NMR structure note: a defective isoform and its activity-improved variant of a type III antifreeze protein from Zoarces elongates Kner
NMR structure note: a defective isoform and its activity-improved variant of a type III antifreeze protein from Zoarces elongates Kner
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DOI：
10.1007/s10858-012-9703-9
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发表时间：
2013-02-01
影响因子：
2.7
通讯作者：
Tsuda, Sakae
中科院分区：
文献类型：
--
作者：
Kumeta, Hiroyuki;Ogura, Kenji;Tsuda, Sakae
方法用pKK 223 - 3UC为载体构建含nfeAFP 11或nfeAFP 11-tri基因的表达质粒，转化大肠杆菌JM 105细胞。将每株大肠杆菌在M9基本培养基中培养。然后，向该培养基中加入15 N标记的NH 4Cl，以表达15 N标记的nfeAFP 11和nfeAFP 11-tri蛋白。分别地，将15 N-标记的NH 4Cl和13 C-标记的葡萄糖两者添加到培养基中以获得13 C/15 N-标记的蛋白质。nfeAFP 11和nfeAFP 11-tri的15 N标记蛋白的最终产量分别为52.5和13.5 mg/L，13 C/15 N标记蛋白的最终产量分别为47.3和19.4 mg/L。将所有蛋白质溶解在含有25 mMKCl的50 mM磷酸盐缓冲液（pH= 6.8）中，以制备最终蛋白质浓度为2 mM的溶液。所有NMR实验在4 ℃下在Varian Unity INOVA 600光谱仪上进行。使用NMRPipe（Delaglio等，1995）软件处理二维和三维（分别为2D和3D）NMR光谱，并在Sparky程序（戈达德和Kneller 1997）的帮助下进行数据分析。使用以下一组光谱进行1H-、13 C-和15 N-共振的归属;[1H-15 N]杂原子单量子相干（HSQC）、[1H-13 C] HSQC、HNCO、HN（CO）CA、HNCA、CBCA（CO）NH、C（CO）NH、HBHA（CO）NH、HC（C）H-TOCSY（总相关光谱）。所有化学位移值均以4，4-二甲基-4-硅戊烷-1-磺酸（DSS）为参考，并使用频率比：（15 N/1H）= 0.101329118，（13 C/1H）= 0.251449519（Wishart等，1995）测定。结构计算的质子间距离限制是使用75 ms混合时间从13 C编辑的NOESY-HSQC和15 N编辑的NOESY-HSQC光谱获得的。的骨干phi和psi扭转角的限制来自使用TALOS的骨干原子的化学位移？程序（Shen et al. 2009）。使用CYANA 2.1软件包（Güntert 2004）计算结构。作为3D结构最终计算的输入，nfeAFP 11共使用2072个距离和78个角度约束，nfeAFP 11-tri共使用2124个距离和75个角度约束（表1）。在每个阶段，使用30，000步模拟退火计算100个结构，并基于CYANA目标函数值选择20个结构的最终系综。原子坐标已存入蛋白质数据库（PDB代码：nfeAFP 11为21 x2，nfeAFP 11-tri为21 x3）。还使用nfeAFP 11和nfeAFP 11-tri的13 C/15 N标记的蛋白进行氢-氘（H-D）交换实验。在溶解冻干样品后，每40分钟记录一次HSQC光谱，持续14小时，这为我们提供了关于酰胺基团的氢键形成的信息。
MethodsEscherichia coli JM105 cells were transformed with a pKK223–3UC-based expression plasmid that contains synthesized DNA encoding either nfeAFP11 or nfeAFP11-tri. Each transformant was cultured in M9 minimal media. Then, 15N-labeled NH4Cl was added to this media for the expression of 15N-labeled proteins of nfeAFP11 and nfeAFP11-tri. Separately, both 15N-labeled NH4Cl and 13C-labeled glucose were added to the media to obtain 13C/15N-labeled proteins. The final yields of the 15N-labeled proteins of nfeAFP11 and nfeAFP11-tri were 52.5 and 13.5 mg/L, and those of the 13C/15N-labeled proteins were 47.3 and 19.4 mg/L, respectively. All of the proteins were dissolved in a 50 mM phosphate buffer (pH= 6.8) containing 25 mM KCl to prepare solutions with a final protein concentration of 2 mM. All NMR experiments were carried out at 4 C on a Varian Unity INOVA 600 spectrometer. Two-and three-dimensional (2D and 3D, respectively) NMR spectra were processed using NMRPipe (Delaglio et al. 1995) software, and the data analysis was performed with the help of the Sparky program (Goddard and Kneller 1997). The assignment of the 1H-, 13C-, and 15N-resonances was carried out using the following set of spectra;[1H-15N] heteronuclear single quantum coherence (HSQC),[1H-13C] HSQC, HNCO, HN (CO) CA, HNCA, CBCA (CO) NH, C (CO) NH, HBHA (CO) NH, HC (C) H-TOCSY (total correlation spectroscopy). All chemical shift values were referenced to 4, 4-dimethyl-4-silapentane-1-sulfonic acid (DSS) and determined with using the frequency ratios:(15N/1H)= 0.101329118,(13C/1H)= 0.251449519 (Wishart et al. 1995). The inter-proton distance restraints for the structural calculations were obtained from 13C-edited NOESY–HSQC and 15N-edited NOESY–HSQC spectra using a 75 ms mixing time. The restraints for the backbone phi and psi torsion angles were derived from the chemical shifts of the backbone atoms using the TALOS? program (Shen et al. 2009). The structure was calculated using the CYANA 2.1 software package (Güntert 2004). As an input for the final calculation of the 3D structures, a total of 2072 distances and 78 angle restraints were used for nfeAFP11, and 2124 distances and 75 angle restraints were used for nfeAFP11-tri (Table 1). At each stage, 100 structures were calculated using 30,000 steps of simulated annealing, and a final ensemble of 20 structures was selected on the basis of the CYANA target function values. The atomic coordinates have been deposited in the Protein Data Bank (PDB code: 2lx2 for nfeAFP11, 2lx3 for nfeAFP11-tri). The hydrogen–deuterium (H–D) exchange experiment was also performed using the 13C/15N-labeled proteins of nfeAFP11 and nfeAFP11-tri. The HSQC spectra were recorded in every 40 min at 4 C for 14 h after dissolving the lyophilized samples, which gave us information about hydrogen-bond formation of the amide groups.