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AMINO ACID LABELING FOR LARGER PROTEINS METHYL GROUP LABELING

AMINO ACID LABELING FOR LARGER PROTEINS METHYL GROUP LABELING
较大蛋白质的氨基酸标记 甲基组标记
批准号:
6120852
负责人:
RODOLFO MARTINEZ
金额:
$7.79万
依托单位国家:
美国
项目类别:
财政年份:
1999
资助国家:
美国
项目状态:
已结题
起止时间:
1999-01-15 至 2000-01-14

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中文摘要
翻译
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。
英文摘要
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.
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AMINO ACID LABELING FOR LARGER PROTEINS METHYL GROUP LABELING
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