PROTEIN EXPRESSION, SELECTIVE ISOTOPIC LABELING, AND ANALYSIS OF HYPERFINE-SHIFTED NMR SIGNALS OF ANABAENA-7120 VEGETATIVE [2FE-2S]FERREDOXIN
PROTEIN EXPRESSION, SELECTIVE ISOTOPIC LABELING, AND ANALYSIS OF HYPERFINE-SHIFTED NMR SIGNALS OF ANABAENA-7120 VEGETATIVE [2FE-2S]FERREDOXIN
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DOI：
10.1006/abbi.1995.1082
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发表时间：
1995-01-10
影响因子：
3.9
通讯作者：
MARKLEY, JL
中科院分区：
文献类型：
--
作者：
CHENG, H;WESTLER, WM;MARKLEY, JL
两种替代的 T7 RNA 启动子/聚合酶系统已用于在大肠杆菌中高水平（类似于 20 毫克/升培养物）异源表达植物型 [2Fe-2S] 铁氧还蛋白（Anabaena 7120 营养铁氧还蛋白）。以(NH4Cl)-N-15为氮源培养大肠杆菌，对铁氧还蛋白进行N-15统一标记时使用一种系统；另一种与营养缺陷型宿主菌株结合使用，通过掺入 H-2、C-13 和 N-15 标记的氨基酸来选择性富集蛋白质。通过 H-1、H-2、C-13 和 N-15 NMR 光谱研究了标记的铁氧还蛋白样品。对含有 [H-2(α)]Cys、[H-2(beta 2,beta 3)]Cys、[C-13(beta)]-Cys 和 [N-15]Cys 的样品进行 H-1 和 H-2 NMR 研究的结果证实了先前的半胱氨酰质子共振归属（L. Skjeldal、W. M. Westler、B.-H. Oh、A. M. Krezel、H. M.， Holden、B. L. Jacobson、I. Rayment 和 J. L. Markley (1991) 生物化学 30, 7363-7368)。来自四个半胱氨酰β-碳的所有四个C-13 NMR 峰和来自四个半胱氨酰氮的所有四个N-15 NMR 峰在氧化和还原铁氧还蛋白的光谱中得到解析。 Cys(46) 位于独特的 (Ala-Cys) 二肽中，通过检测掺入 [C-13']Ala 和 [N-15]Cys 的铁氧还蛋白样品中的 C-13(i)-N-15(i+1) 偶联来确定 Cys(46) 的氮共振。 Cys(41) 的氮信号根据其化学位移和 T-1 弛豫时间初步确定。还原态的半胱氨酰β-碳共振已根据与其（先前指定的）β-质子的相关性被指定给各个残基。氧化态 Cys(46) 的 β-碳共振已通过其与还原态相应共振的相关性来确定；这是通过在甲基紫精存在下，通过连二亚硫酸盐还原蛋白质样品进行逐步空气氧化来实现的。与 Fe(III) 配位的两个半胱氨酸的 β-碳的自旋晶格弛豫时间在氧化态和还原态下相似。这表明还原团簇中存在的反铁磁耦合对 Fe(III) 的电子弛豫时间影响很小。对 [2Fe-2S] 簇的半胱氨酰配体的 H-1、C-13 和 N-15 信号的温度依赖性的研究表明，对于给定残基内的不同原子类型，温度依赖性的斜率 (Delta delta/Delta T-1) 可能不同。例如，在还原型铁氧还蛋白中，虽然 Delta delta/Delta T-1 对 Cys(49) H-1(beta 2) 和 H-1(beta 3) 呈阳性，但对 Cys(49) C-13(beta) 呈阴性。尽管 Delta delta/Delta T-1 对于与 Fe(II) 连接的半胱氨酸的质子呈阴性，而对于与 Fe(III) 连接的半胱氨酸的质子呈阳性，但它对于所有半胱氨酰氮均呈阳性。 Arg(42) 自旋系统的近乎完整的归属源自对三个选择性标记样品的 NMR 研究：包含 [U-N-15]Arg、[26% U-C-13]Arg 和 [H-2(α,β 2,β 3)]Arg 的铁氧还蛋白。主链酰胺氮产生的共振在氧化态下在 201.6 ppm 处表现出不寻常的化学位移，但在还原态下未解决。NMR 结果表明，在氧化铁氧还蛋白晶体的 X 射线结构中，Arg(42) 主链氮和铁硫簇的硫化物之间观察到氢键（W. R. Rypniewski、W. R. Breiter、M. M. Benning、 G. Wesenberg、B.-H. Oh、J. L. Markley、I. Rayment 和 H. M. Holden (1991) Biochemistry 30, 4126-4131) 以氧化和还原形式存在于溶液中。结果表明，还原铁氧还蛋白光谱中的非半胱氨酰超精细位移峰（峰“K”）并非如先前假设的那样由 Arg(42) 的 H-1(α) 产生。 (C) 1995 学术出版社
Two alternative T7 RNA promoter/polymerase systems have been employed for the heterologous expression of a plant-type [2Fe-2S]ferredoxin, Anabaena 7120 vegetative ferredoxin, in Escherichia coli at high levels (similar to 20 mg/liter of culture). One system was used when N-15-labeling the ferredoxin uniformly by growing E. coli with (NH4Cl)-N-15 as the nitrogen source; the other was used in conjunction with auxotrophic host strains to enrich the protein selectively by incorporating H-2-, C-13-, and N-15-labeled amino acids. The labeled ferredoxin samples were studied by H-1, H-2, C-13 and N-15 NMR spectroscopy. Results from H-1 and H-2 NMR studies of samples containing [H-2(alpha)]Cys, [H-2(beta 2,beta 3)]Cys, [C-13(beta)]-Cys, and [N-15]Cys have confirmed previous cysteinyl proton resonance assignments (L. Skjeldal, W. M. Westler, B.-H. Oh, A. M. Krezel, H. M., Holden, B. L. Jacobson, I. Rayment, and J. L. Markley (1991) Biochemistry 30, 7363-7368). All four C-13 NMR peaks arising from the four cysteinyl beta-carbons and all four N-15 NMR peaks from the four cysteinyl nitrogens were resolved in spectra of both the oxidized and reduced ferredoxins. The nitrogen resonance of Cys(46), which is located in a unique (Ala-Cys) dipeptide, was assigned by detection of C-13(i)-N-15(i+1) coupling in a ferredoxin sample with incorporated [C-13']Ala and [N-15]Cys. The nitrogen signal of Cys(41) was assigned tentatively on the basis of its chemical shift and T-1 relaxation time. The cysteinyl beta-carbon resonances in the reduced state have been assigned to individual residues on the basis of correlations with their (previously assigned) beta-protons. The beta-carbon resonance from Cys(46) in the oxidized state has been assigned by its correlation with the corresponding resonance in the reduced state; this was accomplished by following the progressive air oxidation of a protein sample reduced by dithionite in the presence of methyl viologen. The spin-lattice relaxation times of the beta-carbons of the two cysteines coordinated to Fe(III) were similar in the oxidized and reduced states. This suggests that the antiferromagnetic coupling present in the reduced cluster has little influence on the electronic relaxation time of the Fe(III). Studies of the temperature dependence of the H-1, C-13, and N-15 signals of the cysteinyl ligands to the [2Fe-2S] cluster show that the slope of the temperature dependence (Delta delta/Delta T-1) can be different for different atom types within a given residue. For example, in the reduced ferredoxin, although Delta delta/Delta T-1 is positive for Cys(49) H-1(beta 2) and H-1(beta 3), it is negative for Cys(49) C-13(beta). Although Delta delta/Delta T-1 is negative for protons of cysteines ligated to Fe(II) and positive for protons of cysteines ligated to Fe(III), it is positive for all the cysteinyl nitrogens. Nearly complete assignments for the spin system of Arg(42) were derived from NMR studies of three selectively labeled samples: ferredoxin incorporating [U-N-15]Arg, [26% U-C-13]Arg, and [H-2(alpha,beta 2,beta 3)]Arg. The resonance arising from the backbone amide nitrogen exhibited an unusual chemical shift at 201.6 ppm in the oxidized state but was unresolved in the reduced state.The NMR results indicate that the hydrogen bond observed between the Arg(42) backbone nitrogen and a sulfide of the iron-sulfur cluster in the X-ray structure of the oxidized ferredoxin crystal (W. R. Rypniewski, W. R. Breiter, M. M. Benning, G. Wesenberg, B.-H. Oh, J. L. Markley, I. Rayment, and H. M. Holden (1991) Biochemistry 30, 4126-4131) is present in solution in both the oxidized and reduced forms of the protein. The results show that the noncysteinyl, hyperfine-shifted peak (peak ''K'') in the spectrum of the reduced ferredoxin does not arise from H-1(alpha) of Arg(42) as previously postulated. (C) 1995 Academic Press, Inc.