Zouni Athina, Kern Jan, Frank Joachim, Hellweg Thomas, Behlke Joachim, Saenger Wolfram, Irrgang Klaus-Dieter
Max-Volmer-Laboratorium, Technische Universität Berlin, Strasse des 17. Juni 135, D-10623 Berlin, Germany.
Biochemistry. 2005 Mar 22;44(11):4572-81. doi: 10.1021/bi047685q.
The oxygen-evolving photosystem II core complexes (PSIIcc) from the thermophilic cyanobacterium Thermosynechococcus elongatus (PSIIccTe) and the higher plant Spinacia oleracea (PSIIccSo) have been isolated from the thylakoid membrane by solubilization with n-dodecyl-beta-d-maltoside, purified and characterized by gel permeation chromatography (GPC), dynamic light scattering (DLS), and analytical ultracentrifugation (AUC). DLS suggests that PSIIcc from both organisms exists as a monomer in dilute solution and aggregates with increasing protein concentration. In contrast to DLS, GPC and AUC showed that PSIIcc of both organisms occur as monomers and dimers, and it became clear from our studies that calibration of GPC columns with soluble proteins leads to wrong estimates of the molecular masses of membrane proteins. At a PSIIcc protein concentration of 0.2 mg/mL, molar masses, M, of 756 +/- 18 kDa and 710 +/- 15 kDa for dimeric PSIIccTe and PSIIccSo, respectively, were determined by analytical ultracentrifugation. At very low protein concentrations, at or below 0.05 mg/mL, the dimeric form of PSIIccTe partially dissociates (20-30%) to form monomers. On the basis of these studies 3-dimensional crystals of PSIIccTe were obtained that contain dimers in the asymmetric unit [Zouni, A. et al. (2001) Nature 409, 739-743]. Using synchrotron radiation the crystals diffract to a resolution of 3.8 A, which has been improved recently to 3.2 A [Biesiadka, J., et al. (2004) Phys. Chem. Chem. Phys. 6, 4733-4736].
嗜热蓝藻细长聚球藻（PSIIccTe）和高等植物菠菜（PSIIccSo）的析氧光系统II核心复合物（PSIIcc）已通过用正十二烷基-β-D-麦芽糖苷增溶从类囊体膜中分离出来，经凝胶渗透色谱法（GPC）、动态光散射（DLS）和分析超速离心法（AUC）进行纯化和表征。DLS表明，来自这两种生物体的PSIIcc在稀溶液中以单体形式存在，并随着蛋白质浓度的增加而聚集。与DLS相反，GPC和AUC显示这两种生物体的PSIIcc以单体和二聚体形式存在，并且我们的研究清楚地表明，用可溶性蛋白质对GPC柱进行校准会导致对膜蛋白分子量的错误估计。在PSIIcc蛋白质浓度为0.2 mg/mL时，通过分析超速离心法分别测定了二聚体PSIIccTe和PSIIccSo的摩尔质量M为756±18 kDa和710±15 kDa。在非常低的蛋白质浓度下，即0.05 mg/mL及以下，PSIIccTe的二聚体形式会部分解离（20 - 30%）形成单体。基于这些研究，获得了PSIIccTe的三维晶体，其不对称单元中包含二聚体[祖尼，A.等人（2001年）《自然》409，739 - 743]。利用同步辐射，这些晶体的衍射分辨率达到3.8 Å，最近已提高到3.2 Å[比西亚德卡，J.等人（2004年）《物理化学化学物理》6，4733 - 4736]。
