Littérature scientifique sur le sujet « Proteinaceou »

La present tesi està formada per sis treballs de recerca independents que estan situats en el camp dels inhibidors de metal·locarboxipeptidases: s'estudia el seu plegament, estabilitat, estructura i funció. <br/>El primer treball inclou l'aïllament i el clonatge de l'ADNc d'un nou inhibidor de carboxipeptidases de la paparra, anomenat TCI. Els estudis realitzats sobre la forma recombinant del TCI indiquen que aquesta proteïna està fortament limitada pels seus ponts disulfur, sent molt estable enfront un ampli rang de pH i altament resistent a condicions desnaturalitzants. El TCI recombinant és un inhibidor que s'uneix fortament a TAFIa, estimulant així la fibrinòlisi in vitro, fet que li confereix potencial en aplicacions de prevenció o tractament de desordres trombòtics.<br/>El segon treball presenta l'estructura cristal·lina del TCI unit a la carboxipeptidasa A bovina i a la carboxipeptidasa B humana. El TCI està format per dos dominis que són estructuralment molt semblants a pesar de tenir un baix grau d'homologia seqüencial. Cada domini inclou una hèlix a curta seguida per una petita fulla b antiparal·lela girada i presenta una alta homologia estructural enfront proteïnes de la família de les b-defensines. El TCI s'ancora a la superfície de les carboxipeptidases de mamífer en un mode d'unió doble que no s'havia observat abans per cap altre inhibidor de carboxipeptidases.<br/>En el tercer treball s'examina la contribució de cadascun dels aminoàcids del lloc d'unió secundari de l'inhibidor de carboxipeptidases de patata (PCI) de cara a les propietats generals d'aquesta proteïna. Els estudis estructurals i enzimàtics demostren que el posicionament correcte del lloc d'unió primari per una unió i inhibició eficient de la carboxipeptidasa A depèn significativament del lloc d'unió secundari. A més, l'estudi comparatiu del plegament oxidatiu d'una sèrie de mutants del PCI utilitzant una aproximació de captura dels ponts disulfur indiquen que les forces no covalents dirigeixen el replegament d'aquesta petita proteïna rica en ponts disulfur a l'etapa de "reshuffling", que és el pas limitant del procés de plegament del PCI.<br/>En el quart treball s'ha determinat els camins de plegament oxidatiu i desplegament reductor de l'inhibidor de carboxipeptidases de sangonera (LCI). L'LCI reduït i desnaturalitzat és replega ràpidament a través d'un flux seqüencial d'espècies amb un, dos, tres i quatre ponts disulfur fins assolir la forma nativa. Dins dels intermediaris de plegament de l'LCI hi trobem dues espècies predominants de tres ponts disulfur (anomenades III-A i III-B) i una població heterogènia d'isòmers "scrambled" (quatre ponts disulfur no natius) que s'acumulen consecutivament al llarg de la reacció de plegament. Aquest estudi revela que les formes III-A i III-B contenen exclusivament ponts disulfur natius i corresponen a espècies estables i parcialment estructurades que s'interconverteixen entre elles assolint l'equilibri abans que la formació dels isòmers "scrambled" tingui lloc. <br/>En el cinquè treball s'ha purificat l'intermediari III-A directament de la reacció de plegament i se l'ha caracteritzat estructuralment per RMN. Els resultats mostren que aquesta espècie presenta una estructura força nativa, tot i que li manquen alguns elements d'estructura secundària per la qual cosa és més flexible que l'LCI natiu. III-A representa el primer exemple d'un intermediari "disulfide insecure" ("ponts disulfur insegurs") determinat estructuralment. L'oxidació directa d'aquesta espècie cap a la proteïna totalment nativa sembla estar restringida per l'enterrament de les seves dues cisteïnes dins d'una estructura semblant a la nativa. També s'han utilitzat aproximacions teòriques basades en constriccions topològiques que prediuen força bé la presència d'aquest intermediari de plegament. <br/>L'últim treball d'aquesta tesi tracta sobre l'altre intermediari majoritari de plegament de l'LCI. En aquest treball s'ha construït i analitzat extensivament un anàleg d'aquest intermediari. Els resultats obtinguts mostren que aquesta proteïna presenta una estructura global i una activitat semblants a les de la forma salvatge de l'LCI. A més, presenta un procés de plegament més ràpid i eficient que l'LCI salvatge. Tanmateix, aquest anàleg està fortament desestabilitzat, fet que indica que el quart pont disulfur dóna alta estabilitat i especificitat estructural a l'LCI.<br>The present thesis consists of six independent research works that are situated in the field of metallocarboxypeptidase inhibitors: their folding, stability, structure and function are studied. <br/>The first work comprises the isolation and cDNA cloning of a new carboxypeptidase inhibitor from ticks, named TCI. The studies performed on the recombinant form of TCI indicate that this protein is strongly constrained by its disulfide bonds, being unusually stable over a wide pH range and highly resistant to denaturing conditions. As a tight binding inhibitor of TAFIa, TCI stimulates fibrinolysis in vitro and thus may have potential for applications to prevent or treat thrombotic disorders.<br/>The second work presents the crystal structure of TCI in complex with either bovine carboxypeptidase A or human carboxypeptidase B. The structure of TCI consists of two domains that are structurally similar despite the low degree of sequence homology. The domains, each consisting of a short a-helix followed by a small twisted antiparallel b-sheet, show high structural homology to proteins of the b-defensin-fold family. Also, TCI anchors to the surface of mammalian carboxypeptidases in a double-headed manner not previously seen for carboxypeptidase inhibitors. <br/>In the third part, the role of each residue of the secondary binding site of potato carboxypeptidase inhibitor (PCI) in the folding, structure and function of this protein is determined. Structural and enzymatic studies demonstrate that the proper positioning of the primary site for efficiently binding and inhibition of carboxypeptidase A is significantly dependent on such a secondary contact region. In addition, a comparative study of the oxidative folding of several PCI mutants indicates that noncovalent forces drive the refolding of this small disulfide-rich protein at the reshuffling stage, the rate-limiting step of the process. <br/>The fourth work elucidates the oxidative folding and reductive unfolding pathways of leech carboxypeptidase inhibitor (LCI). Reduced and denatured LCI refolds rapidly through a sequential flow of 1-, 2-, 3-, and 4-disulfide (scrambled) species to reach the native form. Folding intermediates of LCI comprise two predominant 3-disulfide species (III-A and III-B) and a heterogeneous population of scrambled isomers that consecutively accumulate along the folding reaction. Our study reveals that forms III-A and III-B exclusively contain native disulfide bonds and correspond to stable and partially structured species that inter-convert, reaching an equilibrium prior to the formation of the scrambled isomers. <br/>In the fifth work, the III-A intermediate is directly purified from the folding reaction and structurally characterized by NMR spectroscopy. The data shows that this species displays a highly native-like structure although it lacks some secondary structure elements being more flexible than native LCI. III-A represents the first structurally determined example of a disulfide insecure intermediate; direct oxidation of this species to the fully native protein seems to be restricted by the burial of its two free cysteine residues inside a native-like structure. We show also that theoretical approaches based on topological constrains predict with good accuracy the presence of this folding intermediate. <br/>The last work of this thesis deals with the other major folding intermediate of LCI: III-B intermediate. In this work, an analog of this intermediate is constructed and extensively analyzed. The derived data shows that this protein displays the same overall structure, a similar activity, a faster and more efficient folding process than wild-type LCI, but a lower stabilization, which indicates that the fourth disulfide bond provides LCI with both high stability and structural specificity.

La maturation des ARNt en 5' est réalisée par RNase P. C'est un ribozyme chez les bactéries, les fungi et les nuclei des mammifères et un enzyme protéique dans les plantes ou des organelles des mammifères qui s’appelle PRORP. Il y a trois PRORP dans A. thaliana. PRORP contiennent deux domaines : un domaine PPR qui reconnaît spécifiquement des séquences d'ARN et un domaine nucléase qui assure la coupure endonucléolytique 5' des précurseurs d’ARNt. Pendant ma thèse j'ai pu montré par des méthodes biophysiques et structurales comme SRCD et SAXS que PRORP1 et 2 sont composées en majorité des hélices alpha Elles ont un rayon de giration de 33 Å et contiennent deux domaines distincts avec et une dimension maximale de 110 Å. Pour le complex entre un substrat d'ARNt et PRORP une constante de dissociation de 1 uM a pu être confirmé par la microcalorimétrie, la thermophorèse et l'ultracentrifugation analytique. Ces analyses nous ont permis de construire un modèle PRORP et un substrat d'ARNt<br>RNase P cleaves 5’ leaders of precursor tRNAs. RNase P is a ribozyme in bacteria, fungi and animal nuclei and a protein in animal organelles, plants and many other organism. There are three PRORPs in A. thaliana. MALS, SRCD and SAXS provided first structural information: 1) PRORPs are monomers in solution. 2) PRORP 1-2 have a high alpha-helical content. 3) PRORPs are composed of two distinct domains with a radius of gyration of 33 A. These results together with homology modelling enabled us to build a first model of PRORPs in complex with tRNA. Using three different methods, isothermal titration calorimetry, microscale thermophoresis and analytical ultracentrifugation, a binding constant of about 1 µM could be determined for the system PRORP2mDD and L5T0 tRNA. This helped us conducting a SAXS experiment taking into account the low resolution affinity and designed to provide the direct structural data of a complex of proteinaceous RNase P with a substrate tRNA

Soil carbon (C) and nitrogen (N) are predominantly in organic form. Proteins/ peptides, as an important organic form of N, constitute a substantial part of soil organic matter. On one hand, proteins/peptides are an important N source for plants and microorganisms, particularly in soils where inorganic N is limited. On the other hand, their stabilization in soils by forming organo-mineral associates or macromolecule complex reduces the C loss as CO2 into the atmosphere. Therefore, studies on the turnover, abundance, composition, and stability of proteins/peptides are of crucial importance to agricultural productivity and environmental sustainability. In the first part of this study, the bioavailability and distribution of amino acids, (building block of proteins/peptides), were investigated, in soils across the North-South and West-East transects of continental United States. The second part of this study aimed to understand the variations of organic C speciation in soils of continental United States. Previous investigations of the interactions between soil minerals and proteins/peptides were mostly limited to batch sorption experiments in labs, seldom of which gave the details at the molecular scales. Therefore, in the third part of this study, the molecular orientation of self-assembled oligopeptides on mineral surfaces was investigated by employing synchrotron based polarization-dependent Near Edge X-ray Adsorption Fine Structure Spectroscopy (NEXAFS) techniques. Specific aims of this study were: 1) to assess potentially bioavailable pool of proteinaceous compounds and the immediately bioavailable pool of free amino acids in surface and subsurface soils of various ecosystems; 2) to evaluate the relationship between environmental factors and levels/composition of the two pools; 3) to investigate the organic C speciation in soils of various land use; and 4) to understand molecular level surface organization of small peptides on mineral surfaces. The levels of free amino acids and hydrolysable amino acids which represent the potentially bioavailable pool of proteinaceous compounds in A-horizon soils were significantly high than in C-horizon soils due to the accumulation of organic matter in surface. On average, free amino acids accounted for less than 4 % of hydrolysable amino acids which represent the total proteinaceous compounds in soils. The composition of free amino acids was significantly different between surface soil and subsurface soil and was significantly influenced by mean annual temperature and precipitation. A relatively uniform composition of hydrolysable amino acids was observed irrespective of a wide range of land use. Significant variations were observed for the levels of free and hydrolysable amino acids along mean annual temperature and precipitation gradients, as well as among vegetation types of continental USA, suggesting levels of free and hydrolysable amino acids were associated with the above-ground biomass and root distribution. Organic C speciation investigation revealed the presence of carboxylic-C (38%), aliphatic-C (~ 22%), aromatic-C (~ 18%), O/N-alkyl-C (~ 16%), and phenolic-C (< 6%). Factors such as temperature and vegetation cover were revealed in this study to account for the fluctuations of the proportions of aromatic-C and phenolic-C, in particular. Phenolic-C may serve as a good indicator for the effect of temperature or vegetation on the composition of SOC. The average composition of soil organic C, over the continental scale, was relatively uniform over various soil ecosystems and between two soil horizons irrespective of surface organic C content. Polarization dependent NEXAFS analysis showed the oligopeptides tend to orient on mineral surface with an average tilt angle of 40 ° between the molecular chain and the mineral surface.<br>Ph. D.

The emerging evidence of preferential accumulation and long residence time of proteinaceous compounds in soil are counter to the traditional view that their structure is readily broken down through microbial activity. The shift in thinking of their residence time is, however, heavily influenced by physical and chemical protections in soil, representing an important change for understanding global biogeochemical carbon and nitrogen cycling. We investigated the accumulation patterns of proteinogenic amino acids for a long term (thousands of years) related to their sources and sinks. We found clear patterns of change in the amino acids in a 4000 year-chronosequence adjacent to Lake Michigan, USA (Michigan chronosequence) and they were tightly related to the shifts in their biological sources, namely aboveground vegetative community (r2=0.66, p<0.0001) and belowground microbial community (r2=0.71, p<0.0001). Results also showed great variations of approximately 49% between seasons (summer and winter). Moreover, seasonal dynamic patterns (22% variations) of the amino acids in soil mineral associated fraction were rather counter to the conceptual view that it represents a slow soil organic pool with long residence times. The amino acids enriched in the mineral associated fraction, (e.g., positively charged, aromatic, and sulfur containing amino acids), tended to preferentially accumulate in whole soil pool during the 4000 years of ecosystem development. Their interaction with soil minerals, therefore, may play a critical role in the long-term sink and selective accumulation of proteinaceous compounds with some degree of the displacement. This was further confirmed by another chronosequence system near Haast River, New Zealand, which is geologically separated and climatically- and ecologically- different from the Michigan chronosequence. Common trends between two chronosequences suggested that either polar interactions or redox reactions may be relatively more important in the mineral interaction of amino acids than non-polar interactions. The consistency of results at two disparate locations in the southern and northern hemispheres is strong evidence that the processes of pedogenesis and ecosystem development are parsimonious and predictable. Our research demonstrated fundamental understanding of behavior of proteinaceous compounds at the molecular species level, and further provided their partitioning mechanisms associated with soil components.<br>Ph. D.

Organic nitrogen containing compounds represent the vast majority of natural N inputs to soils and of these, proteins, peptides and amino acids are the most significant. In order to facilitate the elucidation of both the nature and rates of the processes by which these vital nutrients are transformed in soils, an understanding of their fate at a mole; level is vital. This thesis aimed to utilise soil incubation experiments with dual (¹⁵N- and ¹³C-) labelled substrates, i.e. a suite of amino acids, individual amino acids and more complex substrates, i.e. protein and cow dung, as isotopically labelled trace enabling their N and C to be followed over time. A molecular level approach was adopted which included the application of chemically and isotopically defined substrates to soil and the use of compound-specific stable isotope (N and C) analysis to trace the fate of label into the soil amino acids (and in selected cases phospholipids, C only).