In this research project, structural aspects of monomeric NADP+-dependent isocitrate dehydrogenase from Corynebacterium glutamicum (CgIDH) are investigated together with site-directed mutagenesis and fluorescence spectroscopy studies. CgIDH, one of the enzymes of the Krebs cycle, catalyzes the decarboxylation of isocitrate into α-ketoglutarate, which in some bacteria and plants regulates the flow of carbon into either the Krebs cycle or the glyoxylate bypass depending on the available carbon source. The structure of CgIDH complexed with Mg2+ has been determined at 1.75 Å resolution using X-ray crystallography. In contrast to the closed conformation of published structures of monomeric NADP+-dependent IDH from <i> Azotobactor vinelandii </i> complexed with either isocitrate-Mn2+ or NADP+, the structure of CgIDH complexed with Mg2+ demonstrates the open conformation. The superimposed structure of CgIDH complexed with Mg2+ onto the structures of AvIDH complexes reveals that Domain II is rotated ~24° or ~35º, respectively, relative to Domain I when isocitrate-Mn2+ or NADP+ is bound, resulting in the closure of the active site between the two domains. Fluorescence spectroscopic studies support the proposal that the presence of isocitrate or NADP+ could mediate the conformational changes in CgIDH. <p>In addition, three CgIDH mutants (S130D, K253Q, and Y416T) were created based on the structural analysis and previous mutagenesis studies of homodimeric NADP+-dependent IDH. Both the specific activities and the fluorescence spectra of these CgIDH mutants elucidate the roles of these active site residues in CgIDH catalysis. It has been suggested that the conformational changes observed in the presence of the substrate(s) may regulate enzymatic activity in CgIDH, in contrast to homodimeric NADP+-dependent IDH in Escherichia coli, where the phosphorylation cycle controls activity. It is also presumed that both Lys253 and Tyr416 may play critical roles in CgIDH activity, as do the equivalent residues in homodimeric IDH from porcine heart mitochondria. Similar structural features and conformational changes among monomeric CgIDH and homodimeric NADP+-dependent IDH enzymes suggest the phylogenetic relationships among various monomeric and homodimeric NADP+-dependent IDH from different sources.

In this research project, structural aspects of monomeric NADP+-dependent isocitrate dehydrogenase from Corynebacterium glutamicum (CgIDH) are investigated together with site-directed mutagenesis and fluorescence spectroscopy studies. CgIDH, one of the enzymes of the Krebs cycle, catalyzes the decarboxylation of isocitrate into α-ketoglutarate, which in some bacteria and plants regulates the flow of carbon into either the Krebs cycle or the glyoxylate bypass depending on the available carbon source. The structure of CgIDH complexed with Mg2+ has been determined at 1.75 Å resolution using X-ray crystallography. In contrast to the closed conformation of published structures of monomeric NADP+-dependent IDH from <i> Azotobactor vinelandii </i> complexed with either isocitrate-Mn2+ or NADP+, the structure of CgIDH complexed with Mg2+ demonstrates the open conformation. The superimposed structure of CgIDH complexed with Mg2+ onto the structures of AvIDH complexes reveals that Domain II is rotated ~24° or ~35º, respectively, relative to Domain I when isocitrate-Mn2+ or NADP+ is bound, resulting in the closure of the active site between the two domains. Fluorescence spectroscopic studies support the proposal that the presence of isocitrate or NADP+ could mediate the conformational changes in CgIDH. <p>In addition, three CgIDH mutants (S130D, K253Q, and Y416T) were created based on the structural analysis and previous mutagenesis studies of homodimeric NADP+-dependent IDH. Both the specific activities and the fluorescence spectra of these CgIDH mutants elucidate the roles of these active site residues in CgIDH catalysis. It has been suggested that the conformational changes observed in the presence of the substrate(s) may regulate enzymatic activity in CgIDH, in contrast to homodimeric NADP+-dependent IDH in Escherichia coli, where the phosphorylation cycle controls activity. It is also presumed that both Lys253 and Tyr416 may play critical roles in CgIDH activity, as do the equivalent residues in homodimeric IDH from porcine heart mitochondria. Similar structural features and conformational changes among monomeric CgIDH and homodimeric NADP+-dependent IDH enzymes suggest the phylogenetic relationships among various monomeric and homodimeric NADP+-dependent IDH from different sources.

The work presented in my dissertation focuses on the conformational studies of bio-molecules including proteins and DNA using computational approaches. Conformational changes are important in numerous molecular bioprocesses such as recognition, transcription, replication and repair, etc. Proteins recognize specific DNA sequences and upon binding undergo partial or complete folding or partial unfolding in order to find the optimal conformational fit between molecules involved in the complex. In addition to sequence specific recognition, proteins are able to distinguish between subtle differences in local geometry and flexibility associated with DNA that may further affect their binding affinities. Experimental techniques provide high-resolution details to the static structures but the structural dynamics are often not accessible with these methods; but can be probed using computational tools. Various well-established molecular dynamics methods are used in this work to study differences in geometry and mechanical properties of specific systems under unmodified and modified conditions. Briefly, the studies of several protein and DNA systems investigated the importance of local interactions and modifications for the stability, geometry and mechanical properties using standard and enhanced molecular dynamics simulations. In addition to the conformational studies, the development of a new method for enhanced sampling of DNA step parameters and its application to DNA systems is discussed. Chapter 1 reviews the importance of the conformational changes in bioprocesses and the theory behind the computational methods used in this work. In the project presented in chapter 2 unbiased molecular dynamics and replica exchange molecular dynamics are employed to identify the specific local contacts within the inhibitory module of ETS-1. ETS-1 is a human transcription factor important for normal but also malignant cell growth. An increased concentration of this protein is related to a negative prognosis in many cancers. A part of the inhibitory module, inhibitory helix 1 (HI-1) is located on the site of the protein opposite to the DNA binding site and although loosely packed, stays folded in the apo state and unfolds upon ETS-1 binding to DNA. Our study investigated the character and importance of contacts between HI-1 and neighboring helices of the inhibitory module: HI-2 and H4. We also identified a mutant of HI-1, which possessed the higher helical propensity than the original construct. This study supported the experimental findings and enhanced the field by the identification of new potential target for experimental tests of the system, which plausibly inhibits binding to DNA. In the studies discussed in chapters 3-5 the conformational dynamics of DNA under normal conditions and upon specific epigenetic modifications are presented. Since DNA conformation can be accurately described by six base pair step parameters: twist, tilt, roll, shift, slide and rise, these were extensively analyzed and the results elucidated insights into the properties of the systems. In order to enhance unbiased simulations and allow for easier crossing of the energy barriers, we developed and implemented a novel method to control DNA base pair step parameters. With this approach we obtained the free energy estimates of e.g. DNA rearrangements in a more efficient manner. This advanced computational method, supported by standard and additional enhanced techniques, was then applied in the studies of DNA methylation on cytosine or adenine bases and oxidative damage of cytosine.

The present study exploits two different molecules as biorecognition elements for biosensing. In the first case, a protein biosensor was performed using maltose-binding protein (MBP). The ability to manipulate protein function rationally also offers the possibility of creating new proteins of biotechnological value. Our design has been used to test the understanding of allosteric transitions in proteins. Here we examined a simple conformational change that can represent the biorecognition principle for a reagentless biosensor. Previously, modular strategies for transducing ligand-binding events into fluorescent and electrochemical responses have been reported. Starting with a study of the conformational changes of MBP this research will further develop electrochemical maltose biosensors. The responses of four individual mutations (K46C-MBP-MT, N282C MBP-MT, Q72C-MBP-MT; and K25C-MBP-MT) were evaluated using square wave voltammetry. The possibility of using this type of transduction mechanism for sensor configurations and analyte specificity is discussed.The second part of this work involves SELEX (systematic evolution of ligands by exponential enrichment) and aptamers as biorecognition molecules. As a result of the SELEX method, we can obtain oligonucleotide sequences (aptamers) with recognition properties similar to antibodies. These synthetic elements play an important role in molecular recognition because of their capability for specifically binding of a target molecule. A new approach for the separation step has been performed, termed Soluble-SELEX. This new SELEX method uses hybridization as partitioning mechanism for separating the bound and unbound DNA members from the target-molecule. Hybridization procedure has been evaluated by fluorescence studies as partitioning mechanism for SELEX method. Herein, we exploited the incorporation of an aptamer for biosensing detection of a specific target molecule. Three different transduction methods such as fluorescence, electrochemistry and surface plasmon resonance (SPR) were evaluated. In all three cases, the biosensing procedure was successful.In conclusion, this research has evaluated the translation of a fluorescent biosensor into an electrochemical biosensor using maltose-binding protein as biorecognition element. On the other hand, a new SELEX method has been developed. However, future improvements are required in order to optimize the method. As result of SELEX a new avidin-aptamer was selected and three different transduction systems were employed to construct fluorescent, surface Plasmon resonance and electrochemical biosensors. / El presente estudio utiliza dos moléculas diferentes como elementos de bioreconocimiento. En el primer caso, un biosensor basado en proteínas fue desarrollado utilizando la proteína periplasmica de unión a maltosa (MBP = maltose-binding protein). La habilidad para manipular racionalmente la función de una proteína también ofrece la posibilidad de crear nuevas proteínas con valor biotecnológico. Nuestro diseño proteico ha sido usado para evaluar cambios alostéricos en proteínas. Este estudio evalúa un simple cambio conformacional el cual puede ser usado como el principio transductivo para un biosensor. Diferentes estrategias de transducción usando fluorescencia y electroquímica en eventos de reconocimiento entre la proteínas periplasmicas de unión y el ligando, han sido previamente reportadas. Esta investigación inicia con el estudio de los cambios conformacionales de MBP, continuando con el desarrollo de un biosensor electroquímico para maltosa. La señal de cuatro diferentes mutantes (K46C-MBP-MT, N282C MBP-MT, Q72C-MBP-MT; y K25C-MBP-MT) fue evaluada usando voltimetría de onda cuadrada. La posibilidad de usar este tipo de transducción mecánic (distancia) para la configuración de biosensores y la respectiva especificidad analítica es discutida. La segunda parte de este trabajo incluye el método SELEX (systematic evolution of ligands by exponential enrichment) y aptameros como moléculas de bioreconocimiento. Como resultado de el método SELEX, podemos obtener secuencias de oligonucleótidos (aptameros) con propiedades de reconocimiento similares a los anticuerpos. Estos elementos sintéticos, tienen un importante rol en el reconocimiento molecular por su capacidad de unión específica a la molécula blanco. Un nuevo mecanismo para el paso de separación ha sido realizado, y llamado SELEX-Soluble. Este nuevo método SELEX usa la hibridización como mecanismo de separación para dividir los oligonucleótidos de DNA que no se unen y los que se unen a la molécula blanco. El procedimiento de hibridización y su uso como mecanismo de separación en el método SELEX ha sido evaluado a través de estudios de fluorescencia. Este estudio también explora la incorporación de un aptamero como elemento de reconocimiento en un biosensor. Tres diferentes mecanismos de transducción has sido evaluados: fluorescencia, electroquímica y resonancia de plasmon superficial (SPR). En los tres casos una excelente señal fue reportada. En conclusión, esta investigación ha evaluado la transferencia de una biosensor de fluorescencia a un biosensor electroquímico, utilizando la proteína periplásmica de unión a maltosa como elemento de bioreconocimiento. De otro lado, un nuevo método SELEX ha sido desarrollado. Sin embargo, futuras mejoras son requeridas para optimizar el método. Como resultado del método SELEX realizado un nuevo aptamero para avidita ha sido seleccionado y tres diferentes sistemas de transducción ha sido empleado para construir tres diferentes biosensores (fluorescencia, electroquímica y SPR).

The main focus of my dissertation is on the conformational motion of DNA, studied by applying tools from the computational chemistry field. In addition, studies of relative α- and 310 helical stabilities in peptides/mini-proteins, and a molecular flooding study of the retinoid X-receptor as part of a continuing drug design effort are presented. In molecular biology, it has been well known that sequence determines structure, and structure controls function. For proteins or DNA to work properly, the correct configuration is required. Mutations may alter the structure, which can cause malfunction. Non-mutational effects, such as a change in environment may also cause a configurational change and in turn change the functionality of the protein or DNA. Many experimental technics have been developed to investigate the structural or configurational aspects of biological systems, and molecular dynamics simulation has been proven to be a useful complementary tool to gain insights into this problem due to its ability to explore the dynamics and energetics of biomolecular processes at high spatial and time resolution. Molecular dynamics simulations are constrained by the available computational power, but several computational techniques have been developed to reduce computational costs. Also, development of hardware has helped the issue. Years of hard work on force field parameter optimization built a solid foundation for molecular dynamics simulations, so that the computational model can satisfactory describe many biochemical systems in detail. Techniques such as umbrella ix sampling and reweighting methods have allowed researchers to construct free energy landscapes to reveal the relative stabilities of each major configurational state and the free energy barriers between configurations from relatively short simulations, a process which would otherwise require many microseconds of unbiased simulations. My dissertation applies multiple advanced simulation techniques to investigate several DNA conformational problems, including the coupling between DNA bending and base flipping, the anisotropy of DNA bending, and intercalation of the dye in a Cy3 labeled DNA system. The main part of this work addressed a long standing question about DNA bending: does DNA prefer to bend toward the major or minor groove. My simulations not only answered this question, but also identified the mechanism by which the one direction is favored. Another part describes peptide/mini-protein helical transitions and studies benefiting ligand design for the retinoid X-receptor.

Mediante a técnica de espalhamento de raio-X a baixos ângulos por macromoléculas em solução, estudou-se as variações de forma e tamanho da crotamina, proteína básica e neurotóxica do veneno das cascavéis (crotalus durissus terrificus), em diferentes condições de pH e uma concentração de 10% visando a determinação dos valores do raio de giro. Este raio, RG, é definido como RG = (&#8747vr2 &#961 r&#963; dv&#963;) 1 &#8260 2 onde v é o volume da macromolécula, r o módulo do vetor posição r&#963 com origem no centro de massa, &#961 (r&#963) a densidade eletrônica e n o número de elétrons da macromolécula. Este parâmetro não conduz obviamente ao conhecimento completo da estrutura da macromolécula, mas a sua determinação possibilita estudo de variações conformacionais de forma relativamente simples em sistemas com condições semelhantes às biológicas. Os valores do raio de giro foram determinados mediante gráficos de Guinier (Log JN(s) vs s2, onde s é o módulo do vetor posição do espaço recíproco e JN(s) a intensidade normalizada e livre de espalhamento parasita). Preliminarmente, foi feito um estudo com oxi-hemoglobina, Insulina e hemoglobina de caramujo, por apresentarem características semelhantes às da crotamina, isto é, serem do tipo globular e de forma aproximadamente esférica com o objetivo de familiarizar-se com a técnica experimental e testar a aparelhagem. Os valores de raio de giro deduzidos para a oxi-hemoglobina RGOX= 23.9 &#177 0.7 (A &#176), Insulina RGI= 18.4 &#177 0.5 (A &#176), e hemoglobina de caramujo RGhc= 92 &#177 2 (A&#176), concordam satisfatoriamente com resultados previamente encontrados na literatura. Os valores de raio de giro medido para as diferentes soluções de crotamina encontram-se em bom acordo com a teoria e com o valor do raio de giro esperado de crotamina REC= 9.7 A&#176 / The small angle x-ray scattering technique was used in order to determine the radius of gyration of crotamine, a basic and neurotoxic protein from rattle snake venom, in 10% solutions at different PHs. The radius of gryration, RG is defined as: RG = (&#8747vr2 &#961 r&#963; e dv&#963;) 1 &#8260 2 Where: v is the volume of the macromolecule, r is the modulus of the position vector r with origin at the center of mass, &#961 (r&#963) is the electronic density and n the number of electrons of the macromolecule. This parameter does not give a complete information about the structure of the macromolecule but it is possible to get information about conformation to close to the biological ones. The values of the radius of gyration were fdetermined by means of the Guinier graphics (Log JN(s) vs s2, where s is the magnitude of the scattering vetor and JN(s) is the normalized intensity. In order to get familiar wi th the experimental technique and to test the equipment the study of oxihaemoglobin, Insulin and sea-mail hemoglobin was done. This proteins are very similar to crotamine in the sense that they are globular type and almost of the same spherical shape. The values of the radius of gyration obtained agree very well with the reported ones: Oxihaemoglobin RGOX= 23.9 &#177 0.7 (A &#176), Insulin RGI= 18.4 &#177 0.5 (A &#176), and sea-mail hemoglobin RGhc= 92 &#177 2 (A&#176). The values od the radius of gyration measured for the different solution of crotamine are in good agreement with the calculated and with the expected value REC= 9.7 A&#176

The structure, potential energy functions and vibrational spectra of several cyclic and bicyclic molecules have been investigated using several spectroscopic techniques and high-level ab initio and density functional theory (DFT) calculations. Laser induced fluorescence and Raman spectroscopies were used to study the conformation of 2- indanol in the electronic ground and excited states. These, along with detailed ab initio calculations, confirmed the existence of four different stable conformations with the one undergoing an intermolecular hydrogen bonding being the most stable. A theoretical two-dimensional surface in terms of the ring-puckering and the hydroxyl group internal rotation vibrations was constructed. This work was extended to obtain preliminary insights on the conformations and ring-puckering frequencies of 3-cyclopenten-1-ol using ab initio and DFT calculations. Infrared and Raman spectra were also utilized to study the structures and vibrational spectra of -crotonolactone and 2,3-cyclopentenopyridine (pyrindan). Ab initio results showed that -crotonolactone is rigidly planar in the electronic ground state and has a nearly harmonic ring-puckering potential function. The calculated vibrational levels were shown to be in very good agreement with the experimental ring-puckering frequency from vapor-phase Raman observations. The structures, vibrational spectra, and potential energy functions of several cyclic molecules were reinvestigated using high-level ab initio computations, and detailed vibrational analyses based on DFT-B3LYP calculated frequencies were also carried out. A number of new insights were presented by re-evaluating the available experimental data for several cyclopentenes, silacyclobutanes and silacyclopentenes. It was found that the vibrational spectra of some deuterated cyclopentenes possess extensive coupling between several ring modes and other low-frequency modes. Reassignments of these spectra have been proposed. Frequencies from DFT-B3LYP calculations showed very good agreement with the experimental values for silacyclobutane and its derivatives. The presence of silicon and halogen atoms did not affect the accuracy of the DFT calculations. In addition, the ring-puckering potential energy function for silacyclopent-2-ene was studied and alternative assignments of the far-infrared results were proposed. The new assignments are in good agreement with computational results. Silacyclopent-2-ene and its -1,1-d2 isotopomer were shown to be slightly puckered with barriers of less than 50 cm-1.

Mediante a técnica de espalhamento de raio-X a baixos ângulos por macromoléculas em solução, estudou-se as variações de forma e tamanho da crotamina, proteína básica e neurotóxica do veneno das cascavéis (crotalus durissus terrificus), em diferentes condições de pH e uma concentração de 10% visando a determinação dos valores do raio de giro. Este raio, RG, é definido como RG = (&#8747vr2 &#961 r&#963; dv&#963;) 1 &#8260 2 onde v é o volume da macromolécula, r o módulo do vetor posição r&#963 com origem no centro de massa, &#961 (r&#963) a densidade eletrônica e n o número de elétrons da macromolécula. Este parâmetro não conduz obviamente ao conhecimento completo da estrutura da macromolécula, mas a sua determinação possibilita estudo de variações conformacionais de forma relativamente simples em sistemas com condições semelhantes às biológicas. Os valores do raio de giro foram determinados mediante gráficos de Guinier (Log JN(s) vs s2, onde s é o módulo do vetor posição do espaço recíproco e JN(s) a intensidade normalizada e livre de espalhamento parasita). Preliminarmente, foi feito um estudo com oxi-hemoglobina, Insulina e hemoglobina de caramujo, por apresentarem características semelhantes às da crotamina, isto é, serem do tipo globular e de forma aproximadamente esférica com o objetivo de familiarizar-se com a técnica experimental e testar a aparelhagem. Os valores de raio de giro deduzidos para a oxi-hemoglobina RGOX= 23.9 &#177 0.7 (A &#176), Insulina RGI= 18.4 &#177 0.5 (A &#176), e hemoglobina de caramujo RGhc= 92 &#177 2 (A&#176), concordam satisfatoriamente com resultados previamente encontrados na literatura. Os valores de raio de giro medido para as diferentes soluções de crotamina encontram-se em bom acordo com a teoria e com o valor do raio de giro esperado de crotamina REC= 9.7 A&#176 / The small angle x-ray scattering technique was used in order to determine the radius of gyration of crotamine, a basic and neurotoxic protein from rattle snake venom, in 10% solutions at different PHs. The radius of gryration, RG is defined as: RG = (&#8747vr2 &#961 r&#963; e dv&#963;) 1 &#8260 2 Where: v is the volume of the macromolecule, r is the modulus of the position vector r with origin at the center of mass, &#961 (r&#963) is the electronic density and n the number of electrons of the macromolecule. This parameter does not give a complete information about the structure of the macromolecule but it is possible to get information about conformation to close to the biological ones. The values of the radius of gyration were fdetermined by means of the Guinier graphics (Log JN(s) vs s2, where s is the magnitude of the scattering vetor and JN(s) is the normalized intensity. In order to get familiar wi th the experimental technique and to test the equipment the study of oxihaemoglobin, Insulin and sea-mail hemoglobin was done. This proteins are very similar to crotamine in the sense that they are globular type and almost of the same spherical shape. The values of the radius of gyration obtained agree very well with the reported ones: Oxihaemoglobin RGOX= 23.9 &#177 0.7 (A &#176), Insulin RGI= 18.4 &#177 0.5 (A &#176), and sea-mail hemoglobin RGhc= 92 &#177 2 (A&#176). The values od the radius of gyration measured for the different solution of crotamine are in good agreement with the calculated and with the expected value REC= 9.7 A&#176

Using statistical physics principles to solve problems in biology is one of the most promising directions due to the complexity and non-equilibrium fluctuations in biological systems. In this work, we try to describe the dynamics at both cellular and molecular levels. Microtubule dynamics and dynamic disorder of enzyme proteins are two of the examples we investigated. The dynamics of microtubules and the mechanical properties of these polymers are essential for many key cellular processes. However, critical discrepancies between experimental observations and existing models need to be resolved before further progress towards a complete model can be made. We carried out computational studies to compare the mechanical properties of two alternative models, one corresponding to the existing, conventional model, and the other considering an additional type of tubulin lateral interaction described in a cryo-EM structure of a proposed trapped intermediate in the microtubule assembly process. Our work indicates that a class of sheet structures is transiently trapped as an intermediate during the assembly process in physiological conditions. In the second part of the work, we analyzed enzyme slow conformational changes in the context of regulatory networks. A single enzymatic reaction with slow conformational changes can serve as a basic functional motif with properties normally discussed with larger networks in the field of systems biology. The work on slow enzyme dynamics fills the missing gap between studies on intramolecular and network dynamics. We also showed that enzyme fluctuations could be amplified into fluctuations in phosphorylation networks. This can be used as a novel biochemical "reporter" for measuring single enzyme conformational fluctuation rates. / Ph. D.

L'agrégation protéique induite par la surface des matériaux est un phénomène important dans la stabilité des protéines thérapeutiques. En utilisant l'insuline humaine, nous avons étudié les phénomènes agrégation en présence de surfaces neutres hydrophobes ou hydrophiles et avons montré que la nucléation a lieu sur les surfaces hydrophobes que l'on soit à pH 2.5 ou 7.3. Nous avons montré que l'énergie d'activation de la nucléation est abaissée sur surface hydrophobe. De plus, il apparait que l'agitation de la solution a des effets antagonistes. En particulier, les forces hydrodynamiques de cisaillement détachent de la surface les fibres. Par Résonance Plasmonique de Surface, spectroscopie infrarouge et microscopie à fluorescence, nous avons pu définir les étapes moléculaires ayant lieu à l'interface matériaux hydrophobe/solution. L'insuline s'adsorbe tout d'abord rapidement sur la surface, puis s'accumule lentement parallèlement à une transition de la structure α initiale vers une structure β, aboutissant à la formation de fibres amyloïdes. Par la suite, nous avons étudié le mécanisme d'action d'un peptide connu pour accélérer l'agrégation de l'insuline (LVEALYL). Ce peptide s'adsorbe de façon stable sur la surface hydrophobe en structure β et facilite l'accumulation d'insuline. De plus, il apparait que la séquence du peptide n'est pas essentielle à son action car différents peptides adoptant une structure β sur la surface sont également capables d'induire l'agrégation de l'insuline. La présence de prolines aboli cette action. Ces résultats apportent d'importantes informations sur les mécanismes moléculaires d'auto-association de l'insuline. L'hydrophobicité du matériau facilite le dépliement de l'insuline adsorbée, aboutissant à l'exposition du segment LVEALYL. Cette séquence facilite la propagation du changement de conformation vers les molécules nouvellement adsorbées. Agir contre ce phénomène pourrait permettre de stabiliser les solutions protéiques. / Material surface-induced protein aggregation is important for the stability of therapeutic proteins. Using human insulin, we first study its amyloidal aggregation on neutral hydrophobic or hydrophilic surfaces and show that nucleation takes place on the hydrophobic surfaces at both pH 2.5 and 7.3. We show that the activation energy for nucleation is lower on hydrophobic surfaces than in solution. We observed that agitating the solution has several antagonistic effects. In particular, the hydrodynamic shear stress detaches surface-borne fibrils. Using Surface Plasmon Resonance imaging, infrared spectroscopy and fluorescence microscopy we then define the sequence of molecular events that happen at the interface between hydrophobic materials and fluid phase. Insulin first adsorbs rapidly on the surface and then continues to accumulate, in parallel with an alpha-to beta-structural transition leading to amyloid fibril formation. Hereafter, we study the mechanism of action of a small peptide known to accelerate insulin aggregation (LVEALYL). This peptide stably adsorbs in β-conformation on the surface and helps accumulating insulin on the surface. Moreover, it appears that its sequence is not essential for its effectiveness, since several peptides, having a β-sheet structure on the surface, induce insulin aggregation. The presence of prolines abolishes its pro-aggregative activity. These results shed light on the molecular details of insulin self-association. The hydrophobic nature of material surfaces facilitates the unfolding of adsorbed insulin, resulting in the exposure of the LVEALYL peptide segment. This peptide promotes the propagation of conformational changes among incoming proteins. Counteracting this propagation could help stabilizing protein solutions.