Subnanometer Structure of an Asymmetric Model Membrane: Interleaflet Coupling Influences Domain Properties.
Subnanometer Structure of an Asymmetric Model Membrane: Interleaflet Coupling Influences Domain Properties.
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DOI：
10.1021/acs.langmuir.5b04562
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发表时间：
2016-05-24
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影响因子：
--
通讯作者：
Pabst G
中科院分区：
文献类型：
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作者：
Heberle FA;Marquardt D;Doktorova M;Geier B;Standaert RF;Heftberger P;Kollmitzer B;Nickels JD;Dick RA;Feigenson GW;Katsaras J;London E;Pabst G
细胞膜具有复杂的三维结构，包括双层小叶平面内非随机的脂类侧向组织，以及两个小叶之间的成分不对称。因此，描绘膜结构与功能的关系一直是一项极具挑战性的任务。即使在简化的模型系统中，双层小叶之间的相互作用也鲜为人知，部分原因是很难制备不受残留有机溶剂或渗透胁迫影响的不对称模型膜。为了解决这些问题，我们改进了一种通过环糊精介导的脂质交换来制备不对称大单层囊泡(ALUV)的技术，以生产适合于一系列生物物理研究的无张力、无溶剂的aLUV。小叶的成分和结构使用同位素标记策略来表征，这使得我们可以避免使用笨重的标记。核磁共振和气相色谱提供了精确的定量的脂交换和双层不对称性的程度，而小角中子散射(SANS)被用来解析亚纳米分辨率的双层结构特征。等位不对称的POPC囊泡与对称的POPC囊泡具有相同的双层厚度和单位脂面积，表明改进的交换方案保留了天然的双层结构。DPPC部分交换到POPC囊泡的外叶中，产生化学上不对称的囊泡，外层小叶为凝胶/流体相分离，内叶为均一的富含POPC的小叶。SANS能够分别分解共存结构域的每种脂类的厚度和面积，显示与典型凝胶相相比，外层小叶DPPC富含相的脂类堆积密度降低。我们的发现，无序的内叶可以部分流态化有序的外叶结构域，这表明了一定程度的小叶间耦合，并引发了对双层不对称在调节膜横向组织中的作用的推测。
Cell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two leaflets. As a result, delineating the membrane structure–function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress. To address these problems, we have modified a technique for preparing asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange in order to produce tensionless, solvent-free aLUVs suitable for a range of biophysical studies. Leaflet composition and structure were characterized using isotopic labeling strategies, which allowed us to avoid the use of bulky labels. NMR and gas chromatography provided precise quantification of the extent of lipid exchange and bilayer asymmetry, while small-angle neutron scattering (SANS) was used to resolve bilayer structural features with subnanometer resolution. Isotopically asymmetric POPC vesicles were found to have the same bilayer thickness and area per lipid as symmetric POPC vesicles, demonstrating that the modified exchange protocol preserves native bilayer structure. Partial exchange of DPPC into the outer leaflet of POPC vesicles produced chemically asymmetric vesicles with a gel/fluid phase-separated outer leaflet and a uniform, POPC-rich inner leaflet. SANS was able to separately resolve the thicknesses and areas per lipid of coexisting domains, revealing reduced lipid packing density of the outer leaflet DPPC-rich phase compared to typical gel phases. Our finding that a disordered inner leaflet can partially fluidize ordered outer leaflet domains indicates some degree of interleaflet coupling, and invites speculation on a role for bilayer asymmetry in modulating membrane lateral organization.