School of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, GPO Box U1987, Perth, WA 6845, Australia.
School of Life Sciences, University of Technology Sydney, Ultimo, NSW 2007, Australia.
Int J Mol Sci. 2020 Jan 23;21(3):746. doi: 10.3390/ijms21030746.
Melittin is an anti-microbial peptide (AMP) and one of the most studied membrane-disrupting peptides. There is, however, a lack of accurate measurements of the concentration-dependent kinetics and affinity of binding of melittin to phospholipid membranes. In this study, we used surface plasmon resonance spectroscopy to determine the concentration-dependent effect on the binding of melittin to 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayers in vesicles. Three concentration ranges were considered, and when combined, covered two orders of magnitudes (0.04 µM to 8 µM), corresponding to concentrations relevant to the membrane-disrupting and anti-microbial activities of melittin. Binding kinetics data were analysed using a 1:1 Langmuir-binding model and a two-state reaction model. Using in-depth quantitative analysis, we characterised the effect of peptide concentration, the addition of NaCl at physiological ionic strength and the choice of kinetic binding model on the reliability of the calculated kinetics and affinity of binding parameters. The apparent binding affinity of melittin for POPC bilayers was observed to decrease with increasing peptide/lipid (P/L) ratio, primarily due to the marked decrease in the association rate. At all concentration ranges, the two-state reaction model provided a better fit to the data and, thus, a more reliable estimate of binding affinity. Addition of NaCl significantly reduced the signal response during the association phase; however, no substantial effect on the binding affinity of melittin to the POPC bilayers was observed. These findings based on POPC bilayers could have important implications for our understanding of the mechanism of action of melittin on more complex model cell membranes of higher physiological relevance.
蜂毒素是一种抗微生物肽 (AMP)，也是研究最多的破坏膜肽之一。然而，缺乏对蜂毒素与磷脂膜结合的浓度依赖性动力学和亲和力进行准确测量的方法。在这项研究中，我们使用表面等离子体共振光谱法来确定蜂毒素与脂质体中的 1-棕榈酰-2-油酰基-甘油-3-磷酸胆碱 (POPC) 双层结合的浓度依赖性效应。考虑了三个浓度范围，当它们结合在一起时，覆盖了两个数量级（0.04 µM 至 8 µM），对应于与蜂毒素的膜破坏和抗微生物活性相关的浓度。使用 1:1 朗缪尔结合模型和两态反应模型分析结合动力学数据。通过深入的定量分析，我们描述了肽浓度、在生理离子强度下添加 NaCl 以及选择动力学结合模型对计算动力学和结合参数的可靠性的影响。观察到蜂毒素与 POPC 双层的表观结合亲和力随肽/脂质 (P/L) 比的增加而降低，主要是由于缔合速率的显著降低。在所有浓度范围内，两态反应模型更能拟合数据，从而更可靠地估计结合亲和力。添加 NaCl 会显著降低缔合相期间的信号响应；然而，未观察到 NaCl 对蜂毒素与 POPC 双层结合亲和力的实质性影响。这些基于 POPC 双层的发现可能对我们理解蜂毒素在更复杂的、具有更高生理相关性的模型细胞膜上的作用机制具有重要意义。
