Navegando por Palavras-chave "circular dichroism"

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    Biological and structural characterization of new linear gomesin analogues with improved therapeutic indices
    (Wiley-Blackwell, 2007-01-01) Fazio, Marcos A.; Jouvensal, Laurence; Vovelle, Francoise; Bulet, Philippe; Miranda, M. Teresa M.; Daffre, Sirlei; Miranda, Antonio; Universidade Federal de São Paulo (UNIFESP); Univ Orleans; Atheris Labs; Universidade de São Paulo (USP)
    Gomesin (Gm) is a potent antimicrobial peptide isolated from the spider Acanthoscurria gomesiana. the two disulfide bridges Cys(2,15) and Cys(6,11) facilitate the folding of the molecule in a P-hairpin structure, conferring on the peptide a high stability in human plasma. We report herein biological and structural features of new linear Gm analogues, obtained by combining the removal of both disulfide bridges and the incorporation of a D- or L-proline. Regarding their biological properties, two analogues, namely, [D-Thr(2,6,11,15), Pro(9)]-D-Gm and [Thr(2,6,11,15), D-Pro(9)]-Gm, are as potent as Gm against Candida albicans and only fourfold less against Staphylococcus aureus and Escherichia coli. in addition, at 100 mu M they are approximately threefold less hemolytic than Gm. the best therapeutic indices were found for [D-Thr(2,6,11,15), Pro(9)]-D-Gm and for [(Des-pGlu(1), -Thr(2), -Arg(3))], Thr(6,11,15), with a 32-fold increase of their activity against bacteria, and from 128- to 512-fold against yeast when compared with Gm. Regarding the stability, [D-Thr(2,6,11,15), Pro(9)]-D-Gm appeared to be the most resistant in human serum, along with [D-Thr2,6,11,15, Pro(8)]-D-Gm and [Thr-(2,6,11,15), D-Arg(4,16), D-Pro(9)]-Gm. When evaluating their conformation by CD spectroscopy in sodium dodecyl sulfate (SDS), most linear analogues display beta-conformation characteristics. Moreover, considering its high therapeutic index and stability in serum, [D-Thr(2,6,11,15), Pro(9)]-D-Gm was ftirther analyzed by NMR spectroscopy. H-1 NMR expertiments in SDS micelles demonstrated that [D-Thr(2,6,11,15), Pro(9)]-D-Gm presents a conformation very similar to that of Gm. in our search for Gm analogues with enhanced potential for drug development, we demonstrated that designing cysteine-free analogues can improve the therapeutic index of Gm derivatives. (c) 2006 Wiley Periodicals, Inc.
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    Conformational and functional studies of gomesin analogues by CD, EPR and fluorescence spectroscopies
    (Elsevier B.V., 2007-01-01) Moraes, Luis Guilherme Mello [UNIFESP]; Fázio, Marcos Antonio [UNIFESP]; Vieira, Renata de Freitas Fischer; Nakaie, Clovis Ryuichi; Miranda, Maria Teresa Machini; Schreier, Shirley; Daffre, Sirlei; Miranda, Antonio [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
    The aim of this work was to examine the bioactivity and the conformational behavior of some gomesin (Gm) analogues in different environments that mimic the biological membrane/water interface. Thus, manual peptide synthesis was performed by the solid-phase method, antimicrobial activity was evaluated by a liquid growth inhibition assay, and conformational studies were performed making use of several spectroscopic techniques: CD, fluorescence and EPR. [TOAC(1)]-Gm; [TOAC(1), Ser(2,6,11,15)]-Gm; [Trp(7)]-Gm; [Ser(2,6,11,15), Trp(7)]-Gm; [Trp(9)]-Gm; and [Ser(2,6,11,15), Trp(9)]-Gm were synthesized and tested. the results indicated that incorporation of TOAC or Trp caused no significant reduction of antimicrobial activity; the cyclic analogues presented a beta-hairpin conformation similar to that of Gm. All analogues interacted with negatively charged SDS both above and below the detergent's critical micellar concentration (cmc). in contrast, while Gm and [TOAC(1)]-Gm required higher LPC concentrations to bind to micelles of this zwitterionic detergent, the cyclic Trp derivatives and the linear derivatives did not seem to interact with this membrane-mimetic system. These data corroborate previous results that suggest that electrostatic interactions with the lipid bilayer of microorganisms play an important role in the mechanism of action of gomesin. Moreover, the results show that hydrophobic interactions also contribute to membrane binding of this antimicrobial peptide. (c) 2006 Elsevier B.V. All rights reserved.
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    Conformational basis for the biological activity of TOAC-labeled angiotensin II and Bradykinin: Electron paramagnetic resonance, circular dichroism, and fluorescence studies
    (Wiley-Blackwell, 2004-08-05) Schreier, S.; Barbosa, SR; Casallanovo, F.; Vieira, RDF; Cilli, E. M.; Paiva, ACM; Nakaie, C. R.; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)
    N-Terminally and internally labeled analogues of the hormones angiotensin (All, DRVYIHPF) and bradykinin (BK, RPPGFSPFR) were synthesized containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). TOAC replaced Asp(1) (TOAC(1)-AII) and Val(3) (TOAC(3)-AII) in AII and was inserted prior to Arg(1) (TOAC(0)-BK) and replacing Pro(3) (TOAC(3)-BK) in BK. the peptide conformational properties were examined as a function of trifluoroethanol (TFE) content and pH. Electron paramagnetic resonance spectra were sensitive to both variables and showed that internally labeled analogues yielded rotational correlation times (tau(C)) considerably larger than N-terminally labeled ones, evincing the greater freedom of motion of the N-terminus. in TFE, tau(C) increased due to viscosity effects. Calculation of tau(Cpeptide)/ tau(CTOAC) ratios indicated that the peptides acquired more folded conformations. Circular dichroism spectra showed that, except for TOAC(1)-AII in TFE, the N-terminally labeled analogues displayed a conformational behavior similar to that of the parent peptides. in contrast, under all conditions, the TOAC(3) derivatives acquired more restricted conformations. Fluorescence spectra of AII and its derivatives were especially sensitive to the ionization of Tyr(4). Fluorescence quenching by the nitroxide moiety was much more pronounced for TOAC(3)-AII. the conformational behavior of the TOAC derivatives bears excellent correlation with their biological activity, since, while the N-terminally labeled peptides were partially active, their internally labeled counterparts were inactive [Nakaie, C. R., et al., Peptides 2002, 23, 65-70]. the data demonstrate that insertion of TOAC in the middle of the peptide chain induces conformational restrictions that lead to loss of backbone flexibility, not allowing the peptides to acquire their receptor-bound conformation. (C) 2004 Wiley Periodicals, Inc.
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    Conformational changes of Loxosceles venom sphingomyelinases monitored by circular dichroism
    (Elsevier B.V., 2005-02-04) Andrade, S. A. de; Pedrosa, MFF; Andrade, RMG de; Oliva, MLV; van den Berge, C. W.; Tambourgi, D. V.; Inst Butantan; Universidade Federal de São Paulo (UNIFESP); Cardiff Univ
    Envenomation by arachnids of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and hemolysis. We have previously identified in L. intermedia venom two highly homologous proteins with sphingomyelinase activity, termed P1 and P2, responsible for all these pathological events, and also an inactive isoform P3. the toxins P1 and P2 displayed 85% identity with each other at the amino acid level and showed a 57% identity with SMase I, an active toxin from L. laeta venom. Circular dichroism was used to determine and compare the solution structure of the active and inactive isoforms. Effects of pH and temperature change on the CD spectra of the toxins were investigated and correlated with the biological activities. This study sheds new light on the structure-function relationship of homologous proteins with distinct biological properties and represents the first report on the structure-function relationship of Loxosceles sphingomyelinases D. (C) 2004 Elsevier Inc. All rights reserved.
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    Conformational flexibility of three cytoplasmic segments of the angiotensin II AT(1A) receptor: A circular dichroism and fluorescence spectroscopy study
    (Wiley-Blackwell, 2002-01-01) Pertinhez, Thelma A.; Krybus, Regina; Cilli, Eduardo Maffud [UNIFESP]; Paiva, Antonio Cechelli de Mattos [UNIFESP]; Nakaie, Clovis Ryuichi [UNIFESP]; Franzoni, Lorella; Sartor, Giorgio; Spisni, Alberto; Schreier, Shirley; Univ Parma; LNLS; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)
    The conformation of three synthetic peptides encompassing the proximal and distal half of the third intracellular loop (Ni3 and Ci3) and a portion of the cytoplasmic tail (fCT) of the angiotensin II AT(1A) receptor has been studied using circular dischroism and fluorescence spectroscopies. the results show that the conformation of the peptides is modulated in various ways by the environmental conditions (pH, ionic strength and dielectric constant). Indeed, Ni3 and fCT fold into helical structures that possess distinct stability and polarity due to the diverse forces involved: mainly polar interactions in the first case and a combination of polar and hydrophobic interactions in the second. the presence of these various features also produce distinct intermolecular interactions. Ci3, instead, exists as an ensemble of partially folded states in equilibrium. Since the corresponding regions of the angiotensin II AT(1A) receptor are known to play an important role in the receptor function, due to their ability to undergo conformational changes, these data provide some new clues about their different conformational plasticity. Copyright (C) 2002 European Peptide Society and John Wiley Sons, Ltd.
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    Divalent metal requirements for catalysis and stability of the RNA triphosphatase from Trypanosoma cruzi
    (Elsevier B.V., 2006-11-01) Kikuti, Carlos Massayuki; Tersariol, Ivarne Luis S.; Schenkman, Sergio; Universidade Federal de São Paulo (UNIFESP); Univ Mogi Cruzes
    RNA triphosphatases act in the first step of the mRNA capping process, removing the gamma-phosphoryl group from the 5' end of nascent RNA. A metal-dependent catalysis is found in the enzymes from trypanosomes and several other lower eukaryotes. This contrasts with the cysteine-dependent activity of the corresponding enzymes of mammals, a difference that points to these enzymes as potential targets for drug design. This work describes the identification, expression, purification, enzyme kinetics, and the role of divalent metal in the ATPase activity of the RNA triphosphatase from Trypanosoma cruzi, the agent of Chagas' disease, and compares it with the previously characterized enzyme from Trypanosoma brucei. Sequence similarity of the T cruzi enzyme with the RNA triphosphatase of Saccharomyces cerevisiae indicates that a tunnel domain containing the divalent metal forms its active site. Based on enzyme kinetics, circular dichroism, and intrinsic fluorescence analysis, a kinetic mechanism for the ATPase activity of the T cruzi tunnel triphosphatase is proposed. A single metal is sufficient to interact with the enzyme through the formation of a productive MnATP-enzyme complex, while free ATP inhibits activity. Manganese is also required for the tunnel stability of the T cruzi enzyme, while the T brucei homologue remains stable in the absence of metal, as shown for other triphosphatases. These findings may be useful to devise specific triphosphatase inhibitors to the T cruzi enzyme. (c) 2006 Elsevier B.V. All rights reserved.
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    In vitro evaluation of leptin fragments activity on the ob receptor
    (Wiley-Blackwell, 2008-05-01) Oliveira, Vani Xavier de; Fazio, Marcos Antonio [UNIFESP]; Santos, Edson Lucas [UNIFESP]; Pesquero, Joao Bosco [UNIFESP]; Miranda, Antonio [UNIFESP]; Universidade Federal do ABC (UFABC); Universidade Federal de São Paulo (UNIFESP)
    In an attempt to identify regions in the leptin molecule responsible for its bioactivity, we tested six related-leptin peptide fragments denoted: Ae-hLEP(23-47)-NH(2) (I), Ac-hLEP(48-71)-NH(2) (II), Ac-hLEP(72-88)-NH(2) (III), Ac-hLEP(92-115)-NH(2) (IV), Ac-[Ser(117)]-hLEP(116-140)-NH(2) (V), Ac-hLEP(141-164)-NH(2) (VI) and their correspondent disulfide bridged dimer forms. the activity of the fragments was evaluated in comparision to leptin, by their ability to interact with leptin receptor using a cytosensor microphysiometer. Our results indicated that the fragments IV and V and [D-Leu(4)]-OB(3) and its human sequence analog were recognized by leptin receptor present in HP-75 cells, in agreement with the results obtained by other workers, validating that this region of the molecule contain the functional epitope of the leptin molecule. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.
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    Molecular dynamics and circular dichroism studies of human and rat C-peptides
    (Elsevier B.V., 2006-12-01) Mares-Guia, Thiago Renno; Maigret, Bernard; Martins, Natalia Florencio; Turchetti Maia, Ana Luiza; Vilela, Luciano; Inacio Ramos, Carlos Henrique; Neto, Luiz Juliano; Juliano, Maria Aparecida; Mares-Guia, Marcos Luiz dos; Santoro, Marcelo Matos; Universidade Federal de Minas Gerais (UFMG); Henri Poincare Univ; Universidade de Brasília (UnB); Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA); Biomm SA; Lab Nacl Luz Sincrotron; Universidade Federal de São Paulo (UNIFESP)
    Proinsulin C-peptide has been recently described as an endogenous peptide hormone, responsible for important physiological functions others than its role in proinsulin processing. Accumulating evidences that C-peptide exerts beneficial effects in the treatment of long term complications of patients with type I diabetes mellitus indicate that this molecule may be administered together with insulin in future therapies. Despite its clear pharmacological interest, the secondary and three-dimensional (3D) structures of human C-peptide are still points of controversy. in the present work we report molecular dynamics (MD) simulations of human, rat I and rat II C-peptides. A common experimental strategy applied to all peptides consisted of homology building followed by multinanosecond MD simulations in vacuum and water. Circular dichroism (CD) experiments of each peptide in the absence and presence of 2,2,2-trifluoroethanol (TFE) were performed to support validation of the theoretical models. A multiple sequence alignment of 23 known mammalian C-peptides was constructed to identify significant conserved sites that would be important for the maintenance of secondary and tertiary structures. the analysis of the molecular dynamics trajectories for the human, rat I and rat II molecules have shown quite different general behavior, being the human C-peptide more flexible than the two others. Human and rat C-peptides exhibit very stable turn-like structures at the middle and C-terminal regions, which have been described as potential active sites of C-peptides. Human C-peptide also presented a short alpha-helix throughout the MD, which was not found in the rat molecules. CD data is in very good agreement with the MD results and both methods were able to identify a greater structural stability and potential in rat C-peptides when compared to the human C-peptide. the simulation results are discussed and validated in the light of multiple sequence alignment, recent experimental data from the literature and our own CD experiments. (c) 2006 Published by Elsevier Inc.
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    Molecular modeling of the human eukaryotic translation initiation factor 5A (eIF5A) based on spectroscopic and computational analyses
    (Elsevier B.V., 2006-09-01) Costa-Neto, Claudio M.; Parreiras-e-Silva, Lucas T.; Ruller, Roberto; Oliveira, Eduardo B.; Miranda, Antonio; Oliveira, Laerte; Ward, Richard J.; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)
    The eukaryotic translation initiation factor 5A (eIF5A) is a protein ubiquitously present in archaea and eukarya, which undergoes a unique two-step post-translational modification called hypusination. Several studies have shown that hypusination is essential for a variety of functional roles for eIF5A, including cell proliferation and synthesis of proteins involved in cell cycle control. Up to now neither a totally selective inhibitor of hypusination nor an inhibitor capable of directly binding to cIF5A has been reported in the literature. the discovery of such an inhibitor might be achieved by computer-aided drug design based on the 3D structure of the human eIF5A. in this study, we present a molecular model for the human eIF5A protein based on the crystal structure of the eIF5A from Leishmania brasiliensis, and compare the modeled conformation of the loop bearing the hypusination site with circular dichroism data obtained with a synthetic peptide of this loop. Furthermore, analysis of amino acid variability between different human eIF5A isoforms revealed peculiar structural characteristics that are of functional relevance. (c) 2006 Elsevier Inc. All rights reserved.
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    A proteinase inhibitor from Caesalpinia echinata (pau-brasil) seeds for plasma kallikrein, plasmin and factor XIIa
    (Walter de Gruyter & Co, 2004-11-01) Cruz-Silva, Ilana [UNIFESP]; Gozzo, Andrezza Justino [UNIFESP]; Nunes, Viviane A. [UNIFESP]; Carmona, Adriana K. [UNIFESP]; Faljoni-Alario, Adelaide; Oliva, Maria Luiza V; Sampaio, Misako U. [UNIFESP]; Sampaio, Claudio A M [UNIFESP]; Araujo, Mariana S. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
    Caesalpinia echinata is a tree belonging to the Leguminosae family. the red color of the trunk, looking like burning wood ('brasa' in Portuguese), is the origin of the name Brazil. Seeds of leguminous plants contain high amounts of serine proteinase inhibitors that can affect different biological processes. Here we show that a protein isolated from seeds of C. echinata is able to inhibit enzymes that participate in blood coagulation and fibrinolysis. This inhibitor (CeKI) was purified to homogeneity by ion exchange and reversed-phase chromatography. SDS-PAGE indicated a single polypeptide chain with a molecular mass of 20 kDa. CeKI inhibits human plasma kallikrein K-i=3.1 nm), plasmin K-i=0.18 nm), factor XIIa (K-i=0.18 nm), trypsin K-i=21.5 nm) and factor Xa K-i=0.49 mm). CeKI inhibited kinin release from high-molecular-mass kininogen by kallikrein in vitro. the N-terminal sequence, determined by automatic Edman degradation, identified the inhibitor as a member of the Kunitz family. the secondary structure, determined by circular dichroism, is mainly a random coil followed by P-sheet structure. the action of CeKI on enzymes of the blood-clotting intrinsic pathway was confirmed by prolongation of the activated partial thromboplastin time.
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    Structure-activity relationship of Trp-containing analogs of the antimicrobial peptide gomesin
    (Wiley-Blackwell, 2014-06-01) Domingues, Tatiana Moreira [UNIFESP]; Buri, Marcus V. [UNIFESP]; Daffre, Sirlei; Campana, Patricia T.; Riske, Karin A. [UNIFESP]; Miranda, Antonio [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
    Gomesin (Gm) has a broad antimicrobial activity making it of great interest for development of drugs. in this study, we analyzed three Gm analogs, [Trp(1)]-Gm, [Trp(7)]-Gm, and [Trp(9)]-Gm, in an attempt to gain insight into the contributions of different regions of the peptide sequence to its activity. the incorporation of the tryptophan residue in different positions has no effect on the antimicrobial and hemolytic activities of the Gm analogs in relation to Gm. Spectroscopic studies (circular dichroism, fluorescence and absorbance) of Gm and its analogs were performed in the presence of SDS, below and above its critical micelle concentration (CMC) (similar to 8mM), in order to monitor structural changes induced by the interaction with this anionic surfactant (0-15mM). Interestingly, we found that the analogs interact more strongly with SDS at low concentrations (0.3-6.0mM) than close to or above its CMC. This suggests that SDS monomers are able to cover the whole peptide, forming large detergent-peptide aggregates. On the other hand, the peptides interact differently with SDS micelles, inserting partially into the micelle core. Among the peptides, Trp in position 1 becomes more motionally-restricted in the presence of SDS, probably because this residue is located at the N-terminal region, which presents higher conformational freedom to interact stronger with SDS molecules. Trp residues in positions 7 and 9, close to and in the region of the turn of the molecule, respectively, induced a more constrained structure and the compounds cannot insert deeper into the micelle core or be completely buried by SDS monomers. Copyright (c) 2014 European Peptide Society and John Wiley & Sons, Ltd.
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    Structure-activity relationship studies of gomesin: Importance of the disulfide bridges for conformation, bioactivities, and serum stability
    (Wiley-Blackwell, 2006-01-01) Fazio, M. A.; Oliveira, V. X.; Bulet, P.; Miranda, MTM; Daffre, S.; Miranda, A.; Universidade Federal de São Paulo (UNIFESP); Bernex; Universidade de São Paulo (USP)
    Gomesin is an antimicrobial peptide isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana that contains two disulfide bridges Cys(2-15)/Cys(6-11) and presents a beta-hairpin structure. To investigate the role of the disulfide bridges on gomesin conformation, bioactivities, and serum stability, structure-activity relationship (SAR) studies were conducted. Initially, gomesin and variants lacking one or both disulfide bridges were synthesized. CD studies showed that the gomesin structure is very rigid independently of the solvent enviromnent. On the other hand, the linearized analogues adopted secondary structures according to the environment, while the monocyclic disulfide-bridged peptides had a tendency to adopt a turn structure. the absence of one or both bridges resulted in a decrease in the antimicrobial and hemolytic activities. in addition, serum stability studies revealed that, contrasting to gomesin that was stable even after 48 h of incubation, the linearized analogues were rapidly degraded. the replacement of the disulfide bounds by lactam bridges led to monocyclic and bicyclic compounds. SAR studies indicated that the monocyclic lactam-bridged analogues tend to assume a alpha-helical structure being less potent, hemolytic, and serum stable than the wild-type gomesin. On the other hand, the bicyclic lactam/disulfide-bridged analogues displayed a similar conformation and degradation kinetics identical to gomesin. However, the antimicrobial activity appeared to be dependent on the lactam bridge position and size. These findings indicated that (i) the secondary structure plays a pivotal role for the full activity of gomesin; (ii) the antimicrobial and hemolytic activities of gomesin are correlated events; (iii) while at least one of the disulfide bridges is needed for the maintenance of a significant antimicrobial activity of gomesin, both bridges are required for high serum stability and optimal conformation; and finally (iv) the best analogue obtained was the bicyclo (2-15,6-11)[Glu(2), Cys(6.11), Lys(15) J-Gm since it is as stable and potent as gomesin. (c) 2005 Wiley Periodicals, Inc.
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    Unfolding Studies of the Cysteine Protease Baupain, a Papain-Like Enzyme from Leaves of Bauhinia forficata: Effect of pH, Guanidine Hydrochloride and Temperature
    (Mdpi Ag, 2014-01-01) Silva-Lucca, Rosemeire Aparecida [UNIFESP]; Andrade, Sheila Siqueira [UNIFESP]; Ferreira, Rodrigo Silva [UNIFESP]; Sampaio, Misako Uemura [UNIFESP]; Oliva, Maria Luiza Vilela [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Estadual Oeste Parana
    Baupain belongs to the alpha+beta class of proteins with a secondary structure-content of 44% alpha-helix, 16% beta-sheet and 12% beta-turn. the structural transition induced by pH was found to be noncooperative, with no important differences observed in the pH range from 3.0 to 10.5. At pH 2.0 the protein presented substantial non-native structure with strong ANS binding. Guanidine hydrochloride (GdnHCl)-induced unfolding did not change the protein structure significantly until 4.0 M, indicating the high rigidity of the molecule. the unfolding was cooperative, as seen by the sigmoidal transition curves with midpoints at 4.7 +/- 0.2 M and 5.0 +/- 0.2 M GdnHCl, as measured by CD and fluorescence spectroscopy. A red shift of 7 nm in intrinsic fluorescence was observed with 6.0 M GdnHCl. Temperature-induced unfolding of baupain was incomplete, and at least 35% of the native structure of the protein was retained, even at high temperature (90 degrees C). Baupain showed characteristics of a molten globule state, due to preferential ANS binding at pH 2.0 in comparison to the native form (pH 7.0) and completely unfolded (6.0 M GdnHCl) state. Combined with information about N-terminal sequence similarity, these results allow us to include baupain in the papain superfamily.