Adsorption of membrane-associated proteins to lipid bilayers studied with an atomic force microscope:: Myelin basic protein and cytochrome c
Adsorption of membrane-associated proteins to lipid bilayers studied with an atomic force microscope:: Myelin basic protein and cytochrome c
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DOI：
10.1021/jp9940856
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发表时间：
2000-05-11
影响因子：
3.3
通讯作者：
Bamberg, E
中科院分区：
文献类型：
--
作者：
Mueller, H;Butt, HJ;Bamberg, E
用原子力显微镜研究了两种膜结合蛋白在生理缓冲液中吸附于不同载体磷脂双分子层上的结构。目的是（a）开发一种用于在天然条件下以高分辨率研究膜相关蛋白质的制剂，以及（B）获得有关决定吸附过程和吸附蛋白质结构的因素的信息。因此，通过自发的囊泡吸附和铺展，在云母上形成了固体支撑膜。一旦形成均匀的无针孔的双层，就施加含有适当浓度的蛋白质的溶液。选择的两种带正电荷的蛋白质是髓鞘碱性蛋白（MBP）和细胞色素c，髓鞘碱性蛋白在功能性髓鞘的形成中起重要作用。在带电荷的双层上，以0.5-50 μ g/mL的浓度施加的MBP形成限定高度的聚集体（在带负电荷的脂质上为1.9 +/-0.2 nm，在带正电荷的脂质上为2.7 +/-0.2 nm），其在高浓度下覆盖整个双层。这些聚集体可能是MBP的单分子层。吸附在中性脂质上的MBP形成不规则的聚集体。细胞色素c表现出不同的吸附：在带负电荷的脂质。它形成确定的单分子高度（3.3 +/-0.2nm）的聚集体。在中性双层上观察到小聚集体。在带正电荷的脂质上，根本没有观察到吸附。这些结果表明，（a）细胞色素c的吸附可以解释在一个占主导地位的静电相互作用方面;（B）MBP吸附到脂质双层不完全是静电驱动，并取决于特定的脂质双层组合物;（c）吸附的聚集体的结构表明一个强大的蛋白质-蛋白质相互作用。
Atomic force microscopy was used to study the structure of two membrane-associated proteins adsorbed to various supported phospholipid bilayers in physiological buffer. The aim was (a) to develop a preparation for the investigation of membrane-associated proteins at high resolution under native conditions and (b) to obtain information about the factors that determine the adsorption process and the structure of adsorbed proteins. Therefore, solid-supported membranes were formed on mica by spontaneous vesicle adsorption and spreading. Once a homogeneous, pinhole-free bilayer was formed, solutions containing the proteins at appropriate concentrations were applied. The two positively charged proteins chosen were myelin basic protein (MBP), which plays an essential role in the formation of functional myelin, and cytochrome c. On charged bilayers, MBP applied at concentrations of 0.5-50 mu g/mL formed aggregates of defined height (1.9 +/- 0.2 nm on negatively and 2.7 +/- 0.2 nm on positively charged lipids), which at high concentration covered the entire bilayer. These aggregates are probably monomolecular layers of MBP. On neutral lipid adsorbed MBP formed irregular aggregates. Cytochrome c showed a different adsorption: On negatively charged lipid. it formed aggregates of defined, monomolecular height (3.3 +/- 0.2 nm). On neutral bilayers small aggregates were observed. On positively charged lipid no adsorption was observed at all. These results indicate that (a) the adsorption of cytomchrome c can be interpreted in terms of a dominating electrostatic interaction; (b) MBP adsorption to lipid bilayers is not exclusively electrostatically driven and depends on the specific lipid bilayer composition; (c) the structure of adsorbed aggregates indicates a strong protein-protein interaction.