Structural features and assembly of the soluble overexpressed PsaD subunit of photosystem I.
Structural features and assembly of the soluble overexpressed PsaD subunit of photosystem I.
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DOI：
10.1016/s0005-2728(98)00169-8
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发表时间：
1999-01
期刊：
影响因子：
--
通讯作者：
P. Jin;J. Sun;P. Chitnis
中科院分区：
文献类型：
--
作者：
P. Jin;J. Sun;P. Chitnis
PsaD是光系统I（PS I）还原侧的外周蛋白。我们在大肠杆菌中表达了嗜热蓝藻Mastigocladus laminosus的psaD基因，并获得了一个可溶性蛋白，其羧基末端带有多组氨酸标签。可溶性PsaD蛋白通过Ni亲和层析纯化，并通过MALDI-TOF测定其质量为16716 Da。过表达的PsaD的N-末端氨基酸序列与来自M的天然PsaD的N-末端序列相匹配。椎板肌可溶性PsaD可以组装成无PsaD的PS I。通过等温滴定量热法测定，PsaD与PS I的结合位点为1.0个/PS I，结合常数为7.7× 106 M −1，焓变为−93.6 kJ mol−1。这是第一次，结合常数和结合热已被确定在任何光合膜蛋白的组装。为了鉴定表面暴露的结构域，用N-羟基琥珀酰亚胺生物素（NHS-生物素）和生物素-马来酰亚胺处理纯化的PS I复合物和过表达的PsaD，并绘制生物素化的残基。可溶性PsaD表面有Cys 66、Lys 21、Arg 118和Arg 119残基暴露，而Lys 129和Lys 131残基未暴露。与PS I的X射线晶体学研究一致，圆二色性光谱显示PsaD含有小比例的α-螺旋构象。
PsaD is a peripheral protein on the reducing side of photosystem I (PS I). We expressed the psaD gene from the thermophilic cyanobacterium Mastigocladus laminosus in Escherichia coli and obtained a soluble protein with a polyhistidine tag at the carboxyl terminus. The soluble PsaD protein was purified by Ni-affinity chromatography and had a mass of 16716 Da by MALDI-TOF. The N-terminal amino acid sequence of the overexpressed PsaD matched the N-terminal sequence of the native PsaD from M. laminosus. The soluble PsaD could assemble into the PsaD-less PS I. As determined by isothermal titration calorimetry, PsaD bound to PS I with 1.0 binding site per PS I, the binding constant of 7.7×106M−1, and the enthalpy change of −93.6 kJ mol−1. This is the first time that the binding constant and binding heat have been determined in the assembly of any photosynthetic membrane protein. To identify the surface-exposed domains, purified PS I complexes and overexpressed PsaD were treated with N-hydroxysuccinimidobiotin (NHS-biotin) and biotin-maleimide, and the biotinylated residues were mapped. The Cys66, Lys21, Arg118and Arg119residues were exposed on the surface of soluble PsaD whereas the Lys129and Lys131residues were not exposed on the surface. Consistent with the X-ray crystallographic studies on PS I, circular dichroism spectroscopy revealed that PsaD contains a small proportion of α-helical conformation.