Artificial membranes as model systems for the investigation of lipid protein interactions in the thylakoid membranes of vascular plants and the green alga Mantoniella squamata

申请书中概述的实验被认为是对DFG项目头两年成果的补充。为了进一步研究类囊体脂质MGDG对lhcii相关紫黄质（Vx）去环氧化的重要性，我们希望将不同的lhcii纳入双层脂质体（DGDG， PC）和倒六边形相形成脂质体（MGDG， PE）中。这些脂质体将用不同分离的紫黄质去环氧酶进行去环氧化试验。该实验对于了解Vx在体内的去环氧化具有重要意义，因为所定义的脂质体类似于天然类囊体膜中的情况。我们还将从中性脂质（MGDG、DGDG）和带负电荷的脂质（SQDG、PG）构建脂质体，进一步研究脂质对LHCII结构的影响。这些实验应该支持我们的观察，即带负电荷的脂质导致LHCII的强烈分解，而中性脂质稳定高阶结构，从而表明在天然膜中存在特殊的脂质结构域。我们还将重点关注原始绿藻M. squamata，我们想在那里进行类似的实验，如上所述的维管植物LHCII。项目的第三年还将包括对M. squamata的定量脂质分析，特别关注类囊体脂质的脂肪酸组成和新发现的DGTA。最后，制备天然的鳞状芽孢杆菌MGDG，分析不同饱和度脂肪酸对Vx脱环氧化反应的影响。

The experiments outlined in the application are thought to complement the results of the first two years of the DFG project. To further study the importance of the thylakoid lipid MGDG for the de-epoxidation of LHCII-associated violaxanthin (Vx), we want to incorporate different LHCIIs into liposomes constructed of bilayer (DGDG, PC) and inverted hexagonal phase forming lipids (MGDG, PE). These liposomes will be tested in de-epoxidation assays with different isolated violaxanthin de-epoxidases. The experiments are of importance for the understanding of Vx de-epoxidation in-vivo, as the defined liposomes resemble the situation in the natural thylakoid membrane. Liposomes will also be constructed from neutral (MGDG, DGDG) and negatively charged lipids (SQDG, PG) to further study the lipid effect on the structure of the LHCII. These experiments should support our observation that negatively charged lipids lead to a strong disaggregation of the LHCII, whereas neutral lipids stabilize the higher order structure, thus implying that in the native membrane special lipid domains exist. We will also focus on the primitive green alga M. squamata, where we want to perform comparable experiments as outlined above for the vascular plant LHCII. The third year of the project will also include a quantitative lipid analysis of M. squamata, with a special focus on the fatty acid composition of thylakoid lipids and the newly found DGTA. Finally, native MGDG from M. squamata will be prepared to analyse the effect of fatty acids with a different degree of saturation on the Vx de-epoxidation.