Backbone dynamics of trp repressor studied by 15N NMR relaxation.
Backbone dynamics of trp repressor studied by 15N NMR relaxation.
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通过 15N NMR 弛豫研究色氨酸阻遏物的主链动力学。
DOI：
10.1021/bi00015a035
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Jardetzky,O
中科院分区：
文献类型：
--
作者：
Zheng,Z;Czaplicki,J;Jardetzky,O
方法样品制备。从含有高产质粒pJPR2的大肠杆菌菌株CY15070中分离到了富含15N的色氨酸阻遏蛋白(Paleh&Yanovsky，1986)。该菌株在含有[15N]NH4Cl(99个原子%15N，Isotec Inc.)的M9微量培养基中生长。作为唯一的氮源。细胞生长条件和蛋白质纯化程序已经在前面描述过(Gryk等人，1995年)。然后在核磁共振缓冲液(500 mM氯化钠，50 mM磷酸二氢钠，90%水/10%D20)中将蛋白质浓缩至4.6 mM(以蛋白质单体计)。将摩尔过量3倍的L-色氨酸加入到去抑制剂溶液中，最终得到了全息升压剂的核磁共振样品。用溶液pH为7.2的样品进行了15N7)、R2和{*H}-15N NOE的测定。用相同的样品在pH 6下重复NOE实验，通过添加少于5Pl的1M HCl进行滴定(滴定时蛋白质浓度的变化应该可以忽略不计)。
METHODSSample Preparation. The uniformly 15N-enriched trp repressor was isolated from E. coli strain CY15070 containing the overproducing plasmid pJPR2 (Paluh & Yanovsky, 1986). The strain was grown in M9 minimal medium with [15N] NH4C1 (99 atom% 15N, Isotec Inc.) as the sole nitrogen source. The cellgrowth conditions and protein purification procedures have been described previously (Gryk et al., 1995). The protein was then concentrated to 4.6 mM (in terms of proteinmonomer) in the NMR buffer (500 mM NaCl, 50 mM NaH2P04, 90% H2O/10% D20). The NMR sample of holorepressor was finally obtained by adding a 3-fold molar excess of L-tryptophan into the aporepressor solution. The sample with the solution pH found to be 7.2 was used for the 15N 7), r2, and {* H}—15N NOE measure-ments. The NOE experiments were repeated with the same sample at pH 6 titrated by adding less than 5 pL of 1 M HC1 (the changes in the protein concentration upon titration should be negligible).