Casallanovo Fábio, de Oliveira Felipe J F, de Souza Fernando C, Ros Uris, Martínez Yohanka, Pentón David, Tejuca Mayra, Martínez Diana, Pazos Fabiola, Pertinhez Thelma A, Spisni Alberto, Cilli Eduardo M, Lanio María E, Alvarez Carlos, Schreier Shirley
Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP, Brazil.
Biopolymers. 2006;84(2):169-80. doi: 10.1002/bip.20374.
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 environment, 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 behavior. 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 P1-30 was estimated by measuring the permeability to PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St II 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.
为了研究N端区域在成孔毒素刺参溶细胞素II（St II）的裂解机制中的作用，我们研究了包含该蛋白前30个残基的肽段的构象和功能特性。合成了包含残基1 - 30（P1 - 30）和11 - 30（P11 - 30）的肽段，并在水溶液中研究了它们的构象特性，作为肽浓度、pH、离子强度以及添加二级结构诱导溶剂三氟乙醇（TFE）的函数。圆二色光谱表明，浓度、pH和离子强度的增加导致P1 - 30聚集；结果，该肽获得了β折叠构象。相反，在相同条件下P11 - 30几乎没有构象变化，基本上保持无结构状态。此外，该肽没有发生聚集。这些差异清楚地表明前10个疏水残基对肽的聚集和构象特性具有调节作用。在TFE中，前十个疏水肽都获得了α螺旋构象，尽管程度不同，P11 - 30的α螺旋含量较低。在TFE中，P1 - 30处于α螺旋构象的残基比例比该蛋白该部分在St II晶体结构中发现的比例更大。由于TFE模拟膜环境，毒素与膜结合时也可能发生这种螺旋含量的增加，这代表了孔形成机制中的一个步骤。肽的构象特性与其功能行为密切相关。因此，P1 - 30表现出比P11 - 30高得多的溶血活性。此外，P11 - 30能够阻断毒素的溶血活性。通过测量不同分子量聚乙二醇的通透性来估计P1 - 30在红细胞中形成的孔的大小。孔半径（0.95±0.01 nm）与毒素形成的孔非常相似。结果表明，合成肽P1 - 30是St II构象和功能的良好模型，并强调了毒素N端区域，特别是疏水残基1 - 10对孔形成的贡献。
