Clinical Diagnostics Department, Medical School, Southeast University, Nanjing, China; Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany.
Physical Chemistry Department, Heidelberg University, Germany.
Biochim Biophys Acta Biomembr. 2018 Feb;1860(2):617-623. doi: 10.1016/j.bbamem.2017.10.025. Epub 2017 Oct 26.
Most antimicrobial peptides exert their rapid bactericidal activity through a unique mechanism of bacterial membrane disruption. However, the molecular events that underlie this mechanism remain partly unresolved. In this study, the frequency shift (ΔF) obtained through quartz-crystal microbalance with dissipation (QCM-D) indicated that the initial binding of Ib-AMP4 within the lipid membrane started at a critical Ib-AMP4 concentration that exceeded 100μg/ml. Circular dichroism measurements provided evidence that Ib-AMP4 occurs in a β-sheet configuration which is adapted for insertion into the lipid membrane. Monolayer experiments and the value of dissipation alteration (ΔD) obtained through QCM-D showed that the pressure increased within the phospholipid bilayer upon peptide insertion, and the increase in pressure subsequently forced the bilayer to wrinkle and form pores. However, D continued to increase, indicating that the membrane surface underwent a dramatic morphological transition: the membrane surface likely became porous and uneven as Ib-AMP4 projected from the external surface of the lipid bilayer. Intensive peptide insertion, however, soon plateaued 1min after the addition of Ib-AMP4. This behaviour corresponded with the results of bactericidal kinetics and liposome leakage assays. A sudden decrease in D accompanied by a negligible decrease in F occurred after replacing the Ib-AMP4 solution with HEPES buffer. This result implied that the bilayer surface rearranged and that poration and wrinkling decreased without further peptide insertion. Transmission electron microscopy results indicated that pore formation occurred during Ib-AMP4 insertion but eventually subsided. Therefore, the mode of action of AMP in bacterial membranes could be elucidated through QCM-D.
大多数抗菌肽通过破坏细菌膜的独特机制发挥其快速杀菌活性。然而，这一机制背后的分子事件仍部分未解决。在这项研究中，石英晶体微天平耗散（QCM-D）获得的频率偏移（ΔF）表明，Ib-AMP4 在脂质膜中的初始结合始于超过 100μg/ml 的临界 Ib-AMP4 浓度。圆二色性测量提供的证据表明，Ib-AMP4 呈 β-折叠构象，适用于插入脂质膜。单层实验和 QCM-D 获得的耗散变化（ΔD）值表明，肽插入后磷脂双层内的压力增加，压力的增加随后迫使双层起皱并形成孔。然而，D 继续增加，表明膜表面发生了剧烈的形态转变：由于 Ib-AMP4 从脂质双层的外表面伸出，膜表面可能变得多孔且不均匀。然而，肽的密集插入在加入 Ib-AMP4 1 分钟后很快达到平台期。这种行为与杀菌动力学和脂质体渗漏测定的结果相对应。用 HEPES 缓冲液替换 Ib-AMP4 溶液后，D 的突然下降伴随着 F 的可忽略不计的下降。这一结果意味着双层表面重新排列，穿孔和起皱减少而没有进一步的肽插入。透射电子显微镜结果表明，在 Ib-AMP4 插入过程中发生了孔形成，但最终平息。因此，通过 QCM-D 可以阐明 AMP 在细菌膜中的作用模式。
