Hyperfine-Shifted 13C and 15N NMR Signals from Clostridium pasteurianum Rubredoxin: Extensive Assignments and Quantum Chemical Verification.

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 13C and 15N 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 15N NMR spectroscopy, to nitrogen atoms near the iron center. We refined and extended these 15N assignments to the adjacent carbonyl carbons by means of one-dimensional 13C[15N] decoupling difference experiments. We collected paramagnetic-optimized Su- perWEFT 13C[13C] constant time COSY (SW-CT-COSY) data to complete the assignment of 13C signals of reduced CpRd. By following these 13C 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. [ABSTRACT FROM AUTHOR]