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    题名: 嗜酸熱硫化葉菌的DNA結合蛋白Saci_0101之結構與功能分析;Structural and Functional Analysis of the DNA Binding Protein Saci_0101 from the Hyperthermophile Sulfolobus acidocaldarius
    作者: 謝東霖;Hsieh,Dong-Lin
    贡献者: 生命科學系
    关键词: 嗜酸熱硫化葉菌;DNA結合蛋白;DNA纏繞;Sulfolobus;histone-like protein;Sso7c4;Saci_0101;DNA packing
    日期: 2016-07-27
    上传时间: 2016-10-13 13:04:00 (UTC+8)
    出版者: 國立中央大學
    摘要: 細胞內的DNA需要被蛋白質纏繞,有效地縮小尺寸以便儲存於細胞內,而古生菌分為兩類,一類是似真核生物利用相仿組織蛋白的方式形成四聚體的核小體;另一類則是似原核生物,比如在古生菌硫化葉菌屬胞內,由一群分子量7-10 KDa的染色質蛋白質(Sul10a, Sul10b, Sul7d, Sul7c),將DNA纏繞形成緊密結構。蛋白質Saci_0101通常認為是參與DNA纏繞保存的蛋白質,在此篇研究中,我們解析出了蛋白質Saci_0101兩種不同晶格的結構,解析度分別為1.30 Å和1.40 Å。從結構中可以看出,蛋白質Saci_0101與其在硫磺礦硫化葉菌中同源蛋白Sso7c4之結構相當相似,都藉由-loop-相互交聯形成二聚體的構型,而進行點突變之Saci_0101 I20M也得到解析度1.55 Å的結構,有趣的是,經過點突變之後發現到與野生型的Saci_0101相比,Saci_0101 I20M的1 helix相對較短,B-factor數值也降低至僅有14,可以明顯看出突變過後的動態特性明顯降低。在偏極化螢光分析的實驗中,發現到Saci_0101與雙股DNA的結合力為1.23  0.19 M,這與其他古生菌硫化葉菌屬的其他非特異性的雙鏈DNA結合蛋白相當接近。而利用電子顯微鏡(Electron Microscope)觀察蛋白質與DNA結合機制後,也證明了Saci_0101可以使DNA纏繞,並且具有架橋的功能。;Saci_0101 is commonly believed to be a histone-like protein involved in genomic DNA compaction from Sulfolobus acidocaldarius. Here, to obtain a detailed understanding of its architectural properties, we present two crystal structures of wild type Saci_0101 in different crystal forms at 1.30 Å and 1.40 Å resolution, respectively. The overall crystal structures of both wild type are similar with the homologues of Sso7c4 in S. solfataricus and have a homodimeric DNA-binding fold forming a swapped -loop- ‘Ying-Yang’ topology. The crystal structure of its single mutant, I20M also has been solved at 1.55 Å resolution. Interestingly, the single mutation by replacing Ile with Met leads to the shortening of 1 helix and even makes the mutant structure much more static than the wild type, proved by the very small B-factor of 14 in I20M structure. In fluorescence polarization study, wild type Saci_0101 binds to a 20-bp double-stranded DNA with a binding affinity of 1.23 ± 0.19 M, which is close to other nonspecific dsDNA-binding proteins in Sulfolobus species. The EM studies show Saci_0101 may shape DNA as a wrapper and a briddger, which suggests Saci_0101 play a role in DNA packaging and duplex stabilization at the elevated growth temperatures.
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