THE SOLUTION STRUCTURE OF THE TYR41-]HIS MUTANT OF THE SINGLE-STRANDED-DNA BINDING-PROTEIN ENCODED BY GENE-V OF THE FILAMENTOUS BACTERIOPHAGE M13
THE SOLUTION STRUCTURE OF THE TYR41-]HIS MUTANT OF THE SINGLE-STRANDED-DNA BINDING-PROTEIN ENCODED BY GENE-V OF THE FILAMENTOUS BACTERIOPHAGE M13
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DOI：
10.1006/jmbi.1994.1132
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发表时间：
1994-02-11
影响因子：
5.6
通讯作者：
HILBERS, CW
中科院分区：
文献类型：
--
作者：
FOLKERS, PJM;NILGES, M;HILBERS, CW
通过核磁共振波谱研究了丝状噬菌体M13基因V编码的单链DNA结合蛋白突变体Tyr41→His的溶液结构。二维和三维NMR实验已用于基因V蛋白的多种NMR样品，其中一些样品均匀富集15 N或13 C。使用两阶段程序计算了在溶液中作为对称二聚体出现的M13基因V蛋白的突变体Tyr41→His的结构。该过程的第一步涉及使用距离几何程序 DIANA 计算一组单独的单体结构。然后使用 X-PLOR 程序使用“模拟退火”方案计算二聚体结构。因此，通过使用正确处理亚基内和亚基间对 NOE 峰贡献的目标函数，避免了对称二聚体亚基内和亚基间 NOE 的分配问题。此外，采用伪能量项来限制二聚体的对称性。除了这种新颖的计算策略之外，我们还结合了一组 NOE 的距离信息，这些 NOE 使用基于不对称标记的 NMR 策略明确地鉴定为亚基间 NOE。基于从 NMR 数据得出的约 1000 个实验限制，计算了 M13 基因 V 蛋白突变体 Tyr41→His 的总共 20 个结构。两种单体的残基 1 至 15 和 29 至 87 的结构可以通过平均原子均方根合理确定。各个结构与主链原子的各自平均结构之间的差异为 ≈ 0·9 Å，所有原子的平均结构为 ≈ 1·4 Å。无法确定暴露的反平行 β 环（残基 16 至 28）相对于核心的方向。每个单体的分子结构包括一个包围疏水核心的五链β-桶和两个反向平行的β-环。二聚体结构主要由疏水残基稳定，主要涉及单体的对称相关二元结构域（残基 64 至 82）。之前通过自旋标记寡核苷酸的结合实验鉴定了接近结合单链 DNA 的残基。 M13基因V蛋白的突变Tyr41→His的溶液结构与这些结合数据一致，并提供了该蛋白的单链DNA结合路径的清晰视图。
The solution structure of mutant Tyr41→His of the single-stranded DNA binding protein encoded by gene V of the filamentous bacteriophage M13 has been investigated by nuclear magnetic resonance spectroscopy. Two- and three-dimensional NMR experiments have been employed with a variety of NMR samples of gene V protein, some of which were uniformly enriched with either15N or13C. The structure of mutant Tyr41→His of the M13 gene V protein which occurs in solution as a symmetric dimer was calculated using a two-stage procedure. The first step of the procedure involved the calculation of a set of individual monomer structures using the distance geometry program DIANA. This was then followed by the calculation of dimer structures employing "simulated annealing" protocols with the program X-PLOR. Hereby, the problem of assignment of intra- and inter-subunit NOEs of the symmetric dimer was circumvented through use of a target function that correctly deals with the intra- and inter-subunit contributions to the NOE peaks. Furthermore, a pseudo energy term was employed to restrain the symmetry of the dimer. In addition to this novel calculation strategy, we have incorporated distance information for a set of NOEs which were unambiguously identified as inter-subunit NOEs using an NMR strategy based on asymmetric labelling.A total of 20 structures were calculated for the M13 gene V protein mutant Tyr41→His based on approximately 1000 experimental restraints derived from the NMR data. The structure of residues 1 to 15 and 29 to 87 of both monomers is reasonably well determined with an average atomic r.m.s. difference between the individual structures and the respective mean structure of ≈ 0·9 Å for the backbone atoms and ≈ 1·4 Å for all atoms. The orientation of the exposed anti-parallel β-loop (residues 16 to 28) with respect to the core could not be determined. The molecular architecture of each of the monomers includes a five-stranded β-barrel enclosing a hydrophobic core and two-antiparallel β-loops. The dimer structure is stabilized predominantly by hydrophobic residues primarily involving the symmetry-related dyad domains (residues 64 to 82) of the monomers.Residues which are close to bound single-stranded DNA were identified previously from binding experiments with spin-labelled oligonucleotides. The solution structure of mutant Tyr41→His of the M13 gene V proteins is consistent with these binding data and provides a clear view of the protein's single-stranded DNA binding path.