Graduate Program in Biophysics, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706, USA.
J Am Chem Soc. 2009 Oct 28;131(42):15555-63. doi: 10.1021/ja905928x.
Stable isotope-labeling methods, coupled with novel techniques for detecting fast-relaxing NMR signals, now permit detailed investigations of paramagnetic centers of metalloproteins. We have utilized these advances to carry out comprehensive assignments of the hyperfine-shifted (13)C and (15)N signals of the rubredoxin from Clostridium pasteurianum (CpRd) in both its oxidized and reduced states. We used residue-specific labeling (by chemical synthesis) and residue-type-selective labeling (by biosynthesis) to assign signals detected by one-dimensional (15)N NMR spectroscopy, to nitrogen atoms near the iron center. We refined and extended these (15)N assignments to the adjacent carbonyl carbons by means of one-dimensional (13)C[(15)N] decoupling difference experiments. We collected paramagnetic-optimized SuperWEFT (13)C[(13)C] constant time COSY (SW-CT-COSY) data to complete the assignment of (13)C signals of reduced CpRd. By following these (13)C signals as the protein was gradually oxidized, we transferred these assignments to carbons in the oxidized state. We have compared these assignments with hyperfine chemical shifts calculated from available X-ray structures of CpRd in its oxidized and reduced forms. The results allow the evaluation of the X-ray structural models as representative of the solution structure of the protein, and they provide a framework for future investigation of the active site of this protein. The methods developed here should be applicable to other proteins that contain a paramagnetic center with high spin and slow electron exchange.
稳定同位素标记方法，结合用于检测快速弛豫 NMR 信号的新技术，现在允许对金属蛋白的顺磁中心进行详细研究。我们利用这些进展，对来自巴氏梭菌（CpRd）的氧化还原态 rubredoxin 的超精细位移（13）C 和（15）N 信号进行了全面的分配。我们使用残基特异性标记（通过化学合成）和残基类型选择性标记（通过生物合成）来分配通过一维（15）N NMR 光谱检测到的信号，这些信号位于铁中心附近的氮原子上。我们通过一维（13）C[(15)N]去耦差实验，将这些（15）N 分配进一步扩展到相邻的羰基碳上。我们收集了顺磁优化的 SuperWEFT（13）C[(13)C]恒时间 COSY（SW-CT-COSY）数据，以完成还原 CpRd 的（13）C 信号的分配。通过跟踪随着蛋白质逐渐氧化而产生的这些（13）C 信号，我们将这些分配转移到氧化状态下的碳上。我们将这些分配与从 CpRd 的氧化和还原形式的可用 X 射线结构计算的超精细化学位移进行了比较。结果允许评估 X 射线结构模型作为蛋白质溶液结构的代表性模型，并为该蛋白质活性位点的未来研究提供了框架。这里开发的方法应该适用于其他含有高自旋和慢电子交换的顺磁中心的蛋白质。
