Probing protein-chromophore interactions in Cph1 phytochrome by mutagenesis
Probing protein-chromophore interactions in Cph1 phytochrome by mutagenesis
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DOI：
10.1111/j.1742-4658.2006.05164.x
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发表时间：
2006-04-01
期刊：
影响因子：
5.4
通讯作者：
Hughes, J
中科院分区：
文献类型：
--
作者：
Hahn, J;Strauss, HM;Hughes, J
为了研究发色团-蛋白的相互作用，我们研究了从藻胞菌PCC6803中提取的光敏色素Cph1突变体。Cph1 Delta 2是514残基n端传感器模块，在大肠杆菌中作为重组his6标记载子蛋白生产，在体外自动组装形成与全长产品难以区分的全蛋白。我们产生了122个Cph1 Delta 2的位点导向突变体，重点关注可能参与发色团-蛋白自动组装和光转化的保守残基。利用CD和紫外可见光谱分析了折叠、藻蓝胆素结合和Pr -> Pfr光能转化。MALDI-TOF-MS证实C259为发色团附着位点。C259L不能与发色团共价连接，但仍然自动组装形成红移光致变色全蛋白。H260Q在pH 7.0时显示出与野生型相似的紫外可见性质，但在pH 9.0时Pr和Pfr都（可逆）漂白，表明咪唑侧链缓冲了发色团的质子化。E189的突变干扰了折叠，但残基不是发色团-蛋白自动组装所必需的。在D207A中，基态的红色照射导致红色Pr带像野生型一样褪色，但没有出现类似Pfr的峰，这表明D207是Pfr之前去质子化中间体的质子供体。H260Q在碱性条件下和D207A的紫外可见光谱都表明质子化在Pfr状态下具有特殊的意义，这可能意味着在Pr -> Pfr光转化过程中质子迁移（释放和再摄取）。这些发现与最近发表的细菌色素片段的3D结构有关[Wagner JR， Brunzelle JS, Forest KT & Vierstra RD (2005) Nature 438, 325-331]。
We have investigated mutants of phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 in order to study chromophore-protein interactions. Cph1 Delta 2, the 514-residue N-terminal sensor module produced as a recombinant His6-tagged apoprotein in Escherichia coli, autoassembles in vitro to form a holoprotein photochemically indistinguishable from the full-length product. We generated 122 site-directed mutants of Cph1 Delta 2, focusing on conserved residues which might be involved in chromophore-protein autoassembly and photoconversion. Folding, phycocyanobilin-binding and Pr -> Pfr photoconversion were analysed using CD and UV-visible spectroscopy. MALDI-TOF-MS confirmed C259 as the chromophore attachment site. C259L is unable to attach the chromophore covalently but still autoassembles to form a red-shifted photochromic holoprotein. H260Q shows UV-visible properties similar to the wild-type at pH 7.0 but both Pr and Pfr (reversibly) bleach at pH 9.0, indicating that the imidazole side chain buffers chromophore protonation. Mutations at E189 disturbed folding but the residue is not essential for chromophore-protein autoassembly. In D207A, whereas red irradiation of the ground state leads to bleaching of the red Pr band as in the wild-type, a Pfr-like peak does not arise, implicating D207 as a proton donor for a deprotonated intermediate prior to Pfr. UV-Vis spectra of both H260Q tinder alkaline conditions and D207A point to a particular significance of protonation in the Pfr state, possibly implying proton migration (release and re-Uptake) during Pr -> Pfr photoconversion. The findings are discussed in relation to the recently published 3D structure of a bacteriophytochrome fragment [Wagner JR, Brunzelle JS, Forest KT & Vierstra RD (2005) Nature 438, 325-331].