Navegando por Palavras-chave "TOAC"

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    Analogues containing the paramagnetic amino acid TOAC as substrates for angiotensin 1-converting enzyme
    (Elsevier B.V., 2007-05-29) Teixeira, Luis Gustavo de Deus [UNIFESP]; Bersanetti, Patricia Alessandra [UNIFESP]; Schreier, Shirley; Carmona, Adriana Karaoglanovic [UNIFESP]; Nakaie, Clovis Ryuichi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
    The angiotensin I-converting enzyme (ACE) converts the decapeptide angiotensin I (Ang I) into angiotensin II by releasing the C-terminal dipeptide. A novel approach combining enzymatic and electron paramagnetic resonance (EPR) studies was developed to determine the enzyme effect on Ang I containing the paramagnetic 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) at positions 1, 3, 8, and 9. Biological assays indicated that TOAC(1)-Ang I maintained partly the Ang I activity, and that only this derivative and the TOAC(3)-Ang I were cleaved by ACE. Quenching of Tyr(4) fluorescence by TOAC decreased with increasing distance between both residues, suggesting an overall partially extended structure. However, the local bend known to be imposed by the substituted diglycine TOAC is probably responsible for steric hindrance, not allowing the analogues containing TOAC at positions 8 and 9 to act as substrates. in some cases, although substrates and products differ by only two residues, the difference between their EPR spectral lineshapes allows monitoring the enzymatic reaction as a function of time. (c) 2007 Federation of European Biochemical Societies. Published by 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 Properties of Angiotensin II and Its Active and Inactive TOAC-Labeled Analogs in the Presence of Micelles. Electron Paramagnetic Resonance, Fluorescence, and Circular Dichroism Studies
    (Wiley-Blackwell, 2009-01-01) Vieira, Renata de Freitas Fischer [UNIFESP]; Casallanovo, Fabio; Marin, Nelida; Paiva, Antonio Cechelli de Mattos [UNIFESP]; Schreier, Shirley; Nakaie, Clovis Ryuichi [UNIFESP]; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)
    The interaction between angiotensin II (AII, DRVYIHPF) and its analogs carrying 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and detergents-negatively charged sodium dodecyl sulfate (SDS) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS)-was examined by means of EPR, CD, and fluorescence. EPR spectra of partially active TOAC(1)-AII and inactive TOAC(3)-AII in aqueous solution indicated fast tumbling, the freedom of motion being greater at the N-terminus. Line broadening occurred upon interaction with micelles. Below SDS critical micelle concentration, broader lines indicated complex formation with tighter molecular packing than in micelles. Small changes in hyperfine splittings evinced TOAC location at the micelle-water interface. the interaction with anionic micelles was more effective than with zwitterionic micelles. Peptide-micelle interaction caused fluorescence increase. the TOAC-promoted intramolecular fluorescence quenching was more, pronounced for TOAC(3)-AII because of the proximity between the nitroxide and Tyr(4). CD spectra showed that although both AII and TOAC(1)-AII presented flexible conformations in water, TOAC(3)-AII displayed conformational restriction because of the TOAC-imposed bend (Schreier et al., Biopolymers 2004, 74, 389). in HPS, conformational changes were observed for the labeled peptides at neutral and basic pH. in SDS, all peptides underwent pH-dependent conformational changes. Although the spectra suggested similar folds for All and TOAC(1)-AII, different conformations were acquired by TOAC(3)-AII. the membrane environment has been hypothesized to shift conformational equilibria so as to stabilize the receptor-bound conformation of ligands. the fact that TOAC(3)-AII is unable to acquire conformations similar to those of native AII and partially active TOAC(1)-AII is probably the explanation for its lack of biological activity. (C) 2009 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 92: 525-537, 2009.
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    Conformational studies of TOAC-labeled bradykinin analogues in model membranes
    (Kluwer Academic Publ, 2002-01-01) Vieira, Renata de Freitas Fischer [UNIFESP]; Casallanovo, Fabio; Cilli, Eduardo Maffud [UNIFESP]; Paiva, Antonio Cechelli de Mattos [UNIFESP]; Schreier, Shirley; Nakaie, Clovis Ryuichi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
    Spin-labeled analogues of bradykinin (BK) were synthesized containing the amino acid TOAC ( 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) either before Arg(1) (TOAC(0)-BK) or replacing Pro(3) (TOAC(3)-BK). Whereas the latter is inactive, the former retains about 70% of BK's activity in isolated rat uterus. A combined electron paramagnetic resonance (EPR)-circular dichroism ( CD) approach was used to examine the conformational properties of the peptides in the presence of membrane-mimetic systems ( negatively charged sodium dodecyl sulfate, SDS, and zwitterionic N-hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, HPS). While the peptides bind to both monomeric and micellar SDS, no interaction occurs with HPS, evincing the contribution of electrostatic interactions. TOAC(3)-BK's EPR spectral lineshapes are broader than those of TOAC(0)-BK, indicating a more restricted degree of motion at position 3. Moreover, the motional freedom of both peptides decreased upon binding to SDS. BK and TOAC(0)-BK solution CD spectra indicate highly flexible conformations ( possibly an equilibrium between rapidly interconverting forms), while TOAC(3)-BK's spectra correspond to a more ordered structure. SDS binding induces drastic changes in BK and TOAC(0)-BK spectra, indicating stabilization of similar folds. the micelle interface promotes a higher degree of secondary structure by favoring intramolecular hydrogen bonds. in contrast, TOAC(3)-BK spectra remain essentially unchanged. These results are interpreted as due to TOAC's ring imposing a more constrained conformation. This rigidity is very likely responsible for the inability of TOAC(3)-BK to acquire the correct receptor-bound conformation, leading to loss of biological activity. On the other hand, the greater flexibility of TOAC(0)-BK and the similarity between its conformational behavior and that of the native hormone are probably related to their similar biological activity.
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    Fmoc-POAC: [(9-fluorenylmethyloxycarbonyl)-2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid]: A novel protected spin labeled beta-amino acid for peptide and protein chemistry
    (Pharmaceutical Soc Japan, 2001-08-01) Tominaga, Mineko [UNIFESP]; Barbosa, Simone Reis [UNIFESP]; Poletti, Erick Fernando [UNIFESP]; Zukerman-Schpector, Julio; Marchetto, Reinaldo; Schreier, Shirley; Paiva, Antonio Cechelli de Mattos [UNIFESP]; Nakaie, Clovis Ryuichi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade Federal de São Carlos (UFSCar); UNESP; Universidade de São Paulo (USP)
    The stable free radical 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) is the only spin labeled amino acid that has been used to date to successfully label peptide sequences for structural studies. However, severe difficulty in coupling the subsequent amino acid has been the most serious shortcoming of this paramagnetic marker. This problem stems from the low nucleophilicity of TOAC's amine group towards the acylation reaction during peptide chain elongation. the present report introduces the alternative beta -amino acid 2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid (POAC), potentially useful in peptide and protein chemistry. Investigations aimed at addressing the stereochemistry of this cyclic molecule through X-ray diffraction measurements of crystalline and bulk samples revealed that it consists only of the trans conformer. the 9-fluorenylmethyloxyearbonyl group (Fmoc) was chosen for temporary protection of the POAC amine function, allowing insertion of the probe at any position in a peptide sequence. the vasoactive octapeptide angiotensin II (AII, DRVYIHPF) was synthesized by replacing Pro(7) with POAC. the reaction of Fmoc-POAC with the peptidyl-resin occurred smoothly, and the coupling of the subsequent amino acid showed a much faster reaction when compared with TOAC. POAC(7)-AII was obtained in good yield, demonstrating that, in addition to TOAC, POAC is a convenient amino acid for the synthesis of spin labeled peptide analogues. the present findings open the possibility of a wide range of chemical and biological applications for this novel beta -amino acid derivative, including structural investigations involving its differentiated bend-inducing characteristics.
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    Functional assessment of angiotensin II and bradykinin analogues containing the paramagnetic amino acid TOAC
    (Elsevier B.V., 2008-02-01) Santos, Edson L. [UNIFESP]; Souza, Kely de Picoli [UNIFESP]; Sabatini, Regiane A. [UNIFESP]; Martin, Renan P. [UNIFESP]; Fernandes, Litiam [UNIFESP]; Nardi, Daniela T. [UNIFESP]; Malavolta, Luciana [UNIFESP]; Shimuta, Suma I. [UNIFESP]; Nakaie, Clovis R. [UNIFESP]; Pesquero, Joao B. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    This study characterized pharmacologically the functional responses to agonists angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-Noxyl-4-amino-4-carboxylic acid) spin [abet at the N-terminal (TOAC(1)-AngII and TOAC(0)-BK) and internal (TOAC(3)-AngII and TOAC(3)-BK) positions of these vasoactive peptides. Affinity constants of the ligands for AT(1) and B-2 receptors were evaluated in vitro by binding assays and biological effects by extracellular acidification rates and in vivo by blood pressure responses. in contrast to internally labeled analogues (TOAC(3)-AngII or TOAC(3)-BK), the TOAC(1)-AngII and TOAC(0)-BK derivatives dose-dependently increased the extracellular acidification rate in adherent cultured Chinese hamster ovary (CHO) cells expressing AT, or B2 receptors, respectively. in addition, TOAC(1)-AngII induced an increase in blood pressure when injected intravenously in awaken rats although with a potency four times smaller when compared to native AngII. Similarly to BK, TOAC(0)-BK dose-dependently decreased blood pressure when injected intra-arterially in rats with a lower potency when compared to the native peptide. On the contrary, TOAC(3)-AngII or TOAC(3)-BK did not provoke any alteration in blood pressure levels. in summary, our results confirmed that the insertion of TOAC-probe in the N-terminal region of peptides does not significantly modify the affinity or biological activity in vitro and in vivo conditions and could be an important toool to evaluate peptide-receptor interaction mechanism. Conversely, possibly due to the unique bend-inducing property of the cyclic TOAC probe, its insertion at position 3 in both AngII and BK structures seems to restrict the interaction and the activation of the AT, and B2 receptors. (c) 2007 Elsevier B.V. All rights reserved.
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    A proposed EPR approach to evaluating agonist binding site of a peptide receptor
    (Springer, 2008-06-01) Lopes, Douglas Duarte [UNIFESP]; Poletti, Erick Fernando [UNIFESP]; Vieira, Renata de Freitas Fischer [UNIFESP]; Jubilut, Guita Nicolaewsky [UNIFESP]; Oliveira, Laerte [UNIFESP]; Paiva, Antonio Cechelli de Mattos [UNIFESP]; Schreier, Shirley; Nakaie, Clovis Ryuichi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
    Angiotensin II (Ang II) and its transmembrane AT(1) receptor were selected in order to test an innovative strategy that might allow the assessment of the agonist binding site in the receptor molecule. With the use of the 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) paramagnetic probe, a biologically active agonist (TOAC(1)-Ang II), as well as an inactive control (TOAC(4)-Ang II) analogs were mixed in solution with various synthesized AT(1) fragments. Comparative intermolecular interactions, as estimated by analyzing the EPR spectra of solutions, suggested the existence of an agonist binding site containing a sequence composed of portions of the N-terminal (13-17) and the third extracellular loop (266-278) fragments of the AT(1) molecule. Therefore, this combined EPR-TOAC approach shows promise as an alternative for use also in other applications related to specific intermolecular association processes.