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A primeira parte da discussão de resultados desta tese relata o estudo da isomeria conformacional e das interações eletrônicas dos compostos carbonílicos α-dietoxifosforilsubstituídos (EtO)2P(O)CH2C(O)X (X= Me, Ph, OEt, Net2 e SEt). Este estudo foi realizado através das espectroscopias no infravermelho, Ressonância Magnética Nuclear de 13C e 31P, apoiados por cálculos ab initio nas bases HF/ 3-21G*e HF/6-31G** e difração de raio-X. As freqüências de vCO da única banda no infravermelho dos β-carbonilfosfonatos [XC(O)CH2P(O)(OEt)2: X=Me 1, Ph 2, OEt 3, Net2 4 and SEt 5] (série I), se correlacionaram muito bem com as frequências de vCO do rotâmero gauche das correspondentes β-carbonil sulfonas [XC(O)CH2SO2Et : X=Me 6, Ph 7, OEt 8, Net2 9 and SEt 10] (série II) concordando com os cálculos ab initio 6-31G** de 1ª e 6ª (X=Me) que indicaram a existência de uma única conformação gauche (g1) para a série I em solução. Os deslocamentos negativos anormais de freqüência da carbonila (ΔvCO) para ambas as séries segue aproximadamente a afinidade eletrônica do orbital π*CO dos compostos de referência MeC(O)X 11-15. Estes dados sugerem que a conformação gauche das séries I e II deve possuir uma geometria semelhante. O menor deslocamento positivo de frequência de estiramento assimétrico do grupo sulfonila (ΔvSO2) em comparação com o do grupo fosforila (ΔvPO) e o maior deslocamento negativo da carbonila gauche das β-carbonil sulfonas 6-10 em relação aos valores dos β-carbonil fosfonatos 1-5 correspondentes estão de acordo com os deslocamentos químicos em campo mais alto para a série II em relação à séries I. Estas tendências estão de acordo com o encurtamento da distância O(SO2) .... C(CO) em relação a O(PO) ... C(CO) nos compostos 6ª e 1ª, respectivamente, e são discutidos em termos da interação de transferência de carga O1p→ π* CO o qual é mais forte para a série II que para I. Este comportamento inesperado indica que o átomo de oxigênio do grupo sulfonil do SO2R é melhor doador de elétrons que o oxigênio do grupo fosforila P(O)(OR)2. Contudo, parâmetros geométricos intrínsecos à molécula parecem ser os responsáveis por este comportamento .. De fato, dados de difração de raio-X e cálculos ab initio de metilsulfonilmetil fosfonato de dietila MeSO2CH2P(O)(OR)2 (R= Et, 18, Me 18a) apoiam esta análise. As análises espectroscópicas e cálculos ab initio dos compostos alquil(aril)tiometil-fosfonatos de etila (EtO)2P(O)CH2SOnR (R=Me e Ph) e suas formas mono- (n=1) e di- (n=2) oxigenadas mostraram que os mesmos encontram-se na conformação quasi-gauche. Este estudo mostrou que tanto o deslocamento químico de 31 quanto a freqüência de vPO se correlacionam muito bem com os parâmetros indutivos (σI) do substituinte em α não levando em consideração as interações eletrônicas variáveis que ocorrem entre os substituintes em α e o fósforo do grupo fosforila. Os dados de Ressonância Magnética Nuclear de 13C para os compostos da série I foram interpretados como sendo devido à ação simultânea do efeito -I do grupo dietoxifosforila e da transferência de carga do oxigênio do grupo fosforila ao carbono carbonílico que se encontram na relação espacial gauche. Os estudos de Ressonância Magnética Nuclear de 13C corroboraram os dados de Infravermelho indicando que a transferência de carga O(PO) → π*CO nos rotâmeros gauche da série I é mais fraca que a interação de transferência de carga O(SO2) → π* CO no mesmo rotâmero da série II. Os deslocamentos químicos de 31P mostraram-se praticamente constante ao longo da série estudada (série I). Os espectros ultravioleta dos compostos da série I indicaram que a introdução do grupo dietoxifosforila na posição α à carbonila origina um deslocamento batocrômico da transição n→π*CO em relação aos compostos carbonílicos de referência. Este comportamento pode ser atribuído à ocorrência da interação hiperconjugativa entre os orbitais σ* C-P e da π*CO que estabiliza o orbital π*CO em maior extensão que o efeito -I estabiliza o nível de energia nO(CO). Como decorrência da semelhança conformacional entre os &#946-cetosulfóxidos [MeC(O)CH2S(O)Ph-X (X=Cl, H, Me e OMe)] e &#946-cetosulfonas [MeC(O)CH2SO2Ph-X (X=NO2, H, Me e OMe) e X-Ph-C(O)CH2SO2Et (X=NO2, CN, H, OMe e NH2)] com os correspondentes &#946-fosforil-sulfóxidos [(EtO)2P(O)CH2S(O)R (R= Me ou Ph)] e β-fosforilsulfonas [(EtO)2P(O)CH2SO2R (R= Me ou Ph)] e considerando-se que a série dos β-ceto-sulfóxidos e β-ceto-sulfonas apresentam uma citotoxicidade moderada em relação às células de linhagens leucêmicas humanas K562 (eritroleucemia), tumores de Ehrlich e células de linhagens e U937 (leucemia linfóide), estes dois últimos somente para os β-cetosulfóxidos, achou-se de interesse estudar a atividade citotóxica dos α-metilsulfinil- α-metilsulfonil- metanofosfonatos de dietíla em relação aos mesmos tipos de células tumorais. Os dados da citotoxicidade do α-metilsulfinil- e α-fenil-sulfinil-metanofosfonato de dietila indicaram que os mesmos apresentam um efeito citotóxico bem menor do que aquele observado nos β-cetosulfóxidos. O α-metilsulfonilmetilfosfonato de dietila apresentou uma atividade citotóxica similar em relação às duas séries de β-cetosulfonas. Já o α-fenilsulfonilmetilfosfonato de dietila não apresentou nenhum efeito de citotoxicidade significativo em relação às células tumorais testadas. Finalmente, esta tese relata também, o estudo das reações dos compostos carbonílico α-halossubstituídos X-C(O)CH2-Hal X (X= Me, Ph, OEt, Net2 and SEt) com fosfito de trietila em benzeno anidro à temperatura de refluxo de benzeno. Nessas condições observou-se a formação dos compostos carbonílicos α-dietoxifosforil-substituídos (EtO)2P(O)CH2C(O)X 1-5 e dos enolatos correspondentes CH2=C(X) [P(O)(EtO)2] , havendo mudança na proporção dos isômeros obtidos à medida que se varia o grupo ligado à carbonila. Os substituintes doadores de elétrons levam à formação preferencial dos cetofosfonatos (reação de Michael-Arbuzov) enquanto que substituintes atraentes de elétrons levam à formação preferencial de enolfosfatos (reação de Perkow). Estes dados sugeriram que a adição nucleofílica à carbonila com a formação de enolfosfatos depende da afinidade eletrônica do orbital π* CO. / The first part of the discussion of this thesis reports the conformational and electronic interaction study of some α-diethoxyphosphoryl-substituted carbonyl compounds (EtO)2P(O)CH2-C(O)X (X= Me , Ph , OEt , Net2 and SEt ). This study was performed by means of infrared, 13C and 31P Nuclear Magnetic Resonance spectroscopies supported by 3-21G* and 6-31G** ab initio computations and X-ray diffraction. The IR frequencies of the single vCO band of the β-carbonylphosphonates [XC(O)CH2P(O)(OEt)2: X=Me l, Ph 2, OEt 3, Net2 4 and SEt 5] (series I), which correlate well with the vCO frequencies of the gauche rotamer of the corresponding β-carbonyl sulfones [XC(O)CH2SO2Et : X=Me 6, Ph 7, OEt 8, Net2 9 and SEt 10] (series II) along with the ab initio 6-31G** computations of 1a and 6a (X=Me) have indicated the existence of only a single gauche conformer (g1) for series I in solution. The abnormal negative carbonyl frequency shifts (ΔvCO) for both series follow approximately the electron affinities of the π*CO orbital of the parent carbonyl compounds MeC(O)X 11-15. These data have suggested that the gauche conformations of series I and II should have similar geometry. The less positive asymmetric stretching sulfonyl frequency shifts (ΔvSO2) in comparison with the phosphoryl frequencies shifts (ΔvPO) and the larger negative carbonyl gauche shifts of the β-carbonyl sulfones 6-10 in relation to the corresponding values of the β-carbonyl phosphonates 1-5 are in line with the upfield carbonyl carbon chemical shifts for series II in relation to series I. These trends are in agreement with the shorter O(SO2) ....C(CO) contact in relation to the O(PO) ... C(CO) one in compounds 6a and 1a, respectively, and are discussed in terms of the OIp → &#960*CO charge transfer which is stronger for series II than for I. This unexpected behaviour indicates that the sulfonyl oxygen atom of SO2R group is a better electron donor than the phosphoryl oxygen atom of P(O)(OR)2 group. However the intrinsic geometric parameters of O=S-CH2 and O=P-CH2 moieties seems to be responsible for this behaviour. In fact X-ray and ab initio calculations of dialkyl methylsulfonyl-methylphosphonate MeSO2CH2P(O)(OR)2 (R=Et, 18, Me 18a) gives support to this analysis. The spectroscopic analysis and ab initio computations of the diethyl alkyl(aryl)thiomethyl-phosphonates (EtO)2P(O)CH2SOnR (R=Me and Ph) and their mono- (n=l) and di- (n=2) oxigenated derivatives, showed that the phosphoryl compounds assumes a quasi-gauche conformation. This study showed that both 31P chemical shifts and the vPO frequencies correlate well with the inductive effect (σI) of the α-substituents, not taking into account any electronic interaction which could occur between the α-substituents and the phosphoryl phosphorus atom. The 13C NMR data has been interpreted by the simultaneous occurrence of the -I effect and the charge transfer between the oxygen atom of this group and the carbonyl carbon atom in the gauche rotamer of the title compounds. The 13C NMR studies corroborated the infrared data indicating that the charge transfer O(PO) → π*CO in the gauche rotamers of the serie I is poorer that the charge transfer O(SO2) → π*COfor the serie II. Furthermore, the 31P chemical shifts are almost constant along the série I. The ultraviolet spectra of compounds of the serie I showed that the introduction of the diethoxyphosphoryl group in the α position of the carbonyl compounds originate a bathochromic shift of the n→π*CO transition in relation to their parent carbonyl compounds. This behavior was ascribed to the occurrence of the hyperconjugative interaction between the σ*C-P and π*CO orbitals which stabilize the π*CO orbital in a greater extension than the -I effect of the diethoxyphosphoryl group stabilizes the nO(CO) energy level. Due to conformational similarity between the β-ketosulfoxides MeC(O)CH2S(O)Ph-X (X=Cl, H, Me and OMe) and β-ketosulfones MeC(O)CH2SO2Ph-X (X=NO2, H, Me and OMe) and X-Ph-C(O)CH2SO2Et (X=NO2, CN, H, OMe and NH2) with the β-phosphorylsulfoxides corresponding (EtO)2P(O)CH2S(O)R (R= Me ou Ph) and β-phosphorylsulfones (EtO)2P(O)CH2SO2R (R= Me ou Ph) and considering that the β-ketosulfoxides and β-ketosulfones present a moderate cytotoxicity on human mieloid leukemia K562, on Ehrlich tumor cells and human monocytic cell line (U937), excepting the case of the two latter neoplasic cells which are sensitive to the β-ketosulfoxides, it became also of interest to test the cytotoxicity of the diethyl α-methyl(phenyl)sulfinyl- and α-methyl(phenyl)sulfonylmetane-phosphonates with respect to the above mentioned tumor cells. The cytotoxicity data of the diethyl α-methylsulfinyl- and α-phenylsulfinyl- methanephosphonates indicated that the β-phosphorylsulfoxides exhibit a poorer cytotoxic effect in relation to the β-ketosulfoxides towards the tumor cells tried. The diethyl α-methylsulfonyl methanephosphonates exhibit in relation to the β-ketosulfones a similar cytotoxic effect, while the diethyl α-phenylsulfonyl-methanephosphonate did not show any apparent cytotoxic effect. Finally, this thesis deals with the reactions of the α-halo-substituted carbonyl compounds Hal-CH2C(O)X (X= Me, Ph, OEt, Net2 and SEt) with triethylphosphite in anhydrous benzene under reflux. These reactions gave variable proportions of α-diethoxyphosphoryl-substituted carbonyl compounds (EtO)2P(O)CH2 -C(O)X (X= Me, Ph, OEt, Net2 and SEt) and the corresponding enolphosphates [CH2=C(X)[P(O)(EtO)2] with the changing of the substituent directly bonded to the carbonyl group. The electron-donating substituents originate preferentially the β-ketophosphonates (Michael-Arbuzov product) while electron-withdrawing substituents gave mainly the enolphosphates (Perkow product). These trends strongly suggest that the rate-limiting step of the reactions between the α-halo-substituted carbonyl compounds and triethylphosphite is the nucleophilic addition of the phosphorus lone pair to the carbonyl carbon leading to the obtention of the enolphosphates which in its tum depends on the electron-affinity of the π*CO orbital.

Made available in DSpace on 2017-07-24T19:38:11Z (GMT). No. of bitstreams: 1 Larissa Sens.pdf: 4559561 bytes, checksum: d3bb440abdcd395ceedbf48196d1cee7 (MD5) Previous issue date: 2014-02-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This study presents theoretical results regarding the conformational equilibrium of three diferent chalcones CHALBr (2-propen-1-one, 1-(2-bromophenyl)-3-phenyl), CHALOH (2-Propen-1-one, 3-(4-hydroxyphenyl)-1-phenyl) and CHALBrOH (2-Propen-1-one, 1-(2- bromophenyl)-3-(4-hydroxyphenyl). / Nesse trabalho são apresentados resultados teoricos referentes ao equilibrio conformacional de tres diferentes chalconas, CHALBr [1-(2-bromofenil)-2-propen-3-fenil-1-ona], CHALOH [1-fenil-2-propen-3-(4-hidroxifenil)-1-ona] e CHALBrOH [1-(2-bromofenil)-2-propen-3-(4- hidroxifenil)-1-ona].

Scaffold proteins facilitate many aspects of intracellular signaling. These proteins can regulate two or more proteins in the same pathway, or coordinate signaling from multiple pathways. Scaffold proteins are therefore key control points for the flux of signaling and play essential roles in biological systems. There are four possible mechanisms by which scaffold proteins achieve activation and propagate signaling: 1) rigid protein binding between two or more proteins to co-localize binding partners, 2) ligand-induced activation such as may result from a conformational change, 3) disorder-to-order transition where the scaffold protein folds as a result of a protein-protein interaction, and 4) dynamic processes such as phosphorylation. The scaffold protein NF-κB essential modulator (NEMO) functions via ligand-induced activation and serves as the key control point for canonical NF-κB signaling. The work described in this thesis investigates the role of a previously uncharacterized domain within NEMO that is required for function, which we term the Intervening Domain (IVD). Bioinformatic analysis reveals a high level of sequence conservation across species within this domain. Conformational changes following ligand binding are observed for NEMO and these changes require conserved sequences in the IVD. Additionally, a functional IVD is shown to increase the binding affinity of NEMO for IKKβ, enhance the thermal stability of NEMO, and is required to propagate NF-κB signaling in cells. A fluorescence-based assay is also developed to characterize the formation of a complex composed of NEMO, a zinc ion, and IκBα. A separate fluorescence-based assay is developed to measure IKK activity and is used to determine that NEMO alone or in the presence of linear tetraubiquitin does not enhance the rate of IKKβ phosphorylation of an IκBα-derived peptide. Furthermore, a number of organic small molecules and macrocycles are screened against the NEMO-IKKβ interaction. One small molecule was validated as an inhibitor and its biophysical properties and inhibition kinetics are described in this thesis. These analyses represent the first characterization of a highly conserved domain required for the function of the key control point in NF-κB signaling. The IVD domain of NEMO could be targeted for development of an allosteric effector for therapeutic discovery.

Les récepteurs métabotropes au glutamate (mGluR) sont des RCPG de classe C. Ils sont exprimés dans le système nerveux central où, suite à l'activation par le glutamate, ils participent à la modulation de la transmission nerveuse. En raison de leur rôle essentiel dans la régulation de l'activité synaptique, ils représentent des cibles potentielles pour le développement de médicaments contre les troubles neurologiques et psychiatriques telles que la schizophrénie, l'épilepsie, l'anxiété et la douleur. Mon projet de recherche de doctorat a porté sur l'étude du mécanisme d'activation du domaine extracellulaire de liaison au ligand du mGluR (ECD), avec un accent particulier sur ce qui différencie au niveau moléculaire un agoniste partiel d'un agoniste total. A cette fin, j'ai utilisé une méthode innovante à l'échelle de la molécule unique appelée Transfert d'Energie par Résonance de Forster, développé pour l'étude de la dynamique conformationnelle des molécules individuelles à l'échelle de la nanoseconde. J'ai réussi à montrer que le dimère d'ECD oscille entre une conformation active et une conformation de repos sur une échelle de temps de ~100μsec et que les ligands influencent les vitesses de transition entre ces états avec des vitesses intermédiaires pour les agonistes partiels. Ces résultats sont validés par l'utilisation de mutants spécifiques et indiquent clairement que le rôle des ligands n'est pas de stabiliser une conformation donnée mais de modifier le comportement dynamique du récepteur. L'ensemble de ces résultats contribuent à une meilleure description du mécanisme d'activation des mGluRs, et ouvrent potentiellement la voie à la compréhension des RCPG en général. / Metabotropic Glutamate Receptors (mGluRs) are class C GPCRs, expressed throughout the central nervous system. They participate in the long term modulation of neural transmission following activation by the excitatory neurotransmitter glutamate. This critical role in the regulation of synaptic activity makes them promising targets in the development of drugs for the treatment of various neurologic and psychiatric disorders such as schizophrenia, epilepsy, anxiety and pain relief. My Ph.D. research project has focused on the study of the activation mechanism of the mGluR extracellular ligand binding Venus-Flytrap domain (VFT), with particular emphasis on the differences between partial and full agonists on a molecular level. To this aim, I have used a state-of-the-art single molecule Förster Resonance Energy Transfer (smFRET) approach, developed for the study of conformational dynamics of single molecules on the nanosecond to millisecond timescale. I have managed to show that the VFT-dimer constantly oscillates between an active and a resting conformation on a ~100µsec timescale. I also discovered that the role of ligands is to influence the transition rate between these boundary states, and that partial agonists display intermediate transition rates. My results, supported by the use of specific mutants, clearly indicate that the role of ligands is not to stabilize a given conformation but to modify the overall dynamic of the receptor, which favors a conformational selection mechanism. Altogether, these results represent a most-valuable contribution to the better understanding of the activation mechanism of mGluRs, and potentially GPCRs in general.

A influência da formação de ligações de hidrogênio (LHs) em sistemas moleculares é investigada analisando-se as propriedades de aglomerados próton-ligados e de moléculas em solventes próticos. As estruturas supermoleculares dos aglomerados são obtidas por otimização de geometria com diferentes métodos quânticos ab initio. Por sua vez, as estruturas supermoleculares dos solventes dependem de condições termodinâmicas e, portanto, são obtidas utilizando-se técnicas de simulação Monte Carlo, em que as interações intermoleculares são parametrizadas pelos potenciais de Lennard-Jones e de Coulomb. Os efeitos decorrentes da formação de LHs nos aglomerados moleculares são analisados a partir de suas prioridades: estabilidades, espectros vibracionais, constante rotacionais e dipolos elétricos. Estas propriedades são calculadas usando-se métodos perturbativos (MBPT/CC) e teoria do funcional da densidade(DFT). Para as moléculas solvatadas em meio líquido, os efeitos das LHs são analisadas na interação soluto-solvente duranto o processo de equilíbrio conformacional de fuufural em solventes próticos. A contribuição da interação do soluto com o meio também é investigada no espectro de absorção UV-vis do sistema furfural/água. Os resultados destes estudos mostram que a formação de LHs é importante para explicar o comportamento de algumas propriedades de sistemas moleculares próton-ligados, principalmente de aglomerados. No caso de sistemas em fase líquida, as LHs dependem, basicamente, do caráter prótico e da polaridade do solvente, propriedades que concorrem para a estabilização de possíveis formas conformacionais da molécula do soluto. / The influence of hydrogen bond formation in molecular systems is investigated analyzing the properties of both hydrogen-bonded clusters and molecules in protic solvents. The super-molecular structures of the cluster are obtained by optimizing the geometries with different ab initio quantum methods. On the other hand, the super-molecular structures of the solvents depend on thermodynamic conditions and, so, are obtained by using Monte Carlo simulation techniques, where the intermolecular interactions are accounted for the Lennard-Jones and Coulomb pair-potentials. The effects due to H-bond formation in the molecular clusters are analyzed from their properties: stabilities, vibrational spectra, rotational constants, and electric dipoles. These properties are calculated using perturbation methods (MBPT/CC) and density functional theory (DFT). For the molecules in liquid solvents, the H-bond effects are analyzed in the solute-solvent interaction during the conformational equilibrium process of furfural in protic solvents. The contribution of the interaction of the solute with the medium is also investigated in the UV-vis absorption spectrum of the furfural/water system. The results from these studies show that the H-bonding process is important to account for the behavior of some properties of H-bonded molecular systems, mainly of clusters. In the case of liquid-phase systems, the H-bonds depend basically on both the protic nature and polarity of the solvent, properties that come together to stabilize possible conformational forms of the solute molecules.

Regulation of transcription elongation by P-TEFb is critical for proper gene expression and cell survival. The cell possesses large quantities of P-TEFb, but the vast majority of it is locked away and inactive in the 7SK snRNP. Since the discovery of the 7SK snRNP, research has been conducted to determine how P-TEFb is released from this complex. The goal of the research presented in this thesis is to better understand how the 7SK snRNP regulates P-TEFb and ultimately, gene expression. This work documents the discovery and characterization of the 7SK stability protein LARP7. LARP7 is is associated with 7SK regardless of the presence of P-TEFb and HEXIM1. Stabilization of 7SK is essential for maintenance of the RNP because loss of LARP7 results in an increase in free P-TEFb and a significant reduction in the amount of 7SK. These results indicate that stabilization of the 7SK snRNP by LARP7 is important for regulating P-TEFb homeostasis. Although P-TEFb was first characterized from Drosophila lysates, the conservation of the 7SK snRNP and the mechanisms regulating P-TEFb inhibition have not been described. Here, the Drosophila melanogaster homologues of LARP7 and 7SK are characterized. These studies show that the system of P-TEFb regulation is similar in flies and this makes Drosophila an attractive model system for studying P-TEFb regulation through embryonic and larval development. Finally, factors and modifications involved in releasing P-TEFb directly are explored. An assay was developed for discovering proteins that can bind to and release P-TEFb from the 7SK snRNP. Use of this assay showed that post-translational modification of the components of the 7SK snRNP do not cause P-TEFb release directly. However, HIV Tat and the C-terminal P-TEFb binding region of the bromodomain containing protein, Brd4, are capable of extracting P-TEFb directly. Most importantly, the release of P-TEFb is followed by a conformational change in 7SK RNA that prevents the continued binding of HEXIM1 to the complex. P-TEFb release from the 7SK snRNP is the result of direct extraction of P-TEFb by viral or cellular proteins, and not post-translational modifications or a competition between HEXIM1 and hnRNP proteins for 7SK binding.

By using the technology of molecularly imprinted polymer (MIP), we propose to synthesize a protein conformational imprint that also acts as a plastic enzyme, inducing protein structural transitions. The imprint aims at MIP-induced stabilization and / or formation of bound protein secondary structure and the applications associated with analysis and correction of misfolded proteins. The screening of polymeric functional monomers being able to induce the conformational transitions in proteins is investigated in this report. The fluoro-silanes (3-heptafluoroisopropoxy)propalethoxysilane (7F) and 3,3,3-trifluoropropylmethoxysilane (3F) were employed as functional monomers for synthesis of this catalytic protein conformational imprint via sol-gel reactions. 3F was demonstrated superior to 7F for fluoro-modification of tetraethylorthosilicate (TEOS) gel in terms of retaining gel transparency and increasing hydrophobicity while maintaining a uniform distribution of encapsulated protein. Both hydrolyzed 3F and polymerized 3F exhibited strong influences on structure transitions of three template proteins: bovine serum albumin (BSA), beta-lactoglobulin (BLG), and bovine carbonic anhydrase (BCA). The formation of molten globule intermediates that stabilized by increased alpha-helices was induced by the trifluoro-silane in BLG and BCA. Additionally, 3F was effective at a lower concentration than the benchmark fluoro-alcohol 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), validating the application of 3F as a functional monomer for protein conformational imprinting.

Deposition of protein fibers with a characteristic cross-β sheet structure is the molecular marker associated with human disorders ranging from Alzheimer's disease to type II diabetes and spongiform encephalopathy. Given the large number of non-disease related proteins and peptides that have been shown to form amyloid fibrils in vitro, it has been suggested that amyloid fibril formation represents a generic protein phase transition. In the last two decades it has become clear that the same protein/peptide can assemble into distinct morphologically and structurally amyloid aggregates depending on the solution conditions. Moreover, recent studies have shown that the early stage, oligomeric amyloid assemblies are the main culprit in vivo. We have investigated the amyloid assemblies formed under denaturing conditions for Hen Egg White Lysozyme (HewL) whose human homologue is directly implicated in hereditary non-neuropathic systemic amyloidosis. Our early investigations showed that HewL can aggregate via at least two distinct assembly pathways depending on solution ionic strength at fixed pH, temperature, and protein concentration. By combining Dynamic Light Scattering (DLS), Static Light Scattering (SLS) and Atomic Force Microscopy (AFM) we showed that at low ionic strength, the pathway is characterized by the nucleation and growth of long (several micron), rigid fibrils (RF) via monomers assembly. A second, high ionic strength pathway is characterized by the rapid assembly of monomers into globular oligomers that further polymerize into curvilinear fibrils (aO/CF). At NaCl concentrations above 400 mM, aggregation resulted in precipitate formation. Next, we used Foureir Transform Infrared spectroscopy (FTIR) and an amyloid-specific dye, Thioflavin T (ThT), to show that both RF and (a)O/CF are amyloidogenic species, but they have detectable structural differences. Moreover, we have determined that each assembly pathway has unique SLS, DLS, FTIR and ThT response signatures that help determine the assembly type prior to AFM imaging of aggregates. Taking advantage of the morphological, structural and kinetic signatures for the two distinct HewL amyloid aggregates I mapped out their amyloid aggregates phase diagram spanning over two orders of magnitude in protein concentration and from 50 to 800 mM NaCl in ionic strength. This is the most complete phase diagram for amyloid aggregates of a given protein up to date. The phase diagram has three distinct regions delineated by sharp boundaries. The RF- aO/CF was called Critical Oligomer Concentration, and we commonly refer to “above the COC” as the region were aO/CF are kinetically favored.. In the region of low salt/high protein concentrations, RF were the only amyloid species to nucleate and grow. As both salt and protein concentrations increase, aO/CF become the kinetically favored species, and RF nucleate and grow after several days of incubation. At high protein and high salt concentrations, aO/CF form very fast and eventually lose solubility forming a precipitate (Ppt). Cross-seeding experiments showed that RF is the thermodynamically stable aggregate phase, while the O/CF are the metastable species. Finally, we used the phase diagram to design experiments that would allow us to reveal the RF nucleation mechanism in presence of aO/CF. RF nucleation above the COC can undergo either via internal restructuring of aO/CF (NCC) or through a random coalescence of monomers into a nucleus (NP). The experimental results obtained so far strongly indicate that RF nucleate via NP mechanism both below and above the COC.

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.