Oren Z, Shai Y
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Biochemistry. 1997 Feb 18;36(7):1826-35. doi: 10.1021/bi962507l.
Studies on lipid-peptide interactions of cytolytic polypeptides tend to emphasize the importance of the amphipathic alpha-helical structure for their cytolytic activity. In this study, diasetereomers of the bee venom melittin (26 a.a.), a non-cell-selective cytolysin, were synthesized and investigated for their structure and cytolytic activity toward bacteria and mammalian cells. Similarly to the findings with the diastereomers of the less cytolytic peptide pardaxin (33 a.a.) (Shai & Oren. 1996), the melittin diastereomer, lest their alpha-helical structure, which abrogated their hemolytic activity toward human erythrocytes. However, they retained their antibacterial activity and completely lysed both Gram-positive and Gram-negative bacteria, as revealed by transmission electron microscopy. To understand the molecular mechanism underlying this selectivity, binding experiments utilizing the intrinsic tryptophan of melittin, tryptophan quenching experiments using brominated phospholipids, and membrane destabilization studies were done. The data revealed that the melittin diastereomers bound to and destabilized only negatively-charged phospholipid vesicles, in contrast to native melittin, which binds strongly to both negatively-charged and zwitterionic phospholipids. However, the partition coefficient, the depth of penetration into the membrane, and the membrane-permeating activity of the diastereomers with negatively-charged phospholipids were similar to those obtained with melittin. The results obtained do not support the formation of transmembrane pores as the mode of action of the diastereomers, but rather suggest that these peptides bind to the surface of the bacterial membrane, cover it in a "carpet-like" manner, and dissolve it like a detergent. The results presented here together with those obtained with the cytolytic peptide pardaxin suggest that the combination of hydrophobicity and net positive charge may be sufficient in the design of potent diastereomers of antibacterial polypeptides for the treatment of infectious diseases.
关于溶细胞多肽的脂质 - 肽相互作用的研究往往强调两亲性α - 螺旋结构对其溶细胞活性的重要性。在本研究中，合成了非细胞选择性溶细胞素——蜂毒蜂毒素（26个氨基酸）的非对映异构体，并研究了它们的结构以及对细菌和哺乳动物细胞的溶细胞活性。与细胞毒性较小的肽——豹鳎毒素（33个氨基酸）的非对映异构体的研究结果类似（Shai & Oren，1996），蜂毒素非对映异构体失去了其α - 螺旋结构，这消除了它们对人红细胞的溶血活性。然而，它们保留了抗菌活性，并通过透射电子显微镜显示能完全裂解革兰氏阳性菌和革兰氏阴性菌。为了理解这种选择性背后的分子机制，进行了利用蜂毒素内在色氨酸的结合实验、使用溴化磷脂的色氨酸猝灭实验以及膜去稳定化研究。数据表明，与天然蜂毒素强烈结合带负电荷和两性离子磷脂不同，蜂毒素非对映异构体仅与带负电荷的磷脂囊泡结合并使其去稳定化。然而，非对映异构体与带负电荷磷脂的分配系数、膜渗透深度和膜渗透活性与蜂毒素的相似。所获得的结果不支持跨膜孔的形成作为非对映异构体的作用方式，而是表明这些肽结合到细菌膜表面，以“地毯样”方式覆盖它，并像去污剂一样溶解它。这里呈现的结果与溶细胞肽豹鳎毒素的结果一起表明，疏水性和净正电荷的组合在设计用于治疗传染病的强效抗菌多肽非对映异构体时可能就足够了。
