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)

Model peptides mimic the structure and function of the N-terminus of the pore-forming toxin sticholysin II

To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger-fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane em,, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behaviour. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P 1-30 was estimated by measuring the permeability PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the PEGs of different pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St 11 conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation. (c) 2005 Wiley Periodicals, Inc.

Functional and Topological Studies with Trp-Containing Analogs of the Peptide StII(1-30) Derived From the N-Terminus of the Pore Forming Toxin Sticholysin II: Contribution to Understand its Orientation in Membrane

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The membranotropic activity of N-terminal peptides from the pore-forming proteins sticholysin I and II is modulated by hydrophobic and electrostatic interactions as well as lipid composition

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cellular vacuolation and mitochondrial-associated factors induced by Clostridium perfringens epsilon toxin detected using acoustic flow cytometry

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

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.

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.

EFFECT of DIFFERENT SOLID AMOUNTS IN THE PHYSICAL and MICROSCOPICAL PROPERTIES of SiC POROUS CERAMICS

The presence of pores in ceramics is directly related to the chosen forming process. So, in the starch consolidation method, the ceramics show, after burning, pores with morphology similar to that presented by this organic material. on the other hand, the increase in solid load leads up to alterations in dispersion viscosity, increasing the thermal stresses during drying and sintering processes. In order to verify the solid percentage influence in ceramic final properties, samples were prepared with silicon carbide in different compositions using or not starch as binder agent and pore forming element. The characterization of the ceramic pieces was performed by superficial roughness measurements, porosity besides by optical and scanning electron microscopy. The results showed ceramics with SiC and starch presented physical and microscopic properties slightly higher in relation to those with only ceramic powder in their composition. The presence of organic material, agglomerated and foam during the forming were essential for the final properties of the studied samples.

Alumina porous ceramics: mathematical behavior at different commercial starch concentration

Several researches have been developed in order to verify the porosity effect over the ceramic material properties. The starch consolidation casting (SCC) allows to obtain porous ceramics by using starch as a binder and pore forming element. This work is intended to describe the porous mathematical behavior and the mechanical resistance at different commercial starch concentration. Ceramic samples were made with alumina and potato and corn starches. The slips were prepared with 10 to 50 wt% of starch. The specimens were characterized by apparent density measurements and three-point flexural test associated to Weibull statistics. Results indicated that the porosity showed a first-order exponential equation e(-x/c) increasing in both kinds of starches, so it was confirmed that the alumina ceramic porosity is related to the kind of starch used. The mechanical resistance is represented by a logarithmic expression R = A + B/1+10((Log(x0)-P)C).

Microstructure of ceramics TiO2 obtained by protein consolidation

Porous ceramics can be produced by adding starch (corn, potato) and protein (animal or vegetable) to raw material as pore forming element. In this study, titanium dioxide ceramics were formed by vegetable protein consolidation. Soybean was chosen as the binding agent and pore forming. The samples, which were produced in cylindrical shape, had the following processing: material mixture, gelling, drying, pre-sintering and sintering. Heated platinum microscopy were performed by using suspensions with different compositions in order to verify protein gelling capacity and better know the temperature in which this process occurs. The samples were characterized by apparent porosity and roughness measurement. Besides, imaging by light microscopy was also performed in order to determine the sample morphology and porosity. © (2012) Trans Tech Publications, Switzerland.

Estudo e desenvolvimento de cerâmicas porosas de dióxido de titânio aditivadas com proteínas de soja

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Desenvolvimento de estruturas porosas de polietileno de ultra alto peso molecular (PEUAPM) recobertas com apatitas para substituição e regeneração óssea

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Preparação e caracterização estrutural de aerogéis de sílica com diferentes concentrações de F127

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

Differential Effect of Solution Conditions on the Conformation of the Actinoporins Sticholysin II and Equinatoxin II

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