The crystal structure of the L1 intermediate of halorhodopsin at 1.9 Å resolution
The crystal structure of the L1 intermediate of halorhodopsin at 1.9 Å resolution
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DOI：
10.1562/2006-06-23-ra-947
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发表时间：
2007-03-01
影响因子：
3.3
通讯作者：
Oesterhelt, Dieter
中科院分区：
文献类型：
--
作者：
Gmelin, Walter;Zeth, Kornelius;Oesterhelt, Dieter
对盐生盐杆菌（Halobacterium salinarum）光驱动氯离子泵盐视紫红质突变体T203V进行了结晶，并在1.6埃分辨率下解析了X射线结构。T203V结构被证明与野生型蛋白几乎相同，蛋白骨架的碳α原子的均方根偏差为0.43埃。两个氯离子结合（CB）的网站被证明是由一个取代的氯离子与溴和异常的差异傅立叶变换图的分析。在与野生型结构相同的位置发现了CB1位点。此外，由于突变晶体中的更高分辨率，在Q105周围鉴定了第二个氯离子结合位点CB2。由于T203V显示其光循环中间体L的衰减慢10倍，因此在室温下照射并随后冷却至120 K时，该中间体可以以60%的占有率被捕获。傅立叶变换红外光谱清楚地识别出晶体被捕获在L1中间态，并且X射线结构被解析到1.9埃分辨率。在该中间体中，氯化物在结合位点CBI内移动了0.3埃，如差分傅里叶密度图中的峰所示。第二结合位点CB2中的氯离子保持不变。因此，从细胞外的蛋白质的细胞质部分的蛋白质内的氯化物易位必须发生在反应步骤后的L1中间体的催化循环中的盐视紫红质。
The mutant T203V of the light driven chloride pump halorhodopsin from Halobacterium salinarum was crystallized and the X-ray structure was solved at 1.6 angstrom resolution. The T203V structure turned out to be nearly identical to the wild type protein with a root mean square deviation of 0.43 angstrom for the carbon alpha atoms of the protein backbone. Two chloride binding (CB) sites were demonstrated by a substitution of chloride with bromide and an analysis of anomalous difference Fourier maps. The CB1 site was found at the same position as in the wild type structure. In addition, a second chloride binding site CB2 was identified around Q105 due to higher resolution in the mutant crystal. As T203V showed a 10 times slower decay of its photocycle intermediate L, this intermediate could be trapped with an occupancy of 60% upon illumination at room temperature and subsequent cooling to 120 degrees K. Fourier transform infrared spectroscopy clearly identified the crystal to be trapped in the L1 intermediate state and the X-ray structure was solved to 1.9 angstrom resolution. In this intermediate, the chloride moved by 0.3 angstrom within binding site CBI as indicated by peaks in difference Fourier density maps. The chloride in the second binding site CB2 remained unchanged. Thus, intraproteinous chloride translocation from the extracellular to the cytoplasmic part of the protein must occur in reaction steps following the L1 intermediate in the catalytic cycle of halorhodopsin.