Time-resolved fluorescence analysis of the recombinant photosystem II antenna complex CP29 -: Effects of zeaxanthin, pH and phosphorylation
Time-resolved fluorescence analysis of the recombinant photosystem II antenna complex CP29 -: Effects of zeaxanthin, pH and phosphorylation
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DOI：
10.1046/j.1432-1033.2001.01874.x
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发表时间：
2001-01-01
期刊：
影响因子：
--
通讯作者：
Bassi, R
中科院分区：
文献类型：
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作者：
Crimi, M;Dorra, D;Bassi, R
激发能的非辐射耗散是植物主要的光保护机制。玉米黄质在光系统II的触角中的形成已被证明与体内非光化学猝灭的发生相关。我们已经使用了重组CP 29蛋白，在大肠杆菌中过表达，并在体外用纯化的色素重折叠，以获得与从类囊体中提取的天然复合物难以区分的蛋白质，将紫黄质或玉米黄质与叶黄素结合在一起。这些重组蛋白和天然CP 29用于测量稳态叶绿素荧光发射和荧光衰减动力学。我们发现，与CP 29结合的玉米黄质的存在诱导相对于对照蛋白（天然和无玉米黄质的重构蛋白）荧光产率降低约35%。荧光衰减动力学表明，四种组分总是存在，但两种长寿命组分（tau（3）和tau（4））的寿命（tau）以及相对荧光量子产率（rfqy）被玉米黄质的存在改变。在tau 3的rfqy和平均寿命中观察到最相关的变化（玉米黄质约为2.4 ns，对照蛋白为3.2-3.4 ns）。当在体外研究时，没有观察到酸性pH（5.2-5.3）对叶绿素a荧光产率或动力学的显著影响。呈现的数据显示，重组CP 29能够结合玉米黄质，并且这种蛋白质结合的玉米黄质诱导显著的淬灭效应。
Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, over-expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steady-state chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a approximate to 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin-free reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (tau) as well as relative fluorescence quantum yields (rfqy) of the two long-lived components (tau (3) and tau (4)) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of tau3 and in the average lifetime (approximate to 2.4 ns with zeaxanthin and 3.2-3.4 ns in the control proteins). When studied in vitro, no significant effect of acidic pH (5.2-5.3) is observed on chlorophyll a fluorescence yield or kinetics. The data presented show that recombinant CP29 is able to bind zeaxanthin and this protein-bound zeaxanthin induces a significant quenching effect.