Arbeit war es daher, ebene Oberflächen so mit Motorproteinen zu strukturieren, dass eine kontrollierte und geführte Bewegung von Mikrotubuli-Transportern ermöglicht wird. Der erste Teil der Arbeit war insbesondere darauf fokussiert, Motorprotein-Spuren im Nanometerbereich zu erzeugen. Im zweiten Teil der Arbeit wurde eine Strukturierungsmethode zur Realisierung von benutzerdefinierten Musterdesigns untersucht und die erreichbare Auflösung bestimmt. Für die Nanometerstrukturierung von Oberflächen mit funktionalen Motorproteinen wurde ein neuer Ansatz demonstriert. Mit der Anwendung von Biotemplaten, wie hier der Mikrotubuli, konnte ein hoch-lokalisiertes und orientiertes Anbinden von Proteinen an Oberflächen sowie gleichzeitig geringer Proteindenaturierung erreicht werden. Durch spezifisches Stempeln beziehungsweise Binden von Motoren wurden Muster aus funktionellen Proteinen mit hoher Oberflächendichte hergestellt. Die erzeugten Motor-Spuren haben gezeigt, dass Nanometerstrukturierungen möglich sind und ohne topographische Barrieren zu zuverlässiger Führung von Mikrotubuli führen können. Bisher konnte die nicht-topographische Strukturierung von Oberflächen mit Kinesin-1-Motoren nur im Mikrometerbereich demonstriert werden. Wegen der hohen Steifigkeit der Mikrotubuli war die thermische Energie des Systems in diesen Fällen nicht ausreichend, um die führende Spitze der Mikrotubuli zurück auf das Gebiet mit den strukturierten Motoren zu biegen. Dieses Problem wird durch die kleine Breite der hier demonstrierten Motor-Nanospuren verhindert, da das Auftreffen der Mikrotubuli mit den Grenzlinien auf extrem flache Winkel begrenzt ist. Interessanterweise haben sich Spuren des nicht-prozessiven Motors Kinesin-14 für das Führen und den Transport im Nanometerbereich als noch zuverlässiger herausgestellt als Kinesin-1-Spuren. Es ist zu erwarten, dass nicht-topographisches Führen, wie es in dieser Arbeit gezeigt wurde, das Design und die Herstellung von Mikrotubuli-Transportsystemen deutlich vereinfacht und die Möglichkeit eröffnet, Cargo mit unlimitierter Größe, d.h. ohne Einschränkungen durch die Abmessungen der topographischen Führungskanäle, zu transportieren. Zusätzlich ist die biotemplierte Strukturierung ein viel versprechendes Werkzeug um das individuelle und das kooperative Arbeiten von Motorproteinen in vitro untersuchen und komplexe subzelluläre Maschinerien in synthetischer Umgebung rekonstituieren zu können. Dies wurde am Beispiel des gerichteten Gleitens des biomolekularen Motors Kinesin-14 gezeigt, der ein gerichtetes Gleiten zwischen antiparallelen Mikrotubuli und statisches Vernetzen zwischen parallelen Mikrotubuli hervorruft. Mit dem Ansatz des biotemplierten Strukturierens ist es jedoch nicht einfach möglich, benutzerdefinierte Spuren zu erzeugen. Daher wurde die photothermische Proteinstrukturierung als eine neue Methode für die frei programmierbare, hochauflösende und schnelle Herstellung von strukturierten Proteinoberflächen eingeführt. Auf diese Weise wurden Kinesin-1-Muster durch licht-induziertes Heizen einer licht-absorbierenden Substratschicht erzeugt. Die thermisch schaltbaren poly(N-isopropylacrylamid) (PNIPAM) Moleküle auf der Oberfläche kollabierten lokal und erlaubten es den Motorproteinen, in den beleuchteten Gebieten aus der Lösung an die Oberfläche zu binden. Die Bewegung gleitender Mikrotubuli bestätigte anschließend die erfolgreiche Strukturierung der Kinesin-1-Motoren und deren Funktionalität, da die Mikrotubuli an die strukturierten Motoren banden und ausschließlich in den strukturierten Gebieten transportiert wurden. Neben der Proteinstrukturierung wurde die lokalisierte Licht-zu-Wärme-Umwandlung kombiniert mit einer thermisch schaltbaren Polymerschicht auch für die lokale Aktivierung von Kinesin-1-Motoren auf der Oberfläche genutzt. Ein Vorteil der photothermischen Proteinstrukturierung ist die Möglichkeit, sichtbares Licht zu verwenden, da jede beliebige Wellenlänge angewendet werden kann und sichtbares Licht, im Vergleich zu anderen UV-basierten Photostrukturierungsmethoden, Proteine nicht schädigt. Modellierungen mit Hilfe der Finite-Elemente-Methode (implementiert in der Software COMSOL) haben gezeigt, dass die Lichtintensität und die Oberflächentemperatur speziell eingestellt werden müssen, um definierte Strukturgrößen zu erzielen. Während die derzeitig erzeugten Muster Größen im Bereich von zehn Mikrometern hatten, könnten durch höhere optische Intensitäten kombiniert mit Kühlung der Probe die Größenordnungen signifikant reduziert werden. Die reale experimentelle Auflösung wird jedoch auch von der Schaltcharakteristik des Polymers und der Proteinbindungsdynamik abhängen. Die hergestellten Muster können reversibel bei hohen beziehungsweise niedrigen Temperaturen aktiviert und deaktiviert werden. Zusätzlich können auf die gleiche Weise verschiedene Proteinsorten sequentiell auf einer Oberfläche strukturiert werden, ohne dass spezifische Bindungsmoleküle oder aufwändige Oberflächenpräparationen notwendig wären. Die Möglichkeit, Proteine reversibel an die Oberfläche zu binden, um geschriebene Muster wieder löschen zu können, wäre eine Weiterentwicklung und würde die Anwendungsmöglichkeiten der photothermischen Strukturierungsmethode erweitern. Außerdem würden optisch schaltbare Polymere das direkte Strukturieren von Motoren mit Licht ermöglichen und daher die Methode vereinfachen

Dental erosion is an increasing problem in many countries around the world, and research in this field has increased dramatically in recent years. Dental erosion is the dissolution of tooth tissues by acids that are not of bacterial origin; most commonly these originate from the diet. Methods to reduce erosion are of great import; the application of milk-derived proteins such as casein and casein-derived proteins are of current interest as anti-erosion agents and are the subject of the work presented here in this thesis. The efficacy of casein and casein-derived proteins as agents to inhibit dissolution of hydroxyapatite in simple citric acid solutions are investigated in chapters 3 and 4 with two different in vitro models. It was found that these proteins inhibit hydroxyapatite dissolution over a range of erosion timescales, concentrations and exposure times. The effect of an in vitro formed salivary pellicle is also examined and the proteins were shown to retain their efficacy. The efficacy of these proteins to inhibit the earliest stages of erosion (surface softening) and more progressed stages of erosion (bulk tissue loss) are investigated in chapter 5 using atomic force microscopy nanoindentation and non-contact optical profilometry respectively. It was found that again these proteins inhibit both surface softening and bulk tissue loss of bovine enamel. The nature of the protective mechanism due to casein is investigated in chapter 6 using a range of complementary, inter-disciplinary techniques such as atomic force microscopy, x-ray reflectometry and sodium dodecyl sulphate polyacrylamide gel electrophoresis. The protective effect is ascribed to a thin protein film, of 6.6 nm in thickness, forming on the mineral surface. In conclusion, casein and casein-derived proteins are shown to have anti-erosion properties and potential as oral healthcare products.

Protein microarrays are becoming powerful tools to screen and identify tumor markers for cancer diagnosis, because of the multiplex detection and minute volume of sample requirement. Due to the diversity and variation in different cancers, no single tumor marker is sensitive and specific enough to meet strict diagnostic criteria. Therefore, a combination of tumor markers is required to increase sensitivity and to establish distinct patterns to increase specificity. To obtain reliable tests, the development of reproducible surface chemistry and immobilization procedure are crucial steps in the elaboration of efficient protein microarrays. In this thesis, 3D micro-structured glass slides were functionalized with various surface chemistries like silane monolayer (amino, epoxy and carboxy), and polymer layers of Jeff amine, chitosan, carboxymethyl dextran (CMD), maleic anhydride-alt-methyl vinyl ether copolymer (MAMVE) for physical adsorption or covalent binding with proteins. Surface characterizations, such as X-ray photoelectron spectroscopy (XPS) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), confirmed the monolayer/polymer grafting on the glass slides. Colorimetric assay for determining amine density of three aminated surfaces demonstrated that APDMES had more grafting density than Jeffamine and chitosan. Contact angle measurements show that polymer surfaces were more hydrophilic than monolayer surfaces due to the increasing dosages of polar functional groups. Moreover, the parameters such as additives and pH of spotting buffer, probe concentration, blocking procedures etc, were optimized for tumor marker detection. Under the optimized conditions, antibody microarrays were validated with purified tumor antigens. The best analytical performances obtained for each tumor antigen tested were strongly dependent on functionalized surfaces, e.g. MAMVE exhibited best analytical performances for CEA andHsp60 while NHS leads to best results for PDI and CA19-9. Besides, the implemented antibody microarrays were applied to tumor marker detection from colorectal cancer sera. This evaluation shows the interest to combine several tumor markers on the same surface and the combination of tumor markers on their specific surface lead to remarkably increase the positive responses of tested cancer sera (even up to 100 %). A second type of microarrays (tumor-associated antigens - TAA microarrays) was designed to discriminate breast cancer patients from healthy donors through the detection of tumor autoantibodies. This study included a cohort of 29 breast cancer patients' and 28 healthy donors' sera. A panel of fiveTAAs (Hsp60, p53, Her2, NY-ESO-1 and Hsp70) immobilized on their respective optimized surface chemistry allowed to specifically detect over 82% of breast cancer patients.

Le repliement et la stabilité des protéines dépendent des conditions physico-chimiques de leur environnement. En particulier, le pH, la température, l’agitation et les interactions avec d’autres macromolécules ou avec les interfaces (liquide–surfaces des matériaux ; air-liquide ; etc.) sont connues pour induire des phénomènes de dénaturation et d’agrégation des protéines.Le contrôle de la stabilité des protéines thérapeutiques représente un enjeu médical et économique pour l’industrie pharmaceutique. L’insuline, qui est la protéine thérapeutique la plus produite, est connue in vitro pour former des fibres amyloïdes induites par les surfaces hydrophobes. Les agrégations amyloïdes sont également impliquées dans un certain nombre de pathologies, notamment humaines et animales présentant de forts enjeux de santé publique et économiques.Cette thèse traite en particulier de l’agrégation amyloïde à la surface des matériaux en utilisant l’insuline comme protéine modèle. Les travaux précédents réalisés par notre équipe ont démontré que des peptides de courte longueur avaient la capacité de modifier très significativement la cinétique d’agrégation induite par la surface des matériaux, et ce à des concentrations sub-stœchiométriques par rapport à l'insuline. En particulier les peptides adoptant une conformation secondaire en feuillet beta une fois adsorbés sur les surfaces hydrophobes, induisent une réduction drastique de la durée de nucléation de fibres amyloïdes d’insuline.Dans les travaux présentés ici nous avons découvert des séquences peptidiques présentant, toujours à des concentrations sub-stœchiométriques, deux effets antagonistes sur la cinétique de l’agrégation amyloïde de l’insuline. Le premier effet, coopératif et localisé à la surface de matériaux hydrophobes, résulte en une accélération de la nucléation. A l’inverse le second effet provient des peptides en solution et résulte en une puissante inhibition à la fois de la nucléation et de l’élongation des fibres.Nous avons premièrement caractérisé quantitativement ces effets pour un ensemble de peptides possédant des séquences de type (LK)nL, et investigué les mécanismes à l’origine du phénomène accélérateur. Des mesures quantitatives de fluorescence (Thioflavine T, marquage fluorescent du peptide) ont permis de montrer que l’adsorption coopérative des peptides sur la surface du matériau était responsable de l’accélération de la vitesse de nucléation. Pour l’effet inhibiteur, provenant des peptides en solution, nous avons démontré que cet effet résulte de la liaison des peptides sur l’insuline fibrillaire et qu’il est médié par les charge.De surcroit nous avons étudié la localisation de la nucléation et de l’apparition des premiers agrégats par microscopie à fluorescence. Nous avons observé que les zones situées à l’interface triple matériau-air-solution et subissant une contrainte de cisaillement élevé étaient les sites préférentiels d’apparition des premiers agrégats amyloïdes et donc très probablement les régions dominantes en termes de nucléation.Nous avons enfin développé une technique permettant une croissance localisée, patternable et induite par la lumière d’agrégats amyloïde d’insuline sur une surface de verre. Cette voie d’agrégation singulière ne présente pas de phase de nucléation apparente et dépend strictement de la présence de Thioflavine T. Nous avons montré que la Thioflavine T insérée entre les feuillets béta et qui peut être excitée à 440 nm fournit localement l’énergie nécessaire pour la transition de conformation de l’insuline native adsorbée vers l’état agrégé. Cette méthode permet d’obtenir une croissance différentielle entre des zones de surface hydrophile et hydrophobe
The folding and stability of proteins depend on the physico-chemical conditions of their environment. Especially pH, temperature, stirring and interactions with other macromolecules or with interfaces (liquid-material surfaces; air-liquid; etc.) are known to induce protein denaturation and aggregation phenomena.The control of therapeutic protein stability represents a medical and economic challenge for the pharmaceutic industry. For instance insulin, which is the most s model produced therapeutic protein, is known to form amyloid aggregates in vitro induced by hydrophobic surfaces. Amyloid aggregates are also involved in several pathologies including human and animal diseases of high economic and public health impact.This thesis focuses on amyloid aggregation at material surfaces using insulin as a model protein. Previous work from our team have demonstrated that short peptides have the ability to significantly interfere with the kinetics of surface-driven amyloid aggregation and this at sub-stoichiometric concentrations with respect to insulin. In particular peptides adopting a beta-sheet secondary structure when adsorbed on hydrophobic surfaces, were able to reduce the nucleation time of insulin aggregation.In the present work we have discovered peptide sequences presenting, again at sub-stoichiometric concentrations, two antagonistic effects on insulin aggregation kinetics. The first consists in a cooperative reduction of the nucleation time and operates via peptides bound to the material surface. The second, on the other hand, results in a powerful inhibition of both nucleation and fiber elongation via peptides remaining in solution.We have first quantitatively characterized these effects on a set of peptides presenting alternate primary sequences of the type (LK)nL, and investigated the underlying mechanisms promoting insulin nucleation. Quantitative fluorescence measurements (Thioflavin T, fluorescent labelling of the peptide) have shown that the cooperative adsorption of peptides on hydrophobic material surfaces was responsible for the reduction of the insulin nucleation time. We have then shown that the inhibitory effect results from the binding of peptides in solution to fibrillar insulin aggregates and that this effect is mediated by charges.In addition we studied the localization of the insulin nucleation and of the appearance of the first aggregates using fluorescence microscopy. We observed the preferential appearance of the first ThT positive aggregates at the solid-liquid-air triple interface undergoing high shear stress, making these regions the predominant nucleation sites.We eventually developed a technique allowing a localized and patterned growth of light-induced insulin aggregates on glass surfaces. This atypical aggregation pathway does not present any observable lag time and depends strictly on Thioflavin T. We have shown that the ThT inserted between the cross beta-sheets and which can be excited at 440 nm locally provides the energy required for the conformational transition of the native insulin into the aggregated one. This method can be used to obtain a differential amyloid growth between surface area of different hydrophobicity