Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee.
The Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, Knoxville, Tennessee; Shull Wollan Center-a Joint Institute for Neutron Sciences, Oak Ridge, Tennessee.
Biophys J. 2019 Apr 23;116(8):1495-1506. doi: 10.1016/j.bpj.2019.03.003. Epub 2019 Mar 19.
The plasma membrane (PM) contains an asymmetric distribution of lipids between the inner and outer bilayer leaflets. A lipid of special interest in eukaryotic membranes is the negatively charged phosphatidylserine (PS). In healthy cells, PS is actively sequestered to the inner leaflet of the PM, but PS redistributes to the outer leaflet when the cell is damaged or at the onset of apoptosis. However, the influence of PS asymmetry on membrane protein structure and folding are poorly understood. The pH low insertion peptide (pHLIP) adsorbs to the membrane surface at a neutral pH, but it inserts into the membrane at an acidic pH. We have previously observed that in symmetric vesicles, PS affects the membrane insertion of pHLIP by lowering the pH midpoint of insertion. Here, we studied the effect of PS asymmetry on the membrane interaction of pHLIP. We developed a modified protocol to create asymmetric vesicles containing PS and employed Annexin V labeled with an Alexa Fluor 568 fluorophore as a new probe to quantify PS asymmetry. We observed that the membrane insertion of pHLIP was promoted by the asymmetric distribution of negatively charged PS, which causes a surface charge difference between bilayer leaflets. Our results indicate that lipid asymmetry can modulate the formation of an α-helix on the membrane. A corollary is that model studies using symmetric bilayers to mimic the PM may fail to capture important aspects of protein-membrane interactions.
质膜（PM）在内外双层叶之间存在脂质的不对称分布。真核细胞膜中特别引人关注的脂质是带负电荷的磷脂酰丝氨酸（PS）。在健康细胞中，PS 被主动隔离到 PM 的内叶，但当细胞受损或开始凋亡时，PS 会重新分布到外叶。然而，PS 不对称性对膜蛋白结构和折叠的影响知之甚少。pH 低插入肽（pHLIP）在中性 pH 下吸附在膜表面，但在酸性 pH 下插入膜中。我们之前观察到，在对称囊泡中，PS 通过降低插入 pH 中点来影响 pHLIP 的膜插入。在这里，我们研究了 PS 不对称性对 pHLIP 膜相互作用的影响。我们开发了一种改良的方案来创建含有 PS 的不对称囊泡，并使用用 Alexa Fluor 568 荧光染料标记的 Annexin V 作为新探针来定量 PS 不对称性。我们观察到，带负电荷 PS 的不对称分布促进了 pHLIP 的膜插入，这导致双层叶之间的表面电荷差异。我们的结果表明，脂质不对称性可以调节膜上 α-螺旋的形成。一个推论是，使用对称双层来模拟 PM 的模型研究可能无法捕获蛋白-膜相互作用的重要方面。
