Rabe Martin, Aisenbrey Christopher, Pluhackova Kristyna, de Wert Vincent, Boyle Aimee L, Bruggeman Didjay F, Kirsch Sonja A, Böckmann Rainer A, Kros Alexander, Raap Jan, Bechinger Burkhard
Leiden Institute of Chemistry - Supramolecular and Biomaterials Chemistry, Leiden University, Leiden, the Netherlands.
Université de Strasbourg/CNRS UMR7177, Institut de Chimie, Strasbourg, France.
Biophys J. 2016 Nov 15;111(10):2162-2175. doi: 10.1016/j.bpj.2016.10.010.
A system based on two designed peptides, namely the cationic peptide K, (KIAALKE), and its complementary anionic counterpart called peptide E, (EIAALEK), has been used as a minimal model for membrane fusion, inspired by SNARE proteins. Although the fact that docking of separate vesicle populations via the formation of a dimeric E/K coiled-coil complex can be rationalized, the reasons for the peptides promoting fusion of vesicles cannot be fully explained. Therefore it is of significant interest to determine how the peptides aid in overcoming energetic barriers during lipid rearrangements leading to fusion. In this study, investigations of the peptides' interactions with neutral PC/PE/cholesterol membranes by fluorescence spectroscopy show that tryptophan-labeled K∗ binds to the membrane (K ∼6.2 10 M), whereas E∗ remains fully water-solvated. N-NMR spectroscopy, depth-dependent fluorescence quenching, CD-spectroscopy experiments, and MD simulations indicate a helix orientation of K∗ parallel to the membrane surface. Solid-state P-NMR of oriented lipid membranes was used to study the impact of peptide incorporation on lipid headgroup alignment. The membrane-immersed K∗ is found to locally alter the bilayer curvature, accompanied by a change of headgroup orientation relative to the membrane normal and of the lipid composition in the vicinity of the bound peptide. The NMR results were supported by molecular dynamics simulations, which showed that K reorganizes the membrane composition in its vicinity, induces positive membrane curvature, and enhances the lipid tail protrusion probability. These effects are known to be fusion relevant. The combined results support the hypothesis for a twofold role of K in the mechanism of membrane fusion: 1) to bring opposing membranes into close proximity via coiled-coil formation and 2) to destabilize both membranes thereby promoting fusion.
受SNARE蛋白启发，一种基于两种设计肽的系统被用作膜融合的最小模型，这两种肽分别是阳离子肽K（KIAALKE）及其互补的阴离子对应物肽E（EIAALEK）。尽管通过形成二聚体E/K卷曲螺旋复合物对接单独的囊泡群体这一事实可以得到合理的解释，但肽促进囊泡融合的原因仍无法完全解释。因此，确定这些肽如何在导致融合的脂质重排过程中帮助克服能量障碍具有重要意义。在本研究中，通过荧光光谱对肽与中性PC/PE/胆固醇膜相互作用的研究表明，色氨酸标记的K∗与膜结合（K ∼6.2×10⁻⁶ M），而E∗仍完全溶于水。¹H-NMR光谱、深度依赖荧光猝灭、CD光谱实验和分子动力学模拟表明K∗的螺旋方向与膜表面平行。利用取向脂质膜的固态³¹P-NMR研究了肽掺入对脂质头部基团排列的影响。发现膜中浸泡的K∗会局部改变双层曲率，同时伴随着结合肽附近头部基团相对于膜法线方向的变化以及脂质组成的变化。NMR结果得到了分子动力学模拟的支持，模拟结果表明K会重组其附近的膜组成，诱导正的膜曲率，并增加脂质尾部突出的概率。已知这些效应与融合相关。综合结果支持了K在膜融合机制中具有双重作用的假设：1）通过形成卷曲螺旋使相对的膜紧密靠近；2）使两个膜不稳定从而促进融合。
