Simidjiev Ilian, Várkonyi Zsuzsanna, Lambrev Petar H, Garab Gyozo
Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.
Methods Mol Biol. 2011;684:127-38. doi: 10.1007/978-1-60761-925-3_12.
We describe the method of isolation of loosely stacked lamellar aggregates of LHCII that are capable of undergoing light-induced reversible structural changes, similar to those in granal thylakoid membranes (LHCII, the main chlorophyll a/b light-harvesting antenna complex of photosystem II). This unexpected structural flexibility of the antenna complexes depends largely on the lipid content that is retained during the isolation. As revealed by circular dichroism, in lipid-LHCII aggregates, the pigment-pigment interactions are very similar to those in the thylakoid membranes, while they differ significantly from those in solubilized trimers. The essence of the procedure is to adjust--for the plant material used--the proper conditions of detergent solubilization and purification that are mild enough for the associated lipids but provide sufficient purity. Microcrystals and most other LHCII preparations, which are more delipidated, are not capable of similar changes. The light-induced structural reorganizations can be enhanced by the addition of different thylakoid lipids, which--depending on the lipid species--also lead to the transformation of the lamellar structure. The preparation of different LHCII-lipid macro-assemblies is also described. Both in structurally flexible LHCII preparations and in thylakoids, the changes originate from a thermo-optic effect: fast local thermal transients, T-jumps, due to the dissipation of the (excess) excitation energy, which lead to elementary structural transitions in the close vicinity of the dissipating centers. This can occur because thylakoids and structurally flexible LHCII assemblies, but, e.g., not the microcrystals, exhibit a thermal instability below the denaturation temperature, and thus (local) heating leads to reorganizations without the loss of the molecular architecture of the constituents. We also list the main biochemical and biophysical techniques that can be used for testing the structural flexibility of LHCII, and discuss the potential physiological significance of the structural changes in light adaptation and photoprotection of plants.
我们描述了一种分离LHCII松散堆积层状聚集体的方法，这些聚集体能够发生光诱导的可逆结构变化，类似于基粒类囊体膜中的变化（LHCII是光系统II的主要叶绿素a/b捕光天线复合体）。天线复合体这种意想不到的结构灵活性在很大程度上取决于分离过程中保留的脂质含量。圆二色性显示，在脂质-LHCII聚集体中，色素-色素相互作用与类囊体膜中的非常相似，而与溶解的三聚体中的显著不同。该方法的关键是针对所用植物材料，调整去污剂溶解和纯化的适当条件，这些条件对相关脂质足够温和，但能提供足够的纯度。微晶和大多数其他脂质去除较多的LHCII制剂无法发生类似变化。添加不同的类囊体脂质可以增强光诱导的结构重组，这也会导致层状结构的转变，具体取决于脂质种类。还描述了不同LHCII-脂质宏观组装体的制备方法。在结构灵活的LHCII制剂和类囊体中，这些变化都源于热光效应：由于（过量）激发能的耗散导致快速的局部热瞬变，即T跳变，这会在耗散中心附近引发基本的结构转变。之所以会发生这种情况，是因为类囊体和结构灵活的LHCII组装体，但例如微晶则不会，在低于变性温度时表现出热不稳定性，因此（局部）加热会导致重组，而不会损失成分的分子结构。我们还列出了可用于测试LHCII结构灵活性的主要生化和生物物理技术，并讨论了结构变化在植物光适应和光保护中的潜在生理意义。
