Research Center for Proteinaceous Materials (RCPM), Chosun University, Kwangju, Korea.
PLoS One. 2013 Jul 23;8(7):e67597. doi: 10.1371/journal.pone.0067597. Print 2013.
HP (2-20) is a 19-aa, amphipathic, α-helical peptide with antimicrobial properties that was derived from the N-terminus of Helicobacter pylori ribosomal protein L1. We previously showed that increasing the net hydrophobicity of HP (2-20) by substituting Trp for Gln(17) and Asp(19) (Anal 3) increased the peptide's antimicrobial activity. In hydrophobic medium, Anal 3 forms an amphipathic structure consisting of an N-terminal random coil region (residues 2-5) and an extended helical region (residues 6-20). To investigate the structure-activity relationship of Anal 3, we substituted Pro for Glu(9) (Anal 3-Pro) and then examined the new peptide's three-dimensional structure, antimicrobial activity and mechanism of action. Anal 3-Pro had an α-helical structure in the presence of trifluoroethanol (TFE) and sodium dodecyl sulfate (SDS). NMR spectroscopic analysis of Anal 3-Pro's tertiary structure in SDS micelles confirmed that the kink potential introduced by Pro(10) was responsible for the helix distortion. We also found that Anal 3-Pro exhibited about 4 times greater antimicrobial activity than Anal 3. Fluorescence activated flow cytometry and confocal fluorescence microscopy showed that incorporating a Pro-hinge into Anal 3 markedly reduced its membrane permeability so that it accumulated in the cytoplasm without remaining in the cell membrane. To investigate the translocation mechanism, we assessed its ability to release of FITC-dextran. The result showed Anal 3-Pro created a pore <1.8 nm in diameter, which is similar to buforin II. Notably, scanning electron microscopic observation of Candida albicans revealed that Anal 3-Pro and buforin II exert similar effects on cell membranes, whereas magainin 2 exerts a different, more damaging, effect. In addition, Anal 3-Pro assumed a helix-hinge-helix structure in the presence of biological membranes and formed micropores in both bacterial and fungal membranes, through which it entered the cytoplasm and tightly bound to DNA. These results indicate that the bending region of Anal 3- Pro peptide is prerequisite for effective antibiotic activity and may facilitate easy penetration of the lipid bilayers of the cell membrane.
HP (2-20) 是一种 19 个氨基酸的两亲性 α-螺旋肽，具有抗菌特性，它来源于幽门螺杆菌核糖体蛋白 L1 的 N 端。我们之前的研究表明，通过将 Gln(17)和 Asp(19)替换为色氨酸（Anal 3）增加 HP (2-20) 的净疏水性，可以提高该肽的抗菌活性。在疏水环境中，Anal 3 形成一种两亲性结构，由 N 端的无规卷曲区（残基 2-5）和延伸的螺旋区（残基 6-20）组成。为了研究 Anal 3 的结构-活性关系，我们将 Glu(9)替换为 Pro（Anal 3-Pro），然后检测新肽的三维结构、抗菌活性和作用机制。在三氟乙醇 (TFE) 和十二烷基硫酸钠 (SDS) 存在下，Anal 3-Pro 具有 α-螺旋结构。Anal 3-Pro 在 SDS 胶束中的三级结构的 NMR 光谱分析证实，Pro(10)引入的扭曲势导致了螺旋的扭曲。我们还发现，Anal 3-Pro 的抗菌活性比 Anal 3 大约高出 4 倍。荧光激活流式细胞术和共聚焦荧光显微镜观察表明，在 Anal 3 中引入 Pro 铰链会显著降低其膜通透性，使其在细胞质中积累而不留在细胞膜中。为了研究其转运机制，我们评估了它释放 FITC-葡聚糖的能力。结果表明，Anal 3-Pro 形成的孔直径小于 1.8nm，与 Buforin II 相似。值得注意的是，对白色念珠菌的扫描电子显微镜观察表明，Anal 3-Pro 和 Buforin II 对细胞膜有相似的作用，而 Magainin 2 则有不同的、更具破坏性的作用。此外，在生物膜存在的情况下，Anal 3-Pro 呈现出一个“螺旋-铰链-螺旋”的结构，并在细菌和真菌的膜上形成微孔，通过这些微孔进入细胞质并与 DNA 紧密结合。这些结果表明，Anal 3-Pro 肽的弯曲区域是有效抗生素活性的前提，可能有助于其容易穿透细胞膜的脂质双层。
