Interaction of a type II myosin with biological membranes studied by 2H solid state NMR.
Interaction of a type II myosin with biological membranes studied by 2H solid state NMR.
复制标题
通过 2H 固态 NMR 研究 II 型肌球蛋白与生物膜的相互作用。
作者：
J. Arêas;G. Gröbner;C. Glaubitz;A. Watts
氘核磁共振波谱 (2H NMR) 用于研究肺 II 型肌球蛋白与含有二肉豆蔻酰磷脂酰胆碱 (DMPC) 作为唯一成分的中性双层和含有带负电荷的脂质二肉豆蔻酰磷脂酰甘油 (DMPG) 的混合双层的相互作用。通过取代 α 和 β 位置上的四个质子 (DMPC-d4) 和 γ 位置上的九个质子 (DMPC-d9)，DMPC 在其头部被氘化。 DMPG 在其头部基团 (DMPG-d5) 处进行了全氘化。当将越来越多的肌球蛋白添加到仅由 DMPC-d9 或 DMPC-d4 制成的脂质体中时，没有观察到氘化 DMPC 头基片段的四极分裂或自旋晶格弛豫时间发生变化。然而，当带负电荷的脂质 DMPG 以 1:1 摩尔比插入 DMPC 双层时，发现肌球蛋白与脂质体发生静电相互作用，从而显着影响标记 DMPC 中 α、β 和 γ 氘核的四极分裂和自旋晶格弛豫率。监测混合 DMPC/DMPG 双层中的 DMPG-d5 揭示了 DMPG 与蛋白质的直接静电相互作用，其中位于肌球蛋白尾部结构域的带正电荷的赖氨酸残基为相互作用的发生提供了必要的位点。当 ATP 和 Mg2+ 与肌球蛋白的头域复合时，观察到与带负电荷的双层的相互作用减少。结果清楚地表明，只要双层中存在带负电荷的脂质，II 型肌球蛋白就可以与膜相互作用，而不需要蛋白质分子中的特定疏水结构域或锚定点。
Deuterium nuclear magnetic resonance spectroscopy (2H NMR) has been employed to investigate the interaction of lung type II myosin protein with neutral bilayers containing dimyristoylphosphatidylcholine (DMPC) as the only constituent and mixed bilayers containing the negatively charged lipid dimyristoylphosphatidylglycerol (DMPG). DMPC was deuterated at its headgroup by substituting the four protons at the alpha- and beta-positions (DMPC-d4) and the nine protons at the gamma-position (DMPC-d9). DMPG was perdeuterated at its headgroup (DMPG-d5). No changes were observed in the quadrupole splittings or spin-lattice relaxation times for the deuterated DMPC headgroup segments when increasing amounts of myosin were added to liposomes, made exclusively of DMPC-d9 or of DMPC-d4. However, upon the insertion of the negatively charged lipid DMPG at 1:1 molar ratio into the DMPC bilayers, myosin was found to interact electrostatically with the liposomes, thereby affecting significantly both the quadrupole splittings and spin-lattice relaxation rates of the alpha-, beta-, and gamma-deuterons in labeled DMPC. Monitoring DMPG-d5 in mixed DMPC/DMPG bilayers revealed a direct electrostatic interaction of DMPG with the protein, where positively charged lysine residues located at the tail domain of myosin provide the necessary sites for the interaction to occur. When ATP and Mg2+ were complexed to the head domain of myosin, a reduced interaction with the negatively charged bilayers was observed. The results clearly indicate that a type II myosin can interact with membranes without the need for a specific hydrophobic domain or an anchor in the protein molecule, provided that negatively charged lipids are present in the bilayer.