AMINO ACID LABELING FOR LARGER PROTEINS METHYL GROUP LABELING

SIR得到3，4，6-二氯-5，15-氨基嘧啶; 20 mg 6，2 '脱氧[9 J5 N]腺苷; 20 mg I a碲胱氨酸; 20 mg 93-94%“硒;分析了87 mg含硒蛋氨酸的蛋白质 通过多波长反常衍射（MAD）提供了一种简便的方法， 解决相位问题，其解决方案是必要的 通过X射线晶体学确定蛋白质结构[Hendrickson， W.A.（1991）Science，254，51-58]。 由于该方法需要 在同步辐射下，我们试图将一个真正的重原子纳入 蛋白质，允许解决相位问题， 传统的数据收集方法。 仅含Te-Met的培养基 或低水平Met的Te-Met未能维持 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。 微生物生长至静止 当培养物为 在含有最低Met水平的培养基中启动，并加入Te-Met 用IPTG诱导后。 纯化后的酶具有 类似于天然酶的那些。 原子吸收光谱法 氨基酸分析表明，40%的蛋氨酸是 被Te-Met取代。 序列分析未显示显著性差异。 前三个地点（1、16和20）的更替水平， 这表明Te-Met仅存在于最后两个位点（42和 92).这种酶的晶体在氨甲蝶呤存在下生长 并且与野生型DHFR晶体同晶。 差异 傅立叶映射和约束最小二乘细化显示， 取代在前三个蛋氨酸，而掺入是 在位置42和92处可见。

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.