Lipid-induced conformational switch of apolipophorin III

描述（由申请人提供）：可交换载脂蛋白在脂蛋白代谢中起关键作用。它们可逆地与脂蛋白表面结合，并负责维持脂质稳态。载脂蛋白在心血管疾病中发挥重要作用，但在分子水平上的细节仍然缺乏。本项目旨在研究脂质结合的分子基础，并深入了解载脂蛋白的脂质结合构象。这种认识将有利于心血管疾病的治疗和预防，特别是动脉硬化。本研究建议使用一种特性良好的无脊椎动物载脂蛋白作为模型，载脂蛋白III （apoLp-III）。结合结构分析（圆二色性和荧光光谱）和功能分析使用模型磷脂膜将被采用。该研究计划包含以下目标：(i)研究螺旋束的稳定性作为脂质结合的功能，（ii）鉴定触发载脂蛋白结合的脂质因子，（iii）获得脂质结合形式的高分辨率蛋白质结构。(1)螺旋束稳定性可以提供一种灵活的蛋白质，促进脂质结合时螺旋束的打开。这个开口是必不可少的，因为它暴露了蛋白质的疏水内部，允许与脂质直接相互作用。这将通过改变稳定性的工程突变蛋白和分析它们的脂质结合特性来测试。（ii）对触发载脂蛋白结合的脂质因子知之甚少。由多种脂质组成的双层囊泡模型将用于深入了解这些脂质因素。（iii）已知无脂载脂蛋白的高分辨率结构，但仍不知道脂结合形式。了解这种构象是至关重要的，因为这是蛋白质的生物活性形式。我们的目标是获得高质量的apoLp-III/磷脂复合物晶体，衍射约5埃。这将使单个螺旋可视化，从而深入了解载脂蛋白在脂质表面的螺旋排列。

DESCRIPTION (provided by applicant): Exchangeable apolipoproteins play a critical role in lipoprotein metabolism. They reversibly associate with lipoprotein surfaces, and are responsible for maintaining lipid homeostasis. Apolipoproteins play an important role in cardiovascular disease but details at the molecular level are still lacking. This project aims to investigate the molecular basis of lipid binding, and gain insight in the lipid-bound conformation of the apolipoprotein. This understanding would benefit the treatment and prevention of cardiovascular disease, in particular arteriosclerosis. The present study proposes to use a well characterized invertebrate apolipoprotein as a model, apolipophorin III (apoLp-III). A combination of structural analysis (circular dichroism and fluorescence spectroscopy) and functional analysis using model phospholipid membranes will be employed. The research plan contains the following aims: (i) investigate helix bundle stability as a function of lipid binding, (ii) identification of lipid factors that trigger apolipoprotein binding, and (iii) obtaining a high resolution structure of the protein in the lipid-bound form. (i) Helix bundle stability may provide a flexible protein facilitating helix bundle opening upon lipid binding. This opening is essential as it exposes the protein's hydrophobic interior to allow direct interaction with lipid. This will be tested by engineering mutant proteins with altered stability properties, and analysis of their lipid binding properties. (ii) Not much is known about the lipid factors that trigger apolipoprotein binding. Model bilayer vesicles composed of a variety of lipids will be used to gain insight in these lipid factors. (iii) The high resolution structure of lipid-free apolipoprotein is known, but still not known for the lipid-bound form. It is crucial to gain insight in this conformation as this is the biologically active form of the protein. We aim to obtain high quality crystals of apoLp-III/phospholipid complexes that diffract at approximately 5 angstrom. This would allow visualization of individual helices, thereby gaining insight in the helix arrangement of the apolipoprotein on the lipid surface.