Oren Ziv, Ramesh Jagannathan, Avrahami Dorit, Suryaprakash N, Shai Yechiel, Jelinek Raz
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel; Department of Chemistry, Ben Gurion University of the Negev, Beersheva, Israel.
Eur J Biochem. 2002 Aug;269(16):3869-80. doi: 10.1046/j.1432-1033.2002.03080.x.
The interaction of many lytic cationic antimicrobial peptides with their target cells involves electrostatic interactions, hydrophobic effects, and the formation of amphipathic secondary structures, such as alpha helices or beta sheets. We have shown in previous studies that incorporating approximately 30%d-amino acids into a short alpha helical lytic peptide composed of leucine and lysine preserved the antimicrobial activity of the parent peptide, while the hemolytic activity was abolished. However, the mechanisms underlying the unique structural features induced by incorporating d-amino acids that enable short diastereomeric antimicrobial peptides to preserve membrane binding and lytic capabilities remain unknown. In this study, we analyze in detail the structures of a model amphipathic alpha helical cytolytic peptide KLLLKWLL KLLK-NH2 and its diastereomeric analog and their interactions with zwitterionic and negatively charged membranes. Calculations based on high-resolution NMR experiments in dodecylphosphocholine (DPCho) and sodium dodecyl sulfate (SDS) micelles yield three-dimensional structures of both peptides. Structural analysis reveals that the peptides have an amphipathic organization within both membranes. Specifically, the alpha helical structure of the L-type peptide causes orientation of the hydrophobic and polar amino acids onto separate surfaces, allowing interactions with both the hydrophobic core of the membrane and the polar head group region. Significantly, despite the absence of helical structures, the diastereomer peptide analog exhibits similar segregation between the polar and hydrophobic surfaces. Further insight into the membrane-binding properties of the peptides and their depth of penetration into the lipid bilayer has been obtained through tryptophan quenching experiments using brominated phospholipids and the recently developed lipid/polydiacetylene (PDA) colorimetric assay. The combined NMR, FTIR, fluorescence, and colorimetric studies shed light on the importance of segregation between the positive charges and the hydrophobic moieties on opposite surfaces within the peptides for facilitating membrane binding and disruption, compared to the formation of alpha helical or beta sheet structures.
许多溶菌性阳离子抗菌肽与其靶细胞的相互作用涉及静电相互作用、疏水作用以及两亲性二级结构的形成，如α螺旋或β折叠。我们在先前的研究中表明，将约30%的d-氨基酸掺入由亮氨酸和赖氨酸组成的短α螺旋溶菌肽中，可保留亲本肽的抗菌活性，同时消除溶血活性。然而，掺入d-氨基酸诱导独特结构特征的潜在机制尚不清楚，这些结构特征使短的非对映异构抗菌肽能够保留膜结合和溶菌能力。在本研究中，我们详细分析了模型两亲性α螺旋溶细胞肽KLLLKWLL KLLK-NH2及其非对映异构体类似物的结构，以及它们与两性离子和带负电荷膜的相互作用。基于在十二烷基磷酸胆碱（DPCho）和十二烷基硫酸钠（SDS）胶束中的高分辨率NMR实验进行的计算得出了两种肽的三维结构。结构分析表明，两种肽在两种膜中均具有两亲性组织。具体而言，L型肽的α螺旋结构导致疏水和极性氨基酸分别位于不同表面，从而允许与膜的疏水核心和极性头部基团区域相互作用。值得注意的是，尽管不存在螺旋结构，但非对映体肽类似物在极性和疏水表面之间表现出类似的分离。通过使用溴化磷脂的色氨酸猝灭实验和最近开发的脂质/聚二乙炔（PDA）比色法，进一步深入了解了肽的膜结合特性及其进入脂质双层的深度。与α螺旋或β折叠结构的形成相比，NMR、FTIR、荧光和比色法的联合研究揭示了肽内相反表面上正电荷和疏水部分之间分离对于促进膜结合和破坏的重要性。
