Structural organization of cetyltrimethylammonium sulfate in aqueous solution: the effect of Na2SO4

We used dynamic light scattering (DLS), a steady-state fluorescence, time resolved fluorescence quenching (TRFQ), tensiometry, conductimetry, and isothermal titration calorimetry (ITC) to investigate the self-assembly of the cationic surfactant cetyltrimethylammonium sulfate (CTAS) in aqueous solution, which has SO42- as divalent counterion. We obtained the critical micelled concentration (cmc), aggregation number (N-agg), area per monomer (a(0)), hydrodynamic radius (R-H), and degree of counterion dissociation (alpha) of CTAS micelles in the absence and presence of up to 1 M Na2SO4 and at temperatures of 25 and 40 degrees C. Between 0.01 and 0.3 M salt the hydrodynamic radius of CTAS micelle R-H approximate to 16 angstrom is roughly independent on Na2SO4 concentration; below and above this concentration range R-H increases steeply with the salt concentration, indicating micelle structure transition, from spherical to rod-like structures. R-H increases only slightly as temperature increases from 25 to 40 degrees C, and the cmc decreases initially very steeply with Na2SO4 concentration up to about 10 mM, and thereafter it is constant. The area per surfactant at the water/air interface, a(0), initially increases steeply with Na2SO4 concentration, and then decrases above ca. 10 mM. Conductimetry gives alpha = 0.18 for the degree of counterion dissociation, and N-agg obtained by fluorescence methods increases with surfactant concentration but it is roughly independent of up to 80 mM salt. The ITC data yield cmc of 0.22 mM in water, and the calculated enthalpy change of micelle formation, Delta H-mic = 3.8 kJ mol(-1), Gibbs free energy of micellization of surfactant molecules, Delta G(mic) = -38.0 kJ mol(-1) and entropy T Delta S-mic = 41.7 kJ mol(-1) indicate that the formation of CTAS micelles is entropy-driven. (c) 2006 Elsevier B.V. All rights reserved.

UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation

The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg 2+ as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents. © 2010 Elsevier Inc. All rights reserved.

The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses

Tuberculosis remains as one of the main cause of mortality worldwide due to a single infectious agent, Mycobacterium tuberculosis. The aroK-encoded M. tuberculosis Shikimate Kinase (MtSK), shown to be essential for survival of bacilli, catalyzes the phosphoryl transfer from ATP to the carbon-3 hydroxyl group of shikimate (SKH), yielding shikimate-3-phosphate and ADP. Here we present purification to homogeneity, and oligomeric state determination of recombinant MtSK. Biochemical and biophysical data suggest that the chemical reaction catalyzed by monomeric MtSK follows a rapid-equilibrium random order of substrate binding, and ordered product release. Isothermal titration calorimetry (ITC) for binding of ligands to MtSK provided thermodynamic signatures of non-covalent interactions to each process. A comparison of steady-state kinetics parameters and equilibrium dissociation constant value determined by ITC showed that ATP binding does not increase the affinity of MtSK for SKH. We suggest that MtSK would more appropriately be described as an aroL-encoded type II shikimate kinase. Our manuscript also gives thermodynamic description of SKH binding to MtSK and data for the number of protons exchanged during this bimolecular interaction. The negative value for the change in constant pressure heat capacity (ΔCp) and molecular homology model building suggest a pronounced contribution of desolvation of non-polar groups upon binary complex formation. Thermodynamic parameters were deconvoluted into hydrophobic and vibrational contributions upon MtSK:SKH binary complex formation. Data for the number of protons exchanged during this bimolecular interaction are interpreted in light of a structural model to try to propose the likely amino acid side chains that are the proton donors to bulk solvent following MtSK:SKH complex formation. © 2013 Rosado et al.

RBPI21 interacts with negative membranes endothermically promoting the formation of rigid multilamellar structures

rBPI21 belongs to the antimicrobial peptide and protein (AMP) family. It has high affinity for lipopolysaccharide (LPS), acting mainly against Gram-negative bacteria. This work intends to elucidate the mechanism of action of rBPI21 at the membrane level. Using isothermal titration calorimetry, we observed that rBPI21 interaction occurs only with negatively charged membranes (mimicking bacterial membranes) and is entropically driven. Differential scanning calorimetry shows that membrane interaction with rBPI21 is followed by an increase of rigidity on negatively charged membrane, which is corroborated by small angle X-ray scattering (SAXS). Additionally, SAXS data reveal that rBPI21 promotes the multilamellarization of negatively charged membranes. The results support the proposed model for rBPI21 action: first it may interact with LPS at the bacterial surface. This entropic interaction could cause the release of ions that maintain the packed structure of LPS, ensuring peptide penetration. Then, rBPI21 may interact with the negatively charged leaflets of the outer and inner membranes, promoting the interaction between the two bacterial membranes, ultimately leading to cell death. © 2013 Elsevier B.V.

Caracterização da interação de inositol hexafosfato e cloreto com hemoglobina humana: aspectos energéticos e estruturais

Pós-graduação em Biofísica Molecular - IBILCE

Arteroids: a poética do jogo e o jogo poético em redes digitais

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Interação de poli(etileno glicol) com brometo de dioctadecildimetilamônio e didodecilmetilamônio em dispersões aquosas

Pós-graduação em Biofísica Molecular - IBILCE

Efeitos da dimerização na estrutura e atividade biológica dos peptídeos antimicrobianos Aureína 1.2 e Magainina 2

Pós-graduação em Biotecnologia - IQ

Estudos estruturais de complexos entre fosfolipases A2 homólogas isoladas do veneno de Bothrops moojeni e inibidores da atividade miotóxica

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

Lufaxin, a Novel Factor Xa Inhibitor From the Salivary Gland of the Sand Fly Lutzomyia longipalpis Blocks Protease-Activated Receptor 2 Activation and Inhibits Inflammation and Thrombosis In Vivo

Objective-Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. Methods and Results-Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant approximate to 3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl3-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. Conclusion-Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events. (Arterioscler Thromb Vasc Biol. 2012;32:2185-2196.)

INVESTIGAÇÃO ELETROQUÍMICA E CALORIMÉTRICA DA INTERAÇÃO DE NOVOS AGENTES ANTITUMORAIS BISCATIÔNICOS COM DNA

ELECTROCHEMICAL AND CALORIMETRIC INVESTIGATION OF INTERACTION OF NOVEL BISCATIONIC ANTICANCER AGENTS WITH DNA. Biscationic amidines bind in the DNA minor groove and present biological activity against a range of infectious diseases. Two new biscationic compounds (bis-alpha,omega-S-thioureido, amino and sulfide analogues) were synthesized in good yields and fully characterized, and their interaction with DNA was also investigated. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic properties of binding interactions between DNA and these ligands. A double stranded calf thymus DNA immobilized on an electrode surface was used to study the possible DNA-interacting abilities of these compounds towards dsDNA in situ. A remarkable interaction of these compounds with DNA was demonstrated and their potential application as anticancer agents was furthered.

Interaction between bradykinin potentiating nonapeptide (BPP9a) and beta-cyclodextrin: A structural and thermodynamic study

Herein, we demonstrate the physical and chemical characterizations of the supramolecular complex formed between beta-cyclodextrin (beta CD) and bradykinin potentiating nonapeptide (BPP9a), an endogenous toxin found in Bothrops jararaca. Circular dichroism results indicate a conformational change in the BPP9a secondary structure upon its complexation with beta CD. Nuclear magnetic resonance results, mainly from NOESY experiments, and theoretical calculations showed a favorable interaction between the tryptophan residue of BPP9a and the beta CD cavity. Thermodynamic inclusion parameters were investigated by isothermal titration calorimetry, demonstrating that beta CD/BPP9a complex formation is an exothermic process that results in a reduction in entropy. Additionally, in vitro degradation study of BPP9a against trypsin (37 degrees C, pH 7.2) showed higher stability of peptide in presence of beta CD. This beta CD/BPP9a complex, which presents new chemical properties arising from the peptide inclusion process, may be useful as an antihypertensive drug in oral pharmaceutical formulations. (C) 2011 Elsevier B.V. All rights reserved.

Flavonoid interactions with human transthyretin: Combined structural and thermodynamic analysis

Transthyretin (TTR) is a carrier protein involved in human amyloidosis. The development of small molecules that may act as TTR amyloid inhibitors is a promising strategy to treat these pathologies. Here we selected and characterized the interaction of flavonoids with the wild type and the V30M amyloidogenic mutant TTR. TTR acid aggregation was evaluated in vitro in the presence of the different flavonoids. The best TTR aggregation inhibitors were studied by Isothermal Titration Calorimetry (ITC) in order to reveal their thermodynamic signature of binding to TTRwt. Crystal structures of TTRwt in complex with the top binders were also obtained, enabling us to in depth inspect TTR interactions with these flavonoids. The results indicate that changing the number and position of hydroxyl groups attached to the flavonoid core strongly influence flavonoid recognition by TTR, either by changing ligand affinity or its mechanism of interaction with the two sites of TTR. We also compared the results obtained for ITRwt with the V30M mutant structure in the apo form, allowing us to pinpoint structural features that may facilitate or hamper ligand binding to the V30M mutant. Our data show that the TTRwt binding site is labile and, in particular, the central region of the cavity is sensible for the small differences in the ligands tested and can be influenced by the Met30 amyloidogenic mutation, therefore playing important roles in flavonoid binding affinity, mechanism and mutant protein ligand binding specificities. (C) 2012 Elsevier Inc. All rights reserved.

Investigação eletroquímica e calorimétrica da interação de novos agentes antitumorais biscatiônicos com DNA

Biscationic amidines bind in the DNA minor groove and present biological activity against a range of infectious diseases. Two new biscationic compounds (bis-α,ω-S-thioureido, amino and sulfide analogues) were synthesized in good yields and fully characterized, and their interaction with DNA was also investigated. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic properties of binding interactions between DNA and these ligands. A double stranded calf thymus DNA immobilized on an electrode surface was used to study the possible DNA-interacting abilities of these compounds towards dsDNA in situ. A remarkable interaction of these compounds with DNA was demonstrated and their potential application as anticancer agents was furthered.

Synthese von amphiphilen Bürstenpolymeren mit Kern-Schale-Architektur und Untersuchungen zur Verkapselung von hydrophilen Modellsubstanzen

Die dieser Arbeit zugrundeliegenden Nanopartikel wurden mittels der Makromonomer-Strategie aus polymerisierbaren Polystyrol-b-Poly(2-vinylpyridin) Oligomeren dargestellt. Die Bürstenpolymere besitzen eine polare PS-Schale und einen polaren Kern (P2VP), dessen Polarität durch Quaternisierung deutlich erhöht werden kann. Die Bürstenpolymere weisen bei Molmassen um 400 - 800 kg/mol einen Teilchendurchmesser von ca. 15 - 20 nm auf. Die Nanopartikel eignen sich dazu, hydrophile Farbstoffe in unpolaren Lösungsmitteln zu solubilisieren. Durch spektroskopische Untersuchungen wurden in Abhängigkeit der chemischen Struktur und der Bürstenpolymere Beladungsgrade von über 1 g Farbstoff pro Gramm Polymer ermittelt. Die Beladung der Nanopartikel folgt hierbei einer nichttrivialen Kinetik, was möglicherweise durch eine wasserinduzierte Überstrukturbildung während der Beladung bedingt ist. Mittels isothermer Titrationskalorimetrie konnten die Wechselwirkungen zwischen polymeren Substrat und niedermolekularen Liganden genauer charakterisiert werden. Teilweise werden hierbei zweistufige Titrationsverläufe und "überstöchiometrische" Beladung der Bürstenpolymere beobachtet. Den Hauptbeitrag zur Wechselwirkung liefert hierbei die exotherme Wechselwirkung zwischen basischen Polymer und saurem Farbstoff. Die hohe Farbstoffbeladung führt zur deutlichen Vergrößerung der einzelnen Nanopartikel, was sowohl in Lösung durch Lichtstreu-Techniken als auch auf Oberflächen mit Hilfe des AFM zu beobachten ist. Durch Untersuchungen mit der analytischen Ultrazentrifuge konnte nachgewiesen werden, dass sich der eingelagerte Farbstoff in einem Polaritäts-abhängigen Gleichgewicht mit der Umgebung steht, er somit auch wieder aus den Nanopartikeln freigesetzt werden kann. Darüberhinaus wurden im Rahmen der Arbeit erste Erfolge bei der Synthese von wasserlöslichen Nanopartikeln mit Poly(2-vinylpyridin)-Kern erzielt. Als hierfür geeignet stellte sich eine Synthesestrategie heraus, bei der zunächst ein Bürstenpolymer mit P2VP-Seitenketten dargestellt und dieses anschließend mit geeignet funktionalisierten Polyethylenoxid-Ketten zum Kern-Schale Teilchen umgesetzt wurde. Neben Untersuchungen zum Mizellisierungsverhalten von PEO-b-P2VP Makromonomeren wurden deren Aggregate in Wasser hinsichtlich ihrer Zelltoxizität durch in-vitro Experimente an C26-Mäusekarzinom-Zellen charakterisiert. Die extrem geringe Toxizität macht das PEO-P2VP System zu einem potentiellen Kandidaten für drug-delivery Anwendungen. Besonders die pH-abhängige Löslichkeitsänderung des Poly(2-vinylpyridin) erscheint hierbei besonders interessant.