Gastaldelli Mirko, Canino Giusy, Croce Roberta, Bassi Roberto
Dipartimento Scientifico e Tecnologico, Università di Verona, Strada Le Grazie,15-37134 Verona Italy.
J Biol Chem. 2003 May 23;278(21):19190-8. doi: 10.1074/jbc.M212125200. Epub 2003 Feb 24.
The binding sites for xanthophylls in the CP29 antenna protein of higher plant Photosystem II have been investigated using recombinant proteins refolded in vitro. Despite the presence of three xanthophyll species CP29 binds two carotenoids per polypeptide. The localization of neoxanthin was studied producing a chimeric protein constructed by swapping the C-helix domain from CP29 to LHCII. The resulting holoprotein did not bind neoxanthin, confirming that the N1 site is not present in CP29. Neoxanthin in CP29 was, instead, bound to the L2 site, which is thus shown to have a wider specificity with respect to the homologous site L2 in LHCII. Lutein was found in the L1 site of CP29. For each site the selectivity for individual xanthophyll species was studied as well as its role in protein stabilization, energy transfer, and photoprotection. Putative xanthophyll binding sequences, identified by primary structure analysis as a stretch of hydrophobic residues including an acidic term, were analyzed by site-directed mutagenesis or, in one case, by deleting the entire sequence. The mutant proteins were unaffected in their xanthophyll composition, thus suggesting that the target motifs had little influence in determining xanthophyll binding, whereas hydrophobic sequences in the membrane-spanning helices are important.
利用体外重折叠的重组蛋白，对高等植物光系统II的CP29天线蛋白中叶黄素的结合位点进行了研究。尽管存在三种叶黄素，但CP29每个多肽结合两个类胡萝卜素。通过构建将CP29的C螺旋结构域与LHCII进行交换的嵌合蛋白，研究了新黄质的定位。所得全蛋白不结合新黄质，证实CP29中不存在N1位点。相反，CP29中的新黄质与L2位点结合，因此表明L2位点相对于LHCII中的同源位点L2具有更广泛的特异性。叶黄素存在于CP29的L1位点。对每个位点研究了对单个叶黄素种类的选择性及其在蛋白质稳定、能量转移和光保护中的作用。通过一级结构分析确定为包括酸性末端的一段疏水残基的假定叶黄素结合序列，通过定点诱变或在一种情况下通过删除整个序列进行分析。突变蛋白的叶黄素组成未受影响，因此表明目标基序在决定叶黄素结合方面影响很小，而跨膜螺旋中的疏水序列很重要。
