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Sequence-specific assignment of the 1H and 15N nuclear magnetic resonance spectra of the reduced recombinant high-potential iron-sulfur protein I from Ectothiorhodospira halophila.

A 1H and 15N NMR investigation through two-dimensional and three-dimensional spectroscopy has been performed on the reduced form ([Fe4S4]2+) of the recombinant high-potential iron-sulfur protein (HiPIP) I from Ectothiorhodospira halophila expressed in Escherichia coli. [Fe4S4]2+ clusters in proteins are paramagnetic with a relatively low mu eff of about 0.8 mu B/iron ion, but the paramagnetic effects on nuclear relaxation are so strong as to yield T1 values of a few milliseconds and linewidths of hundreds of hertz for the nuclei closet to the paramagnetic center. Despite these features, 71 out of 73 residues were identified, most of which were assigned completely as far as proton resonances are concerned; as many as 68 residues could be assigned without any reference to the existing X-ray structure. A total of 88% of all protein protons and 58 out of 69 peptide HN nitrogen signals were assigned. To the best of our knowledge, this is the most extensive 1H assignment of a paramagnetic protein to date. Protons sensitive to the proximity of the cluster were assigned through suitable NOE spectroscopy experiments. Three out of the four coordinated cysteines were assigned, and two residues have been identified whose peptide HN protons give rise to H bonds with coordinated sulfur atoms. The inter-residue NOE cross peaks are in qualitative agreement with the secondary and tertiary structure as obtained from the available X-ray crystallographic analysis of the wild-type protein at 250-pm resolution. It is therefore shown that the expressed protein is properly folded and that it is a reliable model for the wild-type protein. These data are meaningful for the detection of structural differences among mutants in future studies.