A refined model for the solution structure of oxidized putidaredoxin.
A refined model for the solution structure of oxidized putidaredoxin.
复制标题
氧化型恶臭氧还蛋白溶液结构的精细模型。
DOI：
10.1021/bi983030b
复制
发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Heymont,J
中科院分区：
文献类型：
--
作者：
Pochapsky,TC;Jain,NU;Kuti,M;Lyons,TA;Heymont,J
氧化putidaredoxin（Pdxo），Cys 4Fe 2S 2铁氧还蛋白的溶液结构的一个改进的模型，已被确定。先前的结构（Pochapsky et al.（1994）Biochemistry 33，6424 - 6432; PDB entry 1 PUT）是使用同源二维NMR实验的结果计算的。新的数据使得有可能计算出原始Pdxo溶液结构的改进。首先，基本上完整的分配抗磁性15 N和13 C共振的Pdxo已使用多维NMR方法，和15 N-和13 C-分辨NOESY实验已允许识别许多新的NOE限制结构计算。立体特异性分配亮氨酸和缬氨酸CH 3共振使用biosynthetically定向fractional 13 C标记，提高涉及这些残基的NOE限制的精度。用二维J-调制的~（15）N，~ 1H HSQC和三维（HN）CO（CO）NH实验相结合的方法得到了骨架二面角约束。第二，已经确定了Pdx的抗磁性形式的溶液结构，即镓putidaredoxin的C85 S变体的溶液结构，其中非配体Cys被Ser取代（Pochapsky等人（1998）J.Biomol.2004：NMR 12，407 - 415），提供了关于天然铁氧还蛋白由于顺磁性而无法观察到的结构特征的信息。第三，一种密切相关的铁氧还蛋白--牛肾上腺素还蛋白的晶体结构已经发表（Müller et al.（1998）Structure 6，269−280）。这种结构已被用来模拟的金属结合位点的结构在Pdx。一个家庭的14个结构，表现出0.51欧姆的骨架重原子和0.83欧姆的所有重原子的RMSD。排除模型化的金属结合环区域将骨架原子的总体rmsd降低至0.30 μ m，并且将所有重原子的rmsd降低至0.71 μ m。
A refined model for the solution structure of oxidized putidaredoxin (Pdxo), a Cys4Fe2S2ferredoxin, has been determined. A previous structure (Pochapsky et al. (1994)Biochemistry33, 6424−6432; PDB entry 1PUT) was calculated using the results of homonuclear two-dimensional NMR experiments. New data has made it possible to calculate a refinement of the original Pdxosolution structure. First, essentially complete assignments for diamagnetic15N and13C resonances of Pdxohave been made using multidimensional NMR methods, and15N- and13C-resolved NOESY experiments have permitted the identification of many new NOE restraints for structural calculations. Stereospecific assignments for leucine and valine CH3resonances were made using biosynthetically directed fractional13C labeling, improving the precision of NOE restraints involving these residues. Backbone dihedral angle restraints have been obtained using a combination of two-dimensional J-modulated15N,1H HSQC and 3D (HN)CO(CO)NH experiments. Second, the solution structure of a diamagnetic form of Pdx, that of the C85S variant of gallium putidaredoxin, in which a nonligand Cys is replaced by Ser, has been determined (Pochapsky et al. (1998)J. Biomol. NMR12, 407−415), providing information concerning structural features not observable in the native ferredoxin due to paramagnetism. Third, a crystal structure of a closely related ferredoxin, bovine adrenodoxin, has been published (Müller et al. (1998)Structure6, 269−280). This structure has been used to model the metal binding site structure in Pdx. A family of fourteen structures is presented that exhibits an rmsd of 0.51 Å for backbone heavy atoms and 0.83 Å for all heavy atoms. Exclusion of the modeled metal binding loop region reduces overall the rmsd to 0.30 Å for backbone atoms and 0.71 Å for all heavy atoms.