Quantifying Lipid Mobility and Peptide Binding for Gram-Negative and Gram-Positive Model Supported Lipid Bilayers
Quantifying Lipid Mobility and Peptide Binding for Gram-Negative and Gram-Positive Model Supported Lipid Bilayers
复制标题
量化革兰氏阴性和革兰氏阳性模型支持的脂质双层的脂质迁移率和肽结合
DOI：
10.1021/acs.jpcb.9b09709
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Smith, Adam W.
中科院分区：
文献类型：
--
作者：
Li, Xiaosi;Smith, Adam W.
模型膜是研究抗生素化合物与细菌膜相互作用机理的有价值的工具。然而，革兰氏阴性和革兰氏阳性细菌的支持脂双层(SLB)模型的发展一直是具有挑战性的，因为组成这些膜的脂类具有高电荷和自发弯曲。在这里，我们描述了一种制备模拟革兰氏阴性内膜和革兰氏阳性膜SLB的方法，包括仅在膜的上部小叶中含有荧光示踪剂的不对称SLB(Easy-SLB)。我们用荧光相关光谱(FCS)定量了这些膜中脂质的动力学，发现在革兰氏阴性SLB/asySLB中，脂质扩散慢于革兰氏阳性SLB/asySLB。与这些膜结合的多肽也用粘菌素进行了表征，粘菌素是一种革兰氏阴性的特异性抗生素。用脉冲交错激发荧光互相关光谱(PIE-FCCS)研究了粘菌素与膜脂磷脂酰乙醇胺(PE)或心磷脂(TOCL)的相互作用。总体而言，我们的数据为脂类在革兰氏阴性膜和革兰氏阳性膜中的扩散动力学提供了独特的见解，并为研究抗菌肽和细菌膜脂之间的相互作用机制提供了一个新的平台。
Model membranes are a valuable tool to investigate the mechanism of interaction between antibiotic compounds and bacterial membranes. However, the development of supported lipid bilayer (SLB) models for Gram-negative and Gram-positive bacteria has been challenging because of the high charge and spontaneous curvature of the lipids that make up these membranes. Here we describe a method for preparing mimetic Gram-negative inner membrane and Gram-positive membrane SLBs, including asymmetric SLBs (asy-SLBs) that contain a fluorescent tracer only in the upper leaflet of the membrane. We quantified the dynamics of the lipids in these membranes with fluorescence correlation spectroscopy (FCS) and found that lipid diffusion is slower in Gram-negative SLBs/asySLBs than in Gram-positive SLBs/asySLBs. Peptide binding to these membranes was also characterized using colistin, a Gram-negative specific antibiotic. Interactions between colistin and membrane lipids phosphatidylethanolamine (PE) or cardiolipin (TOCL) were probed with pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Overall, our data provide unique insight into the diffusion dynamics of lipids in Gram-negative and Gram-positive membranes as well as a novel platform for investigating the mechanism of interaction between antibiotic peptides and bacterial membrane lipids.