AMINO ACID LABELING FOR LARGER PROTEINS METHYL GROUP LABELING

SIR提供3，4，6-二氯-5-15氨基-嘧啶；20毫克 6，2‘脱氧[9J5N]腺苷；20 mg 阿特罗西汀；20毫克 93-94%“硒；87毫克含硒蛋氨酸蛋白质分析 通过多波长反常衍射(MAD)提供了一种简便的手段 解决阶段问题，其解决方案是必要的 用X射线结晶学确定蛋白质结构[Hendrickson， W.A.(1991)科学，254，51-58]。由于此方法需要 同步辐射，我们试图将一个真正的重原子结合到 蛋白质，允许通过更多的方式解决相问题 传统的数据收集方法。仅含Te-Met的介质 或低水平蛋氨酸的Te-Met未能维持A 携带二氢叶酸的大肠杆菌甲硫氨酸营养缺陷型 还原酶表达载体。生物体生长到静止状态 在培养过程中观察到Te-Met的晶相和掺入情况 在含有最低蛋氨酸水平的介质中启动，并添加了Te-Met IPTG诱导后。纯化的酶具有一定的性质 与天然酶相似。原子吸收光谱分析 氨基酸分析表明，40%的蛋氨酸是 替换为Te-Met。序列分析没有显示出显著的 前三个站点(1、16和20)的替换级别， 表明Te-Met只存在于最后两个站点(42和 该酶的晶体是在甲氨蝶呤存在下生长的 并与野生型DHFR的晶体同构。差异化 傅里叶映射和约束最小二乘精化没有 前三个蛋氨酸的取代，而合并是 在42号和92号位置。

The SIR provided 3,4,6 dichloro-5 15 Namino-pyrimidine; 20mg 6,2'deoxy[9J5N]adenosine; 20mg I a tellurocystine; 20mg 93-94% "Selenium; 87mg Selenomethionine containing proteins analyzed by multiwavelength anomalous diffraction (MAD) provide a facile means of addressing the phase problem, whose solution is necessary to determine protein structures by X-ray crystallography [Hendrickson, W.A. (1991) Science, 254, 51-58]. Since this method requires synchrotron radiation, we sought to incorporate a true heavy atom into protein, allowing the solution of the phase problem by more traditional methods of data collection. Media containing Te-Met alone or Te-Met with low levels of Met failed to sustain growth of a methionine auxotroph of Escherichia coli carrying the dihydrofolate reductase expression vector. Growth of the organism to stationary phase and incorporation of Te-Met was observed when the culture was initiated in media containing minimal Met levels and Te-Met was added after induction with IPTG. The purified enzyme exhibited properties similar to those of the native enzyme. Atomic absorption spectroscopy and amino acid analysis indicated that 40% of the methionines were replaced with Te-Met. Sequence analysis did not indicate significant levels of replacement in the first three sites (1, 16, and 20), suggesting that Te-Met was present only in the last two sites (42 and 92).Crystals of this enzyme were grown in the presence of methotrexate and were isomorphous with crystals of wild-type DHFR. Difference Fourier maps and restrained least-squares refinement showed no substitution at the first three methionines, while incorporation was seen at positions 42 and 92.