Phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 -: Purification, assembly, and quaternary structure
Phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 -: Purification, assembly, and quaternary structure
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DOI：
10.1046/j.1432-1327.2001.02395.x
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发表时间：
2001-09-01
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影响因子：
--
通讯作者：
Hughes, J
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文献类型：
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作者：
Lamparter, T;Esteban, B;Hughes, J
蓝细菌的光敏色素Cph 1。集胞藻PCC 6803在体外与胆色素生色团自组装时形成与植物光敏色素光谱相似的全蛋白加合物。Cph 1是一种85 kDa的蛋白质，作为一种光调节组氨酸激酶，似乎参与了“双组分”。信号装置.本文介绍了Cph 1纯化方法的改进、全Cph 1消光系数的估算、组装过程的光谱分析和四级结构的研究。在与天然发色团藻蓝胆素（PCB）组装过程中，观察到一个红移的中间体。当藻红胆素用作发色团时，得到了类似的结果。如SDS/PAGE和Zn 2+荧光所示，PCB的共价连接被1 mM碘乙酰胺（一种半胱氨酸衍生剂）阻断。当PCB与阻断的apo-Cph 1一起孵育时，在较长波长处再次出现肩部。因此，提出长波长吸收形式代表质子化的非共价结合的胆色素。胆绿素，这是既不质子化，也没有共价连接，经历了光谱变化，其蓝色吸收带孵育后与apo-Cph 1。因此，在这些数据的基础上，我们提出了一个三步模型光敏色素自动组装。尺寸排阻色谱显示不同的迁移率的载脂蛋白，红色吸收Cph 1-PCB和远红色吸收Cph 1-PCB。这两个holoprotein加合物的主峰有明显的分子量约为200 kDa，结果在协议的概念，在感官组氨酸激酶的自磷酸化需要二聚化。当通过制备性天然电泳进一步纯化Cph 1-PCB时，在尺寸排阻色谱上的迁移率接近100 kDa，并且发现其已失去激酶活性，结果表明该材料已失去其二聚化能力。
The phytochrome Cph1 from the cyanobacterium. Synechocystis PCC6803 forms holoprotein adducts with close spectral similarity to plant phytochromes when autoassembled in vitro with bilin chromophores. Cph1 is a 85-kDa protein that acts as a light-regulated histidine kinase seemingly involved in 'two-component'. signalling. This paper describes the improvement of Cph1 purification, estimation of the extinction coefficient of holo-Cph1, spectral analyses of the assembly procedure and studies on quaternary structure. During assembly with the natural chromophore phycocyanobilin (PCB), a red-shifted intermediate is observed. A similar result was obtained when phycoerythrobilin was used as chromophore. As shown by SDS/PAGE and Zn2+ fluorescence, the covalent attachment of PCB is blocked by 1 mM iodoacetamide, a cysteine-derivatizing agent. When PCB was incubated with blocked apo-Cph1, again a shoulder at longer wavelengths appeared. It is therefore proposed that the long-wavelength-absorbing form represents the, protonated, noncovalently bound bilin. Biliverdin, which is neither protonated nor covalently attached, undergoes spectral changes in its blue-absorbing band upon incubation with apo-Cph1. On the basis of these data we therefore propose a three-step model for phytochrome autoassembly. Size-exclusion chromatography revealed different mobilities for the apoprotein, red-absorbing Cph1-PCB and far-red-absorbing Cph1-PCB. The major peaks of both holoprotein adducts had apparent molecular masses approximate to 200 kDa, a result in agreement with the notion that autophosphorylation in sensory histidine kinases requires dimerization. When Cph1-PCB was further purified by preparative native electrophoresis, the mobility on size-exclusion chromatography was approximate to 100 kDa, and it was found to have lost its kinase activity, results implying that the material had lost its capacity to dimerize.