Análise estrutural e funcional da interação física entre eIF5A e suas enzimas modificadoras em Saccharomyces cerevisiae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inhibition of Lysozyme by Taurine Dibromamine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Oxidação de lisozima por haloaminas: caracterização química e efeitos biológicos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Síntese, estrutura e atividade de peptídeos derivados de ParD, o antídoto do módulo toxina-antitoxina ParDE

Pós-graduação em Biotecnologia - IQ

Oxidation of Bovine Albumin by Hypochlorous and Hypobromous Acids: Structural and Functional Alterations

Aims: Hypochlorous (HOCl) and hypobromous (HOBr) acids are among the most powerful oxidants produced by the innate immune cells. Albumin is the predominant protein in most body fluids and is considered the most important antioxidant of blood plasma. Study Design: Oxidation of bovine albumin (BSA) and study of its structural and functional alterations. Place and Duration of Study: Faculty of Science and Faculty of Pharmaceutical Science, University of the State of Sao Paulo UNESP, between June and December 2012. Methodology: BSA was oxidized with excess of HOCl or HOBr and its structural and functional alterations were analyzed by spectroscopic techniques as UV-Vis absorption, intrinsic and synchronous fluorescence, fluorescence quenching, Rayleigh scattering and circular dichroism. Results: Both oxidants were able to deplete the intrinsic fluorescence of BSA, but HOBr was more effective than HOCl. The alterations in the synchronous fluorescence, UV-Vis absorption, and the appearance of a fluorescence band centered at 450 nm confirmed the difference between the oxidants. The oxidation did not induce aggregation of BSA as measured by Rayleigh scattering. The far-UV circular dichroism spectra showed a loss in the helical content and the near-UV-circular dichroism showed an alteration in the tertiary structure; HOBr was the more effective of the oxidants in this case. However, the oxidations did not induce significant alterations in the binding capacity of BSA, which was evaluated using hydrophobic (norfloxacin) and hydrophilic (ascorbic acid) drugs. Conclusion: These results suggest that, although highly susceptible to oxidation, the alterations did not inhibit BSA’s physiological function as a transport protein.

Low resolution structural characterization of the Hsp70-interacting protein - Hip - from Leishmania braziliensis emphasizes its high asymmetry

The Hsp70 is an essential molecular chaperone in protein metabolism since it acts as a pivot with other molecular chaperone families. Several co-chaperones act as regulators of the Hsp70 action cycle, as for instance Hip (Hsp70-interacting protein). Hip is a tetratricopeptide repeat protein (TPR) that interacts with the ATPase domain in the Hsp70-ADP state, stabilizing it and preventing substrate dissociation. Molecular chaperones from protozoans, which can cause some neglected diseases, are poorly studied in terms of structure and function. Here, we investigated the structural features of Hip from the protozoa Leishmania braziliensis (LbHip), one of the causative agents of the leishmaniasis disease. LbHip was heterologously expressed and purified in the folded state, as attested by circular dichroism and intrinsic fluorescence emission techniques. LbHip forms an elongated dimer, as observed by analytical gel filtration chromatography, analytical ultracentrifugation and small angle X-ray scattering (SAXS). With the SAXS data a low resolution model was reconstructed, which shed light on the structure of this protein, emphasizing its elongated shape and suggesting its domain organization. We also investigated the chemical-induced unfolding behavior of LbHip and two transitions were observed. The first transition was related to the unfolding of the TPR domain of each protomer and the second transition of the dimer dissociation. Altogether. LbHip presents a similar structure to mammalian Hip, despite their low level of conservation, suggesting that this class of eukaryotic protein may use a similar mechanism of action. (C) 2012 Elsevier Inc. All rights reserved.

Interaction of gramicidin with DPPC/DODAB bilayer fragments

The interaction between the antimicrobial peptide gramicidin (Gr) and dipalmitoylphosphatidylcholine (DPPC)/dioctadecyldimethylammonium bromide (DODAB) 1:1 large unilamellar vesicles (LVs) or bilayer fragments (BFs) was evaluated by means of several techniques. The major methods were: 1) Gr intrinsic fluorescence and circular dichroism (CD) spectroscopy; 2) dynamic light scattering for sizing and zeta-potential analysis; 3) determination of the bilayer phase transition from extrinsic fluorescence of bilayer probes; 4) pictures of the dispersions for evaluation of coloidal stability over a range of time and NaCl concentration. For Gr in LVs, the Gr dimeric channel conformation is suggested from: 1) CD and intrinsic fluorescence spectra similar to those in trifluoroethanol (TFE); 2) KCl or glucose permeation through the LVs/Gr bilayer. For Gr in BFs, the intertwined dimeric, non-channel Gr conformation is evidenced by CD and intrinsic fluorescence spectra similar to those in ethanol. Both LVs and BFs shield Gr tryptophans against quenching by acrylamide but the Stern-Volmer quenching constant was slightly higher for Gr in BFs confirming that the peptide is more exposed to the water phase in BFs than in LVs. The DPPC/DODAB/Gr supramolecular assemblies may predict the behavior of other antimicrobial peptides in assemblies with lipids. (C) 2012 Elsevier B.V. All rights reserved.

Zwei-Photonen-Fluoreszenzmikroskopie und -Spektroskopie von Sauerstofftransportproteinen

In dieser Arbeit wurde die intrinsische Tryptophanfluoreszenz von Proteinen nach Zwei-Photonen-Anregung untersucht. Als interessantes Modellsystem wurde das Sauerstofftransportprotein der Vogelspinne Eurypelma californicum gewählt. Zum einen besitzt das Protein 148 Tryptophan-Seitenketten, so daß deren geringer Absorptionsquerschnitt kompensiert werden kann und eventuell einzelne Proteine aufgrund ihrer Tryptophanfluoreszenz detektiert werden können. Zum anderen signalisiert diese Fluoreszenz die Sauerstoffbeladung, so daß die kooperative Sauerstoffbindung auf Einzelmolekülebene untersucht werden könnte. Als limitierender Faktor hat sich die Photostabilität der Tryptophane nach Zwei-Photonen-Anregung herausgestellt. Im Mittel können von einem Hämocyanin-Molekül drei Photonen detektiert werden, bevor alle 148 Tryptophan-Seitenketten geblichen sind. Dies ist ein für Einzelmolekülspektroskopie äußerst niedriger Wert. Trotz dieser geringen Photostabilität ist es zum erstem Mal gelungen, die Diffusion einzelner Proteine mit Hilfe ihrer intrinsischen Tryptophanfluoreszenz zu beobachten. Wenn in einer geeigneten Umgebung die Photostabilität der Tryptophane höher ist, so reicht die Zahl der detektierten Photonen aus, um einzelne Teilchen abzubilden. Überraschend hat sich ergeben, daß es möglich ist, durch Lichteinstrahlung von außen in das Sauerstoffbindungsgleichgewicht einzugreifen und die Reaktion des Proteins auf die Auslenkung aus dem Gleichgewichtszustand zu beobachten. Das intensitätsabhängige Sauerstoffbindungsverhalten wurde modelliert und an die Messungen angepaßt. Die lichtinduzierte Sauerstoffabgabe führt anscheinend nicht zu einem Konformationswechsel.

Development and applications of hollow fiber flow field-flow fractionation in the bioanalytical field. Studies of aggregation phenomena in complex protein samples

Recent advances in the fast growing area of therapeutic/diagnostic proteins and antibodies - novel and highly specific drugs - as well as the progress in the field of functional proteomics regarding the correlation between the aggregation of damaged proteins and (immuno) senescence or aging-related pathologies, underline the need for adequate analytical methods for the detection, separation, characterization and quantification of protein aggregates, regardless of the their origin or formation mechanism. Hollow fiber flow field-flow fractionation (HF5), the miniaturized version of FlowFFF and integral part of the Eclipse DUALTEC FFF separation system, was the focus of this research; this flow-based separation technique proved to be uniquely suited for the hydrodynamic size-based separation of proteins and protein aggregates in a very broad size and molecular weight (MW) range, often present at trace levels. HF5 has shown to be (a) highly selective in terms of protein diffusion coefficients, (b) versatile in terms of bio-compatible carrier solution choice, (c) able to preserve the biophysical properties/molecular conformation of the proteins/protein aggregates and (d) able to discriminate between different types of protein aggregates. Thanks to the miniaturization advantages and the online coupling with highly sensitive detection techniques (UV/Vis, intrinsic fluorescence and multi-angle light scattering), HF5 had very low detection/quantification limits for protein aggregates. Compared to size-exclusion chromatography (SEC), HF5 demonstrated superior selectivity and potential as orthogonal analytical method in the extended characterization assays, often required by therapeutic protein formulations. In addition, the developed HF5 methods have proven to be rapid, highly selective, sensitive and repeatable. HF5 was ideally suitable as first dimension of separation of aging-related protein aggregates from whole cell lysates (proteome pre-fractionation method) and, by HF5-(UV)-MALS online coupling, important biophysical information on the fractionated proteins and protein aggregates was gathered: size (rms radius and hydrodynamic radius), absolute MW and conformation.

Monitoring of tumor response to Cisplatin using optical spectroscopy

INTRODUCTION Anatomic imaging alone is often inadequate for tuning systemic treatment for individual tumor response. Optically based techniques could potentially contribute to fast and objective response monitoring in personalized cancer therapy. In the present study, we evaluated the feasibility of dual-modality diffuse reflectance spectroscopy-autofluorescence spectroscopy (DRS-AFS) to monitor the effects of systemic treatment in a mouse model for hereditary breast cancer. METHODS Brca1(-/-); p53(-/-) mammary tumors were grown in 36 mice, half of which were treated with a single dose of cisplatin. Changes in the tumor physiology and morphology were measured for a period of 1 week using dual-modality DRS-AFS. Liver and muscle tissues were also measured to distinguish tumor-specific alterations from systemic changes. Model-based analyses were used to derive different optical parameters like the scattering and absorption coefficients, as well as sources of intrinsic fluorescence. Histopathologic analysis was performed for cross-validation with trends in optically based parameters. RESULTS Treated tumors showed a significant decrease in Mie-scattering slope and Mie-to-total scattering fraction and an increase in both fat volume fraction and tissue oxygenation after 2 days of follow-up. Additionally, significant tumor-specific changes in the fluorescence spectra were seen. These longitudinal trends were consistent with changes observed in the histopathologic analysis, such as vital tumor content and formation of fibrosis. CONCLUSIONS This study demonstrates that dual-modality DRS-AFS provides quantitative functional information that corresponds well with the degree of pathologic response. DRS-AFS, in conjunction with other imaging modalities, could be used to optimize systemic cancer treatment on the basis of early individual tumor response.

Seawater carbonate chemistry and combined mechanistic effects of CO2 and light on the N2-fixing cyanobacterium Trichodesmium IMS101, 2010

The marine diazotrophic cyanobacterium Trichodesmium responds to elevated atmospheric CO2 partial pressure (pCO2) with higher N2 fixation and growth rates. To unveil the underlying mechanisms, we examined the combined influence of pCO2(150 and 900 µatm) and light (50 and 200 µmol photons m-2 s-1) on TrichodesmiumIMS101. We expand on a complementary study that demonstrated that while elevated pCO2 enhanced N2 fixation and growth, oxygen evolution and carbon fixation increased mainly as a response to high light. Here, we investigated changes in the photosynthetic fluorescence parameters of photosystem II, in ratios of the photosynthetic units (photosystem I:photosystem II), and in the pool sizes of key proteins involved in the fixation of carbon and nitrogen as well as their subsequent assimilation. We show that the combined elevation in pCO2 and light controlled the operation of the CO2-concentrating mechanism and enhanced protein activity without increasing their pool size. Moreover, elevated pCO2 and high light decreased the amounts of several key proteins (NifH, PsbA, and PsaC), while amounts of AtpB and RbcL did not significantly change. Reduced investment in protein biosynthesis, without notably changing photosynthetic fluxes, could free up energy that can be reallocated to increase N2 fixation and growth at elevated pCO2 and light. We suggest that changes in the redox state of the photosynthetic electron transportchain and posttranslational regulation of key proteins mediate the high flexibility in resources and energy allocation in Trichodesmium. This strategy should enableTrichodesmium to flourish in future surface oceans characterized by elevated pCO2, higher temperatures, and high light.

Yeast actin: polymerization kinetic studies of wild type and a poorly polymerizing mutant.

Wild-type actin and a mutant actin were isolated from yeast (Saccharomyces cerevisiae) and the polymerization properties were examined at pH 8.0 and 20 degrees C. The polymerization reaction was followed either by an increase in pyrene-labeled actin fluorescence or by a decrease in intrinsic fluorescence in the absence of pyrene-labeled actin. While similar to the properties of skeletal muscle actin, there are several important differences between the wild-type yeast and muscle actins. First, yeast actin polymerizes more rapidly than muscle actin under the same experimental conditions. The difference in rates may result from a difference in the steps involving formation of the nucleating species. Second, as measured with pyrene-labeled yeast actin, but not with intrinsic fluorescence, there is an overshoot in the fluorescence that has not been observed with skeletal muscle actin under the same conditions. Third, in order to simulate the polymerization process of wild-type yeast actin it is necessary to assume some fragmentation of the filaments. Finally, gelsolin inhibits polymerization of yeast actin but is known to accelerate the polymerization of muscle actin. A mutant actin (R177A/D179A) has also been isolated and studied. The mutations are at a region of contact between monomers across the long axis of the actin filament. This mutant polymerizes more slowly than wild type and filaments do not appear to fragment during polymerization. Elongation rates of the wild type and the mutant differ by only about 3-fold, and the slower polymerization of the mutant appears to result primarily from poorer nucleation.

The folding of GroEL-bound barnase as a model for chaperonin-mediated protein folding.

We have analyzed the pathway of folding of barnase bound to GroEL to resolve the controversy of whether proteins can fold while bound to chaperonins (GroEL or Cpn60) or fold only after their release into solution. Four phases in the folding were detected by rapid-reaction kinetic measurements of the intrinsic fluorescence of both wild type and barnase mutants. The phases were assigned from their rate laws, sensitivity to mutations, and correspondence to regain of catalytic activity. At high ratios of denatured barnase to GroEL, 4 mol of barnase rapidly bind per 14-mer of GroEL. At high ratios of GroEL to barnase, 1 mol of barnase binds with a rate constant of 3.5 x 10(7) s-1.M-1. This molecule then refolds with a low rate constant that changes on mutation in parallel with the rate constant for the folding in solution. This rate constant corresponds to the regain of the overall catalytic activity of barnase and increases 15-fold on the addition of ATP to a physiologically relevant value of approximately 0.4 s-1. The multiply bound molecules of barnase that are present at high ratios of GroEL to barnase fold with a rate constant that is also sensitive to mutation but is 10 times higher. If the 110-residue barnase can fold when bound to GroEL and many moles can bind simultaneously, then smaller parts of large proteins should be able to fold while bound.

Simultaneous high-resolution pH and spectrophotometric recordings of oxygen binding in native blood microvolumes, link to supplementary material

Oxygen equilibrium curves have been widely used to understand oxygen transport in numerous organisms. A major challenge has been to monitor oxygen binding characteristics and concomitant pH changes as they occur in vivo, in limited sample volumes. Here we report a technique allowing highly resolved and simultaneous monitoring of pH and blood pigment saturation in minute blood volumes. We equipped a gas diffusion chamber with a broad range fibre optic spectrophotometer and a micro-pH optode and recorded changes of pigment oxygenation along PO2 and pH gradients to test the setup. Oxygen binding parameters derived from measurements in only 15 µl of haemolymph from the cephalopod Octopus vulgaris showed low instrumental error (0.93%) and good agreement with published data. Broad range spectra, each resolving 2048 data points, provided detailed insight into the complex absorbance characteristics of diverse blood types. After consideration of photobleaching and intrinsic fluorescence, pH optodes yielded accurate recordings and resolved a sigmoidal shift of 0.03 pH units in response to changing PO2 from 0-21 kPa. Highly resolved continuous recordings along pH gradients conformed to stepwise measurements at low rates of pH changes. In this study we showed that a diffusion chamber upgraded with a broad range spectrophotometer and an optical pH sensor accurately characterizes oxygen binding with minimal sample consumption and manipulation. We conclude that the modified diffusion chamber is highly suitable for experimental biologists who demand high flexibility, detailed insight into oxygen binding as well as experimental and biological accuracy combined in a single set up.

Étude des mécanismes d’extraction lipidique par le peptide mélittine et la protéine BSP1

Les peptides et protéines extracteurs de lipides (PEL) se lient aux membranes lipidiques puis en extraient des lipides en formant de plus petits auto-assemblages, un phénomène qui peut aller jusqu'à la fragmentation des membranes. Dans la nature, cette extraction se produit sur une gamme de cellules et entraîne des conséquences variées, comme la modification de la composition de la membrane et la mort de la cellule. Cette thèse se penche sur l’extraction lipidique, ou fragmentation, induite par le peptide mélittine et la protéine Binder-of-SPerm 1 (BSP1) sur des membranes lipidiques modèles. Pour ce faire, des liposomes de différentes compositions sont préparés et incubés avec la mélittine ou la BSP1. L'association aux membranes est déterminée par la fluorescence intrinsèque des PEL, tandis que l'extraction est caractérisée par une plateforme analytique combinant des tests colorimétriques et des analyses en chromatographie en phase liquide et spectrométrie de masse (LCMS). La mélittine fait partie des peptides antimicrobiens cationiques, un groupe de PEL très répandu chez les organismes vivants. Ces peptides sont intéressants du point du vue médical étant donné leur mode d’action qui vise directement les lipides des membranes. Plusieurs de ceux-ci agissent sur les membranes des bactéries selon le mécanisme dit « en tapis », par lequel ils s’adsorbent à leur surface, forment des pores et ultimement causent leur fragmentation. Dans cette thèse, la mélittine est utilisée comme peptide modèle afin d’étudier le mécanisme par lequel les peptides antimicrobiens cationiques fragmentent les membranes. Les résultats montrent que la fragmentation des membranes de phosphatidylcholines (PC) est réduite par une déméthylation graduelle de leur groupement ammonium. L'analyse du matériel fragmenté révèle que les PC sont préférentiellement extraites des membranes, dû à un enrichissement local en PC autour de la mélittine à l'intérieur de la membrane. De plus, un analogue de la mélittine, dont la majorité des résidus cationiques sont neutralisés, est utilisé pour évaluer le rôle du caractère cationique de la mélittine native. La neutralisation augmente l'affinité du peptide pour les membranes neutres et anioniques, réduit la fragmentation des membranes neutres et augmente la fragmentation des membranes anioniques. Malgré les interactions électrostatiques entre le peptide cationique et les lipides anioniques, aucune spécificité lipidique n'est observée dans l'extraction. La BSP1 est la protéine la plus abondante du liquide séminal bovin et constitue un autre exemple de PEL naturel important. Elle se mélange aux spermatozoïdes lors de l’éjaculation et extrait des lipides de leur membrane, notamment le cholestérol et les phosphatidylcholines. Cette étape cruciale modifie la composition lipidique de la membrane du spermatozoïde, ce qui faciliterait par la suite la fécondation de l’ovule. Cependant, le contact prolongé de la protéine avec les spermatozoïdes endommagerait la semence. Cette thèse cherche donc à approfondir notre compréhension de ce délicat phénomène en étudiant le mécanisme moléculaire par lequel la protéine fragmente les membranes lipidiques. Les résultats des présents travaux permettent de proposer un mécanisme d’extraction lipidique en 3 étapes : 1) L'association à l’interface des membranes; 2) La relocalisation de l’interface vers le cœur lipidique; 3) La fragmentation des membranes. La BSP1 se lie directement à deux PC à l'interface; une quantité suffisante de PC dans les membranes est nécessaire pour permettre l'association et la fragmentation. Cette liaison spécifique ne mène généralement pas à une extraction lipidique sélective. L'impact des insaturations des chaînes lipidiques, de la présence de lysophosphatidylcholines, de phosphatidyléthanolamine, de cholestérol et de lipides anioniques est également évalué. Les présentes observations soulignent la complexe relation entre l'affinité d'un PEL pour une membrane et le niveau de fragmentation qu'il induit. L'importance de la relocalisation des PEL de l'interface vers le cœur hydrophobe des membranes pour permettre leur fragmentation est réitérée. Cette fragmentation semble s'accompagner d'une extraction lipidique préférentielle seulement lorsqu'une séparation de phase est induite au niveau de la membrane, nonobstant les interactions spécifiques PEL-lipide. Les prévalences des structures amphiphiles chez certains PEL, ainsi que de la fragmentation en auto-assemblages discoïdaux sont discutées. Finalement, le rôle des interactions électrostatiques entre les peptides antimicrobiens cationiques et les membranes bactériennes anioniques est nuancé : les résidus chargés diminueraient l'association des peptides aux membranes neutres suite à l'augmentation de leur énergie de solvatation.