Seidler A
Séction de Bioénergétique (CNR URA 1290), Département de Biologie Cellulaire et Moléculaire, CEA Saclay, Gif-sur-Yvette, France.
Eur J Biochem. 1996 Dec 15;242(3):485-90. doi: 10.1111/j.1432-1033.1996.0485r.x.
Intermolecular and intramolecular interactions of the extrinsic 33-kDa protein in photosystem II were investigated by cross-linking with a water-soluble carbodiimide as cross-linking agent. This zero-length cross-linker is known to cross-link the 33-kDa protein to the chlorophyll-alpha-binding protein CP47 [Bricker, T. M., Odom, W. R. & Queirolo, C. B. (1988) FEBS Lett. 231, 111-117; Enami, I., Kaneko, M., Kitamura, N., Koike, H., Sonoike, K., Inoue, Y & Katoh, S. (1991) Biochim. Biophys. Acta 1960, 224-232]. In this work, cross-linking was observed not only to CP47 but also to a small intrinsic subunit. In addition, through the use of a high-resolution SDS-gel system, three intramolecular cross-linked products of the 33-kDa protein were detected. To search for additional cross-linking sites that might not be accessible to the cross-linker in intact photosystem II, the isolated 33-kDa protein was activated for cross-linking and subsequently bound to CaCl2-washed photosystem II. In the complementary experiment, CaCl2-washed photosystem II was activated, then reconstituted with the 33-kDa protein. The results of the cross-linking reactions demonstrated that all carboxylic acid groups involved in cross-linking were located on the 33-kDa protein and all primary amines were located on intrinsic membrane proteins. No cross-linking other than those observed in cross-linking experiments with intact photosystem II were observed. This indicated that the 33-kDa protein is bound to CP47 and a small subunit but not to the photosystem II reaction centre. This observation is consistent with the finding that cross-linking was independent of the presence or absence of the manganese cluster. Possible residues on the 33-kDa protein and CP47 involved in cross-linking are suggested.
通过使用水溶性碳二亚胺作为交联剂进行交联，研究了光系统II中外源33 kDa蛋白的分子间和分子内相互作用。已知这种零长度交联剂可将33 kDa蛋白与叶绿素α结合蛋白CP47交联[布里克，T.M.，奥多姆，W.R. & 奎罗洛，C.B.（1988年）《欧洲生物化学学会联合会快报》231，111 - 117；江见，I.，金子，M.，北村，N.，小池，H.，园池，K.，井上，Y. & 加藤，S.（1991年）《生物化学与生物物理学报》1960，224 - 232]。在这项工作中，不仅观察到与CP47的交联，还观察到与一个小的内在亚基的交联。此外，通过使用高分辨率SDS凝胶系统，检测到了33 kDa蛋白的三种分子内交联产物。为了寻找完整光系统II中交联剂可能无法接近的其他交联位点，将分离的33 kDa蛋白激活以进行交联，随后与用氯化钙洗涤过的光系统II结合。在互补实验中，用氯化钙洗涤过的光系统II被激活，然后用33 kDa蛋白进行重组。交联反应的结果表明，参与交联的所有羧酸基团都位于33 kDa蛋白上，所有伯胺都位于内在膜蛋白上。未观察到与完整光系统II交联实验中观察到的交联以外的其他交联。这表明33 kDa蛋白与CP47和一个小亚基结合，但不与光系统II反应中心结合。这一观察结果与交联与锰簇的存在与否无关的发现一致。提出了33 kDa蛋白和CP47上可能参与交联的残基。
