School of Chemistry and ‡Florey Department of Neuroscience and Mental Health, Centre for Neuroscience Research Chemistry, University of Melbourne , Parkville, Victoria 3010, Australia.
Langmuir. 2013 Nov 26;29(47):14613-21. doi: 10.1021/la403083m. Epub 2013 Nov 14.
The interaction dynamics between a lytic peptide and a biomembrane was studied using time-lapse fluorescence lifetime imaging microscopy. The model membrane was 1,2-dipalmitoyl-sn-glycero-3-phosphochloine giant unilamellar vesicles (GUVs), and the peptide was the K14 derivative of melittin, to which the polarity-sensitive fluorescent probe AlexaFluor 430 was grafted. The interaction of the peptide with the GUVs resulted in a progressive quenching of the fluorescence lifetime over a period of minutes. From previous photophysics characterization of the peptide, we were able to deconvolve the contribution of three distinct peptide states to the lifetime trajectory and use this data to develop a kinetics model for the interaction process. It was found that the peptide-membrane interaction was well described by a two-step mechanism: peptide monomer adsorption followed by membrane surface migration, assembly, and insertion to form membrane pores. There was an equilibrium exchange between pore and surface monomers at all lipid/peptide (L/P) concentration ratios, suggesting that the fully inserted phase was reached, even at low peptide concentrations. In contrast to previous studies, there was no evidence of critical behavior; irrespective of L/P ratio, lytic pores were the dominant peptide state at equilibrium and were formed even at very low peptide concentrations. We suggest that this behavior is seen in GUVs because their low curvature means low Laplace pressure. Membrane elasticity is therefore relatively ineffective at damping the thermal fluctuations of lipid molecules that lead to random molecular-level lipid protrusions and membrane undulations. The transient local membrane deformations that result from these thermal fluctuations create the conditions necessary for facile peptide insertion.
使用时移荧光寿命成像显微镜研究了溶菌肽与生物膜之间的相互作用动力学。模型膜为 1,2-二棕榈酰-sn-甘油-3-磷酸胆碱巨型单层囊泡（GUVs），肽是蜂毒素的 K14 衍生物，其被极性敏感荧光探针 AlexaFluor 430 接枝。肽与 GUV 的相互作用导致荧光寿命在数分钟内逐渐猝灭。通过对肽的先前光物理特性的表征，我们能够对三个不同肽态对寿命轨迹的贡献进行反卷积，并使用此数据为相互作用过程开发动力学模型。结果发现，肽-膜相互作用很好地用两步机制来描述：肽单体吸附，然后是膜表面迁移、组装和插入以形成膜孔。在所有脂质/肽（L/P）浓度比下，孔和表面单体之间都存在平衡交换，这表明即使在低肽浓度下也达到了完全插入相。与先前的研究不同，没有证据表明存在临界行为；无论 L/P 比如何，在平衡时溶孔都是占主导地位的肽态，甚至在肽浓度非常低的情况下也能形成。我们认为这种行为在 GUV 中可见，因为它们的低曲率意味着低拉普拉斯压力。因此，膜弹性在抑制导致随机分子水平脂质突起和膜波动的脂质分子热涨落方面效果相对较差。这些热涨落导致的瞬态局部膜变形为肽的插入创造了必要的条件。
