Abstract
Assignment of hyperfine-shifted resonances in paramagnetic metalloproteins such as Fe2S2ferredoxins poses a major experimental challenge due to hyperfine shifts and/or severe line broadening. We have explored the possibility of using structural data from homologous proteins as part of an assignment strategy for the sequence-specific assignment of hyperfine-shifted backbone carbonyl (13C′) and nitrogen resonances (15N) in Fe2S2ferredoxins. This strategy is based on the assignment of resonances in the paramagnetic region to particular types of amino acid residues using selective isotope labeling. Reduced metal–nuclear distances are then calculated from experimentally determined T1relaxation times for those resonances and the calculated distances aligned with the distances of nuclei at corresponding amino acid sequence positions in the crystal structure of a structurally homologous protein. The comparative assignment approach has met with success in correctly predicting the13C′ and15N assignments in Pdx° from the crystal structure data of two similar and related ferredoxins, namely bovine adrenodoxin andAnabaenaferredoxin. Sequence-specific assignments made in this fashion were verified by selective13C′{15N} decoupling experiments.
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