Repositório Institucional - Unifesp :: Navegando por Palavras-chave "fluorescence"

Navegando por Palavras-chave "fluorescence"

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Acesso aberto (Open Access)
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 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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Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells
(Elsevier B.V., 2008-06-23) Boucas, Rodrigo Ippolito [UNIFESP]; Trindade, Edvaldo S. [UNIFESP]; Tersariol, Ivarne L. S. [UNIFESP]; Dietrich, Carl P. [UNIFESP]; Nader, Helena B. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Fed Parana; Univ Mogi das Cruzes
Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. the structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). the binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. the interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. the apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines, the interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture. (c) 2008 Elsevier B.V. All rights reserved.
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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.
Somente Metadadados
Effects of magnesium ions on recombinant human furin: selective activation of hydrolytic activity upon substrates derived from virus envelope glycoprotein
(Walter de Gruyter & Co, 2010-09-01) Izidoro, Mario A. [UNIFESP]; Assis, Diego M. [UNIFESP]; Oliveira, Vitor [UNIFESP]; Santos, Jorge A. N. [UNIFESP]; Juliano, Maria A. [UNIFESP]; Lindberg, Iris; Juliano, Luiz [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Maryland
Here we report a detailed analysis of magnesium (Mg(2+)) ion effects on furin hydrolysis of fluorescent resonance energy transfer decapeptide substrates derived from canonical R-X-K/R-R furin cleavage motifs within certain viral envelope glycoproteins and eukaryotic proproteins. Using virus-derived sequences a selective activation of furin by Mg(2+) ions was observed as a result of cooperativity between furin subsites. Furin hydrolysis of the peptides Abz-SRRHKR down arrow FAGV-Q-EDDnp (from measles virus fusion protein F(o)) and Abz-RERRRKKR down arrow GLFG-Q-EDDnp (from Asian avian influenza A, H5N1) was activated between 60- and 80-fold by MgCl(2). It appears that virus envelope glycoprotein mutations have been selected to increase their susceptibility to furin within cells, a location where Mg(2+) is present in adequate concentrations for activation. Both the pH profile of furin and its intrinsic fluorescence were modified by Mg(2+) ions, which bind to furin with a K(d) value of 1.1 mM.
Somente Metadadados
Enhancement of europium emission band of europium tetracycline complex in the presence of cholesterol
(Springer, 2008-01-01) Silva, Flávia Rodrigues de Oliveira; Samad, Ricardo Elgul; Gomes, Laercio; Courrol, Lilia Coronato [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); IPEN CNEN SP
We report here the observation, for the first time, of the enhancement of Europium-Tetracycline complex emission in cholesterol solutions. This enhancement was initially observed with the addition of the enzyme cholesterol oxidase, which produces H2O2, the agent driver of the Europium tetracycline complex, to the solution. However, it was found that the enzyme is not needed to enhance the luminescence. A calibration curve was determined, resulting in a simple method to measure the cholesterol quantity in a solution. This method shows that the complex can be used as a sensor to determine cholesterol in biological systems.
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Fluorescent properties of amino acids labeled with ortho-aminobenzoic acid
(Wiley-Blackwell, 1998-01-01) Ito, A. S.; Turchiello, RDF; Hirata, I. Y.; Cezari, MHS; Meldal, M.; Juliano, L.; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); Dept Chem
ortho-Aminobenzoic acid (Abz) has been used as a convenient fluorescent donor group in internally quenched fluorescent peptides, which are employed as substrates for several proteolytic enzymes. As Abz is usually bound to the N-amino terminal of these peptides, it is of interest to investigate the Abz group fluorescent properties bound to different amino acids. We report in this article the optical absorption and fluorescent properties, in aqueous media, of Abz bound to the alpha-amino group of Ala, Gly, Leu, ne, Val, Pro, Phe, Arg, Glu, Met, Asn, Tyr, and Trp, with monomethyl-amidated alpha-carboxyl group, in order to explore the origin of the drastic reduction of Abz attached to N-alpha amino group of prolyl-peptides, we also examined the fluorescence properties of Abz-NHCH3, Abz-N(CH3)(2), and Abz-pyrrolidine. Molecular dynamics simulation and NMR data indicated a lack of periplanarity of the Abz-dimethylamide, which could be the origin of low fluorescence quantum yield of Abz-prolyl-peptides. (C) 1998 John Wiley & Sons, Inc.
Acesso aberto (Open Access)
The Methylene Blue Self-aggregation in Water/Organic Solvent Mixtures: Relationship Between Solvatochromic Properties and Singlet Oxygen Production
(Univ Federal Mato Grosso Sul, Dept Quimica, 2017) Moreira, Leonardo Marmo; Lyon, Juliana Pereira; Lima, Adriana; Codognoto, Lucia [UNIFESP]; Severino, Divinomar; Baptista, Mauricio S.; Tessaro, Andre Luiz; Gerola, Adriana Passarela; Hioka, Noboru; Rodrigues, Maira Regina; Bonacin, Juliano Alves; dos Santos, Sandra Cruz; Romani, Ana Paula; Moises de Oliveira, Hueder Paulo
In this work is presented a spectrophotometric investigation focused on the solvatochromic effects upon Methylene Blue (MB). Measurements were carried out in four different water/organic solvent mixtures: low polarity protic solvent (ethanol), polar non-protic solvent (acetonitrile), highly polar protic solvent (glycerol), and non-polar solvent (dioxane). The results showed that the photophysical behavior of MB is highly affected by self-aggregates formation at 80% of water/organic solvent blends. Besides polarity, the protic character and the coordinating properties of the solvent molecules are the key parameters for its photophysical behavior, since the sulfur atom of the cationic structure can act as a coordination center due to its Lewis acid character. In fact, water and acetonitrile coordinating properties have proved to be important to determine MB interactions intensity and its spectroscopic properties as singlet oxygen emission. It was observed that an increase of the amount of the organic solvent resulted in an enhancement of the singlet oxygen emission intensity. The presence of the water increases the dielectric constant of the medium and favors the self-aggregation process. Besides, the water molecules can act as a quencher and it decreases the quantum yield of the fluorescence of MB.
Somente Metadadados
Mixtures of trifluoroethanol or hexafluoroisopropanol and dimethylformamide are not of general applicability for peptide condensations catalyzed by trypsin
(Munksgaard Int Publ Ltd, 1998-01-01) Bemquerer, Marcelo P.; Liria, Cleber W.; Kitagawa, Kouki; Miranda, M Teresa M; Tominaga, Mineko; Universidade de São Paulo (USP); Niigata Coll Pharm; Universidade Federal de São Paulo (UNIFESP)
Mixtures of a good hydrogen bond donor, 2,2,2-trifluoroethanol (TFE) or 1,1,1,3,3,3-hexafluroisopropanol, and an acceptor, dimethylformamide (DMF) (1:1,v/v), containing 4% buffer have been described as adequate solvent systems for trypsin-catalyzed peptide fragment condensations [Mihara et al. (1993) Int. J. Pept. Protein Res. 41, 405]. Thus, we decided to study the behaviour of trypsin in such solvent systems. We investigated whether this protease would efficiently catalyze condensations between fragments derived from an analogue of the gp-41 capsid protein of HIV virus or from cholecystokinin-22. None of the reactions carried out yielded the desired condensation products. However, when Fmoc-NLQNLDPSHR-OH and cholecystokinin-12 (H-ISDRDYMGWMDF-NH2) were used as substrates, the last had its R-D peptide bond hydrolyzed producing cholecystokinin-8. The proteolytic activity of this enzyme measured against a fluorogenic peptide derivative was 50 times lower in DMF/TFE containing 5% of aqueous phase than in buffer. Steady-state fluorescence studies in DMF/TFE buffer were performed to examine the structure of this protease in these media. Steady-state spectra obtained with increasing proportions of these two organic solvents in buffer showed that the emission intensities built up. Quenching studies with iodide revealed that the I-o/I ratio (where I,and I are the fluorescence emission intensities in the absence and presence of quencher, respectively) changed from 1.2 in aqueous media to 2.2 in DMF/TFE (1:1, v/v) containing 11% 0.2 M Tris-HCl buffer, pH 8.0, for 0.5 M iodide. The complete data indicated a higher exposure of tryptophan residues to the quencher in organic media, probably because of the partial unfolding of the enzyme. (C) Munksgaard 1997.
Somente Metadadados
Photochemistry of anthocyanins and their biological role in plant tissues
(Walter de Gruyter Gmbh, 2009-09-01) Quina, Frank H.; Moreira, Paulo F.; Vautier-Giongo, Carolina [UNIFESP]; Rettori, Daniel [UNIFESP]; Rodrigues, Rita F.; Freitas, Adilson A.; Silva, Palmira F.; Macanita, Antonio L.; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP); Univ Bandeirante São Paulo; Univ Tecn Lisboa
Anthocyanins, the major red, purple, and blue pigments of plants, absorb visible as well as UV radiation and are effective antioxidants and scavengers of active oxygen species. in plant leaves, one of the functional roles proposed for anthocyanins is protection of the photosynthetic apparatus from the effects of excess incident visible or UV-B radiation and photooxidative stress. in essence, a photoprotective role requires that the excited singlet states of both complexed and uncomplexed anthocyanins deactivate back to the ground state so quickly that intersystem crossing, photoreaction, and diffusion-controlled quenching processes cannot compete. Studies of the photochemical properties of synthetic analogs of anthocyanins and of several naturally occurring anthocyanins show that this is indeed the case, uncomplexed anthocyanins decaying back to the ground state via fast (subnanosecond) excited-state proton transfer (ESPT) and anthocyanin-copigment complexes by fast (sub-picosecond) charge-transfer-mediated internal conversion.
Acesso aberto (Open Access)
Photophysical properties of coumarin compounds in neat and binary solvent mixtures: evaluation and correlation between solvatochromism and solvent polarity parameters
(Sociedade Brasileira de Química, 2014-05-01) Moreira, Leonardo Marmo; Melo, Mirian M. de; Martins, Priscila A.; Lyon, Juliana Pereira; Romani, Ana Paula; Codognoto, Lucia [UNIFESP]; Santos, Sandra C. dos; Oliveira, Hueder Paulo Moisés de; Universidade Federal de São João Del Rei Departamento de Zootecnia; Universidade do Vale do Paraíba Instituto de Pesquisa e Desenvolvimento; Universidade Federal de São João Del Rei Departamento de Ciências Naturais; Universidade Federal do ABC Centro de Engenharia, Modelagem e Ciências Sociais Aplicadas; Universidade Federal de São Paulo (UNIFESP); Universidade Federal de Pelotas Centro de Ciências Químicas, Farmacêuticas e de Alimentos; Universidade Federal do ABC Centro de Ciências Naturais e Humanas
This work studied the fluorescent and solvatochromic properties of coumarin and its derivatives aiming to contribute in the elucidation of the excited state behavior of the molecule in solvent mixtures. This approach has been developed through a set of measurements involving different solvent ratios, which present distinct dielectric constants. The excitation and fluorescence maxima were largely blue-shifted with increasing participation of the organic solvent and the Stokes shifts were unusually low in mixtures with lower solvent polarity parameter, Δf. The emission intensity of coumarin was extremely sensitive to the polarity and the different structural conformations of solvents. These results influenced the spectroscopic behavior and explain the deviation from the linearity of the Lippert-Mataga relationship. The use of fluorescence spectroscopy makes the understanding of the behavior of the systems studied when in the presence of different environments being extremely important for the identification and characterization of coumarin in different pharmaceutical formulations.
Somente Metadadados
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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Study of blood porphyrin spectral profile for diagnosis of tumor progression
(Springer, 2007-05-01) Courrol, Lilia Coronato; Silva, Flávia Rodrigues de Oliveira; Coutinho, Enia Lucia; Piccoli, Michelly Franca; Mansano, Ronaldo Domingues; Vieira Junior, Nilson Dias; Schor, Nestor; Bellini, Maria Helena; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); IPEN CNEN SP
Renal cell carcinoma (RCC) accounts for approximately 3% of new cancer incidence and mortality in the United States. Unfortunately many RCC masses remain asymptomatic and nonpalpable until they are advanced. Diagnosis and localization of early carcinoma play an important role in the prevention and curative treatment of RCC. the autofluorescence of blood porphyrin of healthy and tumor induced in male SCID mice was analyzed using fluorescence and excitation spectroscopy. A significant contrast between normal and tumor blood could be established. Blood porphyrin fluorophore showed enhanced fluorescence band (around 630 nm) in function of the tumor growth. This indicates that either the autofluorescence intensity of the blood fluorescence may provide a good parameter for the first approximation characterization of the tumor stage.
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Study of color centers produced in thulium doped YLF crystals irradiated by electron beam and femtosecond laser pulses
(Elsevier B.V., 2007-02-15) Courrol, Lilia C.; Ranieri, Izilda M.; Baldochi, Sonia Licia; Samad, Ricardo E.; Freitas, Anderson Z. de; Gomes, Laercio; Vieira, Nilson D.; Universidade Federal de São Paulo (UNIFESP); IPEN
In this work, we report the influence of the presence of photochromic and color centers in the photobleaching of thulium ions blue emission in YLF (YLiF4) crystals doped with 1 mol% Tm (3+). the samples were irradiated at room temperature both with electron beam and high intensity ultrashort pulses from a Ti:Sapphire CPA laser system. in both irradiations the production of photochromic and color centers was observed via the absorption bands in the UV and visible ranges. Pure LiF and pure and oxygen doped YLF crystals were used to identify the color centers produced and their optical properties. From a phenomenological model it was possible to study the interaction between color centers and thulium ions, and their effect in photobleaching and photodarkening behaviors. Finally, the blue up laser level population was computed using a rate equation analysis. (c) 2006 Elsevier B.V. All rights reserved.
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Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase
(Blackwell Publishing Ltd, 2002-09-01) Oliveira, Vitor [UNIFESP]; Gatti, R; Rioli, V; Ferro, Emer Suavinho [UNIFESP]; Spisni, A.; Camargo, Antonio Carlos Martins de [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Juliano, Luiz [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); CBME; Universidade de São Paulo (USP); Univ Parma; Inst Butantan
We report the recombinant neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R-R or at R-X bonds, and TOP hydrolyzed at R-R or L-R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. the kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. the relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.054.2 M for NaCl, KCl, NH4Cl and NaI, and 0.025-0.1 M for Na2SO4. Concentration higher than 0.2 N NH4Cl and NaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na2SO4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na2SO4, while NH4Cl and NaI have very modest effect. High positive values; of enthalpy (DeltaH*) and entropy (DeltaS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. the effects of low temperature and high NaCl concentration on the hydrolytic activities of neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.
Somente Metadadados
Trifluoroethanol and binding to model membranes stabilize a predicted turn in a peptide corresponding to the first extracellular loop of the angiotensin II AT(1A) receptor
(Wiley-Blackwell, 2002-10-05) Salinas, R. K.; Shida, C. S.; Pertinhez, T. A.; Spisni, A.; Nakaie, C. R.; Paiva, ACM; Schreier, S.; Universidade de São Paulo (USP); Natl Lab Synchrotron Light; Universidade Federal de São Paulo (UNIFESP)
Homology modeling of the angiotensin H AT(1A) receptor based oil rhodopsin's crystal structure has assigned the 92-100 (YRWPFGNHL) sequence of the receptor to its first extracellular loop. Solution and membrane-bound conformational properties of a peptide containing this sequence (EL1) were examined by CD, fluorescence, and H-1-NMR. CD spectra in aqueous solution revealed an equilibrium between less organized and folded conformers. NMR spectra indicated the coexistence of trans and cis isomers of the Trp(3)-Pro(4) bond. A positive band at 226 urn in the CD spectra suggested aromatic ring stacking, modulated by EL1's ionization degree. CD spectra showed that trifluoroethanol (TFE), or binding to detergent micelles and phospholipid bilayers, shifted the equilibrium toward conformers with higher secondary structure content. Different media gave rise to spectra suggestive of different beta-turns. Chemical shaft changes in the NMR spectra corroborated the stabilization of different conformations. Thus, environments of lower polarity or binding to interfaces probably favored the formation of hydrogen bonds, stabilizing beta-turns, predicted for this sequence in the whole receptor. Increases in Trp(3) fluorescence intensity and anisotropy, blue shifts of the maximum emission wavelength, and pK changes also evinced the interaction between EL1 and model membranes. Binding was seen to depend on both hydrophobic and electrostatic interactions, as well cis lipid phase packing. Studies with water-soluble and membrane-bound fluorescence quenchers demonstrated that Trp(3) is located close to the water-membrane interface. the results are discussed with regard to possible implications in receptor folding and function. (C) 2002 Wiley Periodicals, Inc. Biopolymers 65: 21-31, 2002.

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