Tertiary structural changes of the alpha-hemolysin from Staphylococcus aureus on association with liposome membranes

The interaction of alpha-hemolysin (also called alpha-toxin) from Staphylococcus aureus with mixed egg-yolk phosphatidylcholine/cholesterol liposomes has been investigated using the intrinsic tryptophan fluorescence emission (ITFE) signal. The ITFE intensity of alpha-hemolysin, which was obtained using a novel purification protocol, showed a triphasic increase on incubation with liposomes at low protein/lipid ratios. The first, rapid phase results in an increase in ITFE of 10%, which reflects rapid conformation changes in the alpha-hemolysin on association with the liposome membrane, the second phase of the ITFE increase is associated with a red shift from 334 to 339 nm in the maximum emission wavelength, suggesting the transition to a partially unfolded intermediate in the oligomerization process. The third phase of the ITFE intensity change demonstrates a temporal correlation with the appearance of SDS-stable oligomers. The results demonstrate the feasibility of identification of intermediate protein conformations in complex membrane-associated processes by manipulation of the liposomal membrane composition. (C) 1998 Academic Press.

A stability transition at mildly acidic pH in the alpha-hemolysin (alpha-toxin) from Staphylococcus aureus

The effects of mildly acidic conditions on the free energy of unfolding (Delta G(u)(buff)) of the pore-forming alpha-hemolysin (alpha HL) from Staphylococcus aureus were assessed between pH 5.0 and 7.5 by measuring intrinsic tryptophan fluorescence, circular dichroism and elution time in size exclusion chromatography during urea denaturation, Decreasing the pH from 7.0 to 5.0 reduced the calculated Delta G(u)(buff) from 8.9 to 4.2 kcal moI(-1), which correlates with an increased rate of pore formation previously observed over the same pH range, It is proposed that the lowered surface pH of biological membranes reduces the stability of alpha HL thereby modulating the rate of pore formation. (C) 1999 Federation of European Biochemical Societies.

The X-ray crystallographic structure of Escherichia coli branching enzyme

Branching enzyme catalyzes the formation of alpha-1,6 branch points in either glycogen or starch. We report the 2.3-Angstrom crystal structure of glycogen branching enzyme from Escherichia coli. The enzyme consists of three major domains, an NH2-terminal seven-stranded beta-sandwich domain, a COOH-terminal domain, and a central alpha/beta-barrel domain containing the enzyme active site. While the central domain is similar to that of all the other amylase family enzymes, branching enzyme shares the structure of all three domains only with isoamylase. Oligosaccharide binding was modeled or branching enzyme using the enzyme-oligosaccharide complex structures of various alpha-amylases and cyclodextrin glucanotransferase and residues were implicated in oligosaccharide binding. While most of the oligosaccharides modeled well in the branching enzyme structure, an approximate 50degrees rotation between two of the glucose units was required to avoid steric clashes with Trp(298) of branching enzyme. A similar rotation was observed in the mammalian alpha-amylase structure caused by an equivalent tryptophan residue in this structure. It appears that there are two binding modes for oligosaccharides in these structures depending on the identity and location of this aromatic residue.

Activation of Ca2+-independent membrane-damaging activity in Lys49-phospholipase A(2) promoted by amphiphilic molecules

Association of class-II phospholipase A(2) (PLA(2)) with aggregated phospholipid substrate results in elevated levels of the Ca2+-dependent hydrolytic activity. The Asp49 residue participates in coordination of the Ca2+ ion cofactor, however, in Lys49-PLA(2) homologues (Lys49-PLA(2)S), substitution of the Asp49 by Lys results in loss of Ca2+ binding and lack of detectable phospholipid hydrolysis. Nevertheless, Lys49-PLA2S cause Ca2+-independent damage of liposome membranes. Bothropstoxin-I is a homodimeric Lys49-PLA(2) from the venom of Bothrops jararacussu, and in fluorescent marker release and dynamic light scattering experiments with DPPC liposomes we demonstrate activation of the Ca2+-independent membrane damaging activity by similar to4 molecules of sodium dodecyl sulphate (SDS) per protein monomer. Activation is accomparlied by significant changes in the intrinsic tryptophan fluorescence emission (ITFE) and near UV circular dichroism (UVCD) spectra of the protein. Subsequent binding of 7-10 SDS molecules results in further alterations in the ITFE and far UVCD spectra. Reduction in the rate of N-bromosuccinimide modification of Trp77 at the dimer interface suggests that initial binding of SDS to this region accompanies the activation of the membrane damaging activity. 1-anilinonaphthalene-8-sulphonic acid binding studies indicate that subsequent SDS binding to the active site is concomitant with the second structural transition. These results provide insights in the structural basis of amphiphile/protein coupling in class-II PLA(2)s. (C) 2004 Published by Elsevier B.V.

The interaction between heparin and Lys49 phospholipase A(2) reveals the natural binding of heparin on the enzyme

We have studied at a molecular level the interaction of heparins on bothropstoxin-1 (BthTx-1), a phospholipase A(2) toxin. The protein was monitored using gel filtration chromatography, dynamic light scattering (DLS), circular dichroism (CD), attenuated total reflectance Fourier transform infrared (ATR-FTIR) and intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. The elution profile of the protein presents a displacement of the protein peak to larger complexes when interacting with higher concentration of heparin. The DLS results shows two R-h at a molar ratio of 1, one to the distribution of the protein and the second for the action of heparin on BthTx-I structures, and a large distribution with the increase of protein. The interaction is accompanied by significant changes in the CD spectra, showing two common features: a decrease in signal at 208 nm (3 and 6 kDa heparins) and an isodichroic point near 226 nm (3 kDa heparin). FTIR spectra indicate that only a few amino acid residues are involved in this interaction. Alterations in the ITFE by binding heparins suggest that the initial binding occurs on the ventral face of BthTx-1. Together, these results add an experimental and structural basis on the action mechanism of the heparins over the phospholipases A(2) and provide a molecular model to elucidate the interaction of the enzyme-heparin complex at a molecular level. (c) 2005 Elsevier B.V. All rights reserved.

Crystallographic and spectroscopic characterization of a molecular hinge: Conformational changes in bothropstoxin I, a dimeric Lys49 phospholipase A2 homologue

Bothropstoxin I(BthTX-I) from the venom of Bothrops jararacussu is a myotoxic phospholipase A2 (PLA2) homologue which, although catalytically inactive due to an Asp49-->Lys substitution, disrupts the integrity of lipid membranes by a Ca2+-independent mechanism, the crystal structures of two dimeric farms of BthLTX-I which diffract X-rays eo resolutions of 3.1 and 2.1 Angstrom have been determined, the monomers in both structures are related by an almost perfect twofold axis of rotation and the dimer interfaces are defined by contacts between the N-terminal alpha-helical regions and the tips of the beta-wings of partner monomers. Significant differences in the relative orientation of the monomers in the two crystal forms results in open and closed dimer conformations, Spectroscopic Investigations of BthTX-I in solution have correlated these conformational differences with changes in the intrinsic fluorescence emission of the single tryptophan residues located at the dimer interface, the possible relevance of this structural transition in the Ca2+-independent membrane damaging activity is discussed. (C) 1998 Wiley-Liss, Inc.

An ultraviolet photoacoustic spectroscopy study of the interaction between Lys49-phospholipase A(2) and amphiphilic molecules

We have used near ultraviolet photoacoustic spectroscopy (PAS) over the wavelength range 240-320 nm to investigate the complex formed between the homodimeric bothropstoxin-I, a lysine-49-phospholipase A(2) from the venom of Bothrops jararacussu (BthTx-I), with the anionic amphiphile sodium dodecyl sulfate (SDS). At molar ratios > 10, the complex developed a significant light scatter, accompanied by a decrease in the intrinsic tryptophan fluorescence intensity emission (ITFE) of the protein, and an increase in the near UV-PAS signal. Difference PAS spectroscopy at SDS/BthTx-I ratios < 8 were limited to the region 280-290 nm, suggesting initial SDS binding to the tryptophan 77 located at the dimer interface. At SDS/BthTx-I ratios > 10, the intensity between 260 and 320 nm increases demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interaction. PAS signal phase changes at wavelengths specific for each aromatic residue suggest that the Trp77 becomes more buried on SDS binding, and that protein structural changes and dehydration may alter the microenvironments of Tyr and Phe residues. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scatter is significant. (c) 2006 Elsevier B.V. All rights reserved.

Photoacoustic spectroscopy of aromatic amino acids in proteins

This paper concerns the use of photoacoustic spectroscopy (PAS) to study the presence of aromatic amino acid in proteins. We examined the aromatic amino acids in six proteins with well-known structures using absorption spectra of near ultraviolet PAS over the wavelength range 240-320 nm. The fundamental understanding of the physical and chemical properties that govern the absorption of light and a subsequent release of heat to generate a transient pressure wave was used to test the concept of monitoring aromatic amino acids with this method. Second derivative spectroscopy in the ultraviolet region of proteins was also used to study the regions surrounding the aromatics and the percentage area in each band was related in order to determine the contribution in function of the respective molar extinction coefficients for each residue. Further investigation was conducted into the interaction between sodium dodecyl sulphate (SDS) and bothropstoxin-I (BthTx-I), with the purpose of identifying the aromatics that participate in the interaction. The clear changes in the second derivative and curve-fitting procedures suggest that initial SDS binding to the tryptophan located in the dimer interface and above 10 SDS an increased intensity between 260 and 320 nm, demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interactions. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scattering is significant.

Validation of a mutant of the pore-forming toxin sticholysin-I for the construction of proteinase-activated immunotoxins

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

Indole ring oxidation by activated leukocytes prevents the production of hypochlorous acid

Hypochlorous acid (HOCl) released by activated leukocytes has been implicated in the tissue damage that characterizes chronic inflammatory diseases. In this investigation, 14 indole derivatives, including metabolites such as melatonin, tryptophan and indole-3-acetic acid, were screened for their ability to inhibit the generation of this endogenous oxidant by stimulated leukocytes. The release of HOCl was measured by the production of taurine-chloramine when the leukocytes (2 x 10(6) cells/mL) were incubated at 37ºC in 10 mM phosphate-buffered saline, pH 7.4, for 30 min with 5 mM taurine and stimulated with 100 nM phorbol-12-myristate acetate. Irrespective of the group substituted in the indole ring, all the compounds tested including indole, 2-methylindole, 3-methylindole, 2,3-dimethylindole, 2,5-dimethylindole, 2-phenylindole, 5-methoxyindole, 6-methoxyindole, 5-methoxy-2-methylindole, melatonin, tryptophan, indole-3-acetic acid, 5-methoxy-2-methyl-3-indole-acetic acid, and indomethacin (10 µM) inhibited the chlorinating activity of myeloperoxidase (MPO) in the 23-72% range. The compounds 3-methylindole and indole-3-acetic acid were chosen as representative of indole derivatives in a dose-response study using purified MPO. The IC50 obtained were 0.10 ± 0.03 and 5.0 ± 1.0 µM (N = 13), respectively. These compounds did not affect the peroxidation activity of MPO or the production of superoxide anion by stimulated leukocytes. By following the spectral change of MPO during the enzyme turnover, the inhibition of HOCl production can be explained on the basis of the accumulation of the redox form compound-II (MPO-II), which is an inactive chlorinating species. These results show that indole derivatives are effective and selective inhibitors of MPO-chlorinating activity.

Identificação de alguns constituintes químicos de Indigofera hirsuta Linn. (Fabaceae) por CLAE-IES-EM (TOF) e avaliação da atividade antirradicalar

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

Studies on stem cuttings of kiwi (Actinidia chinensis PL. CV Bruno)

O presente trabalho teve como objetivo, estudar o efeito de auxinas sintéticas e do boro, sobre o enraizamento de estacas caulinares de kiwi (Actinidia chinensisPlanch. cv Bruno). As estacas continham dois nós com aproximadamente 10 cm de comprimento, contendo 2 folhas cortadas ao meio. As bases das estacas receberam os seguintes tratamentos: control (H2O); NAA 300 mg.L-1; IBA 300 mg.L-1; NAA 300 mg.L-1 + B; IBA 300 mg.L-1 + B; NAA 0,5%-pó e IBA 0,5%-pó. Após os tratamentos as estacas foram plantadas em bandejas de enraizamento contendo vermiculita pura e colocadas em câmara de nebulização por 120 dias até a coleta das mesmas. Para a avaliação do efeito das auxinas e boro, foram realizadas as seguintes observações: 1. porcentagem de estacas enraizadas; 2. análise de açúcares redutores e açúcares totais (em g/100 g de matéria seca); 3. análise de triptofano (em µg/100 mg de matéria seca). Além disso, foram verificados o efeito dos tratamentos em quatro épocas, que corresponderam às estações do ano (primavera, verão, outono e inverno). Através dos resultados obtidos no processo de enraizamento de estacas caulinares de kiwi (Actinidia chinensis Planch. cv Bruno), conclui-se ser o verão a melhor época de coleta dos ramos para a produção das estacas sem a necessidade do tratamento com auxinas.

Composição química de macrófitas aquáticas flutuantes utilizadas no tratamento de efluentes de aqüicultura

Os objetivos deste trabalho foram avaliar a composição química de duas espécies de macrófitas aquáticas flutuantes (Eichhornia crassipes e Pistia stratiotes), utilizadas no tratamento de efluentes de aqüicultura, e inferir as possibilidades de aproveitamento desses vegetais. E. crassipes apresentou os maiores conteúdos de cálcio (1,51%), magnésio (3.916,67 mg kg-1), manganês (1.233,33 mg kg-1), zinco (81,83 mg kg-1), ferro (5.425,00 mg kg¹) e cobre (25,83 mg kg-1). Na biomassa de P. stratiotes foram obtidos os maiores valores de matéria mineral (18,95%), fósforo (0,38%), nitrogênio (2,40%), proteína bruta (15,02%) e aminoácidos, à exceção de ácido aspártico e triptofano.

Enraizamento de estacas caulinares de kiwi (Actinidia chinensis Planch cv Abbott) tratadas com auxinas e boro

O trabalho teve como finalidade, estudar o efeito de várias auxinas sintéticas em formulações comerciais e do boro, sobre o enraizamento de estacas caulinares de kiwi (Actinidia chinensis Planch, cv Abbott.). As estacas utilizadas continham dois nós e duas folhas cortadas ao meio, com aproximadamente 10 cm de comprimento, onde o corte basal em bisel foi realizado logo abaixo de um nó e o apical acima do outro nó. O efeito das auxinas, sobre o enraizamento de estacas caulinares de kiwi foi verificado mediante os seguintes tratamentos, aplicados sobre as bases das estacas: T1 H(2)0); T2 (NAA 300 ppm); T3 (IBA 300 ppm); T4 (NAA 300 ppm + B); T5 (IBA 300 ppm + B); T6 (NAA 0,5%-pó) e T7 (IBA 0,5%-pó). Após o tratamento das estacas, estas foram plantadas em bandejas de enraizamento, contendo vermiculita pura e colocadas em câmara de nebulização, onde permaneceram por 120 dias, até a sua coleta. Para a avaliação do efeito de auxinas e do ácido bórico, sobre o enraizamento de estacas caulinares de kiwi, foram realizadas as seguintes observações: 1. porcentagem de estacas enraizadas; 2. análise de açúcares redutores e açúcares totais (em g/100 g de matéria seca); 3. análise de triptofano (em µg/100 mg de matéria seca). Os resultados obtidos no processo de enraizamento de estacas caulinares de kiwi (Actinidia chinensis Planch.) variedade Abbott, levou a concluir que o inverno e outono foram as melhores épocas de coleta dos ramos de auxinas para a confecção das estacas. O processo de enraizamento foi ainda incrementado com a aplicação exógena na base das estacas, sendo que o alto teor de açúcares redutores e totais beneficiou a maior porcentagem de enraizamento.

Effects of auxins and boron on rooting of kiwi stem cuttings (Actinidia chinensis Pl cv Matua)

This work studies the effects of some synthetical auxins and boron in the rooting of stem cuttings of kiwi (Actinidia chinensis Planch cv Matua). The stems used had two nodes and two leaves cut in half. The auxin effect was observed through seven different treatments: T1 (H2O); T2 (NAA 300 ppm); T3 (IBA 300 ppm); T4 (NAA 300 ppm + B); T5 (IBA 300 ppm + B); T6 (NAA 0,5%-talc) and T7 (IBA 0,5%-talc), applied to the bases of stem cuttings. After these treatments, the cuttings were placed in suitable rooting dishes, with pure vermiculite in misty nebulization chamber for 120 days until collection day. The evaluation of auxin and boric acid effects were made based on the following observations: 1. The percentage of rooted stem cuttings; 2. reducing sugar and total sugar analyses; and 3. tryptophan analyses. The effects of such treatments were observed in the four seasons. The results showed that winter is best for rooting. Application of IBA talc 0,5% to the cuttings bases increased rooting.