neo Trichosanthin及其突变体Y70A neo Trichosanthin的晶体结构研究  

CRYSTAL STRUCTURE OF neo TRICHOSANTHIN AND ITS SITE-DIRECTED MUTANT-Y70A neo TRICHOSANTHIN

作　　者：颜丽[1,2] 吴伸[1,2] 李会广[1,2] 李建辉[1,2] 黄岳顺[1,2] 施庆利[1,2] 董贻诚[1,2] 

机构地区：[1]中国科学院生物物理研究所 [2]香港浸会大学生物系

出　　处：《生物物理学报》1999年第1期33-39,共7页Acta Biophysica Sinica

基　　金：国家自然科学基金;香港研究基金委员会基金;香港浸会大学院务研究基金

摘　　要：天花粉蛋白（Ｔｒｉｃｈｏｓａｎｔｈｉｎ，ＴＣＳ）的一个亚型，ｎｅｏＴｒｉｃｈｏｓａｎｔｈｉｎ（ｎ－ＴＣＳ），及其突变体Ｙ７０Ａｎ－ＴＣＳ被克隆和表达为重组蛋白。用悬滴汽相扩散法得到ｎ－ＴＣＳ和Ｙ７０Ａｎ－ＴＣＳ的晶体。在ＭａｒＲｅｓｅａｒｃｈ面探测器系统上分别收集了０．２０ｎｍ和０．２０５ｎｍ分辨率的Ｘ射线衍射数据。用同晶差值傅立叶法解析了结构。最后晶体学Ｒ因子分别为０．１８３和０．１８４。键长的ｒ．ｍ．ｓ．偏差分别为０．００１６ｎｍ和０．００１４ｎｍ，键角的ｒ．ｍ．ｓ．偏差分别为２．９３０°和２．７３２°。ｎ－ＴＣＳ虽然与ＴＣＳ有１１个氨基酸的差别，但它们都不是活性口袋区的关键残基。并且这些氨基酸的替换绝大多数属于保守替换。和ＴＣＳ相比，这１１个残基及其附近残基的主链间形成的保守氢键在ｎ－ＴＣＳ中没被破坏，它们所处的二级结构也保持不变。所以ｎ－ＴＣＳ的整体结构与ＴＣＳ十分相近。Ｙ７０Ａｎ－ＴＣＳ的晶体在收集数据前曾用含５＇ＡＭＰ的缓冲液浸泡过，活性口袋区没发现可与Ａｄｅｎｉｎｅ相匹配的密度。用ＨＰＬＣ检测Ｙ７０Ａｎ－ＴＣＳ与５＇ＡＭＰ作用后产物，层析图谱上相应位置也没有出现Ａｄｅｎｉｎｅ。上述结果表明，７?An isoform of trichosanthin(TCS), neo-trichosanthin(n-TCS), as well as its site-directed mutant-Y70A neo-trichosanthin, has been cloned and expressed as recombinant protein. Crystals of n-TCS and Y70A n-TCS were grown by handing drop vapor diffusion technique. X-ray diffraction data were collected to 0.20 nm and 0.205 nm separately on a Mar Research IP. The structures were determined by the difference Fourier method. The final R factor are 0.183 and 0.185 respectively. The r.m.s. deviations of bond length are 0.0016 nm and 0.0014 nm, the r.m.s. deviations of bond angle are 2.930° and 2.732° respectively, Although there are 11 amino acids difference between n-TCS and TCS, all those residues are not situated in the active pocket. In addition, most of those amino acid replacements are conservative substitutions. In comparison with the structure of TCS, the hydrogen bonds form between the main chains of those 11 residues in n-TCS and their neighboring residues at the same positions of TCS 11 residues within the secondary structures remain unaltered. Therefore the overall structure of n-TCS is very similar to TCS. The crystal of Y70A n-TCS was allowed to soak in buffer containing 5'-adenosine monophosphate (5'AMP) (10g/l) prior to data collection. No electron density corresponding to adenine can be detected around the active pocket. Furthermore, no adenine can be detected after incubation of Y70A n-TCS with 5'AMP. Those results suggest that Tyr70 is crucial to n-TCS and TCS for substrate recognition, binding and perhaps hydrolysis of N-glycoside bond.

关 键 词：天花粉蛋白 n-TCS 核糖体 失活蛋白 晶体结构 

分 类 号：Q513[生物学—生物化学] Q617