【作者】 王少雄；

【导师】 隋森芳；

【作者基本信息】 清华大学 ， 生物物理学， 1999， 博士

【摘要】 载脂蛋白 H（apolipoproteinH，简称ApoH）是一种属于载脂蛋白家族的血浆 糖蛋白。目前为止，ApoH的生物学功能并不十分清楚。由于ApoH在体内的 很多生理和病理功能都与它和磷脂膜的相互作用有关，而目前这方面的研究成 果还很少。因此，研究ApoH与脂膜的相互作用及其在膜表面的结构将具有十 分重要的生物学意义和潜在的应用前景。 本文应用单层膜、脂质体以及含有甲醇的有机溶剂体系等模型系统对ApoH 与磷脂膜的相互作用及其膜结合态的构象进行了深入的研究。 首先，我们利用气／液界面脂单层膜的方法在国际上首次证明了ApoH分子 具有较强的两亲性，能够插入带负电荷的磷脂膜内，蛋白的插膜深度约为2±1 A。ApoH的插膜的作用力来自膜脂一蛋白之间的静电作用和疏水作用。 其次，我们应用荧光光谱和圆二色光谱等实验技术研究了与脂质体相结合 的ApoH的构象。实验结果显示当结合到带负电荷的脂质体表面之后，ApoH 分子的二级结构将发生显著变化，蛋白分子被诱导产生出一定比例的α-螺旋 结构成分。通过改变脂质体中磷脂的组成和缓冲液中的离子强度等条件，我们 首次发现膜结合状态的ApoH分子具有两种不同的构象。在低离子强度条件 下，蛋白分子中大部分色氨酸残基吸附于脂膜表面，少部分插入磷脂膜内；随 着体系离子强度的提高，蛋白分子中非插膜的色氨酸残基将首先从脂膜表面脱 附，而插膜片段的脱附要显著滞后。ApoH分子在膜表面的构象变化为其在体 内发挥标记并免疫清除血浆中带负电荷的脂质体，以及在抗磷脂综合症等自身 免疫疾病中的生物学功能提供了重要的结构基础。 第三，我们应用含有一定浓度甲醇的缓冲液模拟膜一水界面区域的微环 境，对ApoH分子的插膜与其膜结合态构象之间的相互关系作了进一步的研 究。在含57％左右甲醇的溶液中ApoH能被诱导产生出与其在负电荷磷脂膜表 面几乎相同的构象；同时经该有机溶剂体系处理后，构象发生变化的ApoH分 子的插膜能力也相应增强。经毛细管电泳技术检测，这时有更多的蛋白质分子吸附于脂膜表面区域。以上实验说明在ApoH与磷脂膜作用的过程中，蛋白的插膜与其特定的膜结合态构象密切相关。 综上，本文揭示了ApoH与磷脂膜相互作用的分子机制，并建立了一个有关ApoH插膜过程及在膜表面发生构象变化的模型。ApoH首先通过与负电荷磷脂之间的静电引力富集于脂膜表面区域。接着，受该区域微观物理化学环境（主要是介电常数和pH值条件）的影响，吸附于膜脂界面的ApoH将发生特定的构象变化，同时蛋白分子的一部分插入到脂膜内部。最后，插膜的ApoH分于将在膜脂界面发生进一步的构象调整井最终稳定于膜脂界面。有关ApoH插膜的模型具有一般性的生物学意义，它的建立为深入研究膜外周蛋白与磷脂膜的相互作用提供了重要的信息。更多还原

【Abstract】 Apolipoprotein H (ApoH) is a plasma glycoprotein belonging to the family of apolipoprotein. Hitherto, the biological function of ApoH is not very clear. Since it is known that the in vivo physiological and pathogenic roles of ApoH are closely related to its interaction with the phospholipid membrane, a more profound understanding on the interaction mechanism, as well as on the membrane-bound conformation of ApoH may have not only theoretically but also practically beneficial consequence. In the dissertation, the interaction of ApoH with phospholipid membranes and the conformation of membrane-bound ApoH was studied by using different model systems of lipid monolayer, liposome and an organic solvent contain water and methanol. Firstly, with the method of phospholipid monolayer at air/water interface, we proved for the first time that ApoH is an amphiphilic protein which is capable of insertion into negatively charged lipid monolayer. The membrane insertion depth of ApoH is about 2± A. In the process of ApoH insertion into membranes, both the electrostatic and the hydrophobic interactions between protein and the lipid are evidently involved. Secondly, we combined techniques of fluorescence and circular dichroism spectroscopy to study the conformation of membrane-bound ApoH. The experimental results showed that after binding to the negatively charged liposome, the secondary structure of ApoH will be significantly changed and some a-helix structure can be induced. By changing the composition of phospholipid in vesicles and the ionic strengths in the buffer, we found that the membrane-bound ApoH molecules can adopt two conformations at the membrane surface. Under conditions of low ionic strength, the water-soluble ApoH will bind to the negatively charged lipid vesicles with most of its tryptophan residues adsorbing on the membrane surface and a small part of them inserting into the membrane. Under conditions of high ionic strength, the membrane-bound ApoH will have a different conformation with its non-inserted tryptophan residues mainly separated from the membrane surface, and its membrane- inserted ones still maintained their association with the membrane. The conformational change of ApoH upon binding to the membrane may provide important structural bases for the protein’s biological function, e.g. in clearing the negatively charged liposome in plasma, and in the autoimmune diseases such as systemic lupus erythematosus. Thirdly, we studied the correlation between the membrane insertion ability and the membrane-bound conformation of ApoH with the model system of water- methanol mixed solvent at moderately low pH that mimics the micro- physicochemical environment near the membrane surface. In a buffer of 57% methanol, ApoI-I can be induced to adopt a special conformation just the same as that bound to the negatively charged membrane. On the other hand, it is observed that the membrane insertion ability of the conformational changed ApoH increased, too. With the method of capillary electrophoresis, it is found that in this case, there are more ApoH molecules adsorbing on the monolayer surface. The above experiments suggest the close relationship between the membrane insertion ability and the membrane- bound conformation of ApoH. In all, we partially revealed the molecular mechanism of the interaction of ApoH with phospholipid membranes and obtained a model for the membrane insertion and conformational change of ApoH at the membrane surfa更多还原