SEQUENCE-SPECIFIC ASSIGNMENT OF THE H-1 AND N-15 NUCLEAR-MAGNETIC-RESONANCE SPECTRA OF THE REDUCED RECOMBINANT HIGH-POTENTIAL IRON-SULFUR PROTEIN-I FROM ECTOTHIORHODOSPIRA-HALOPHILA

A H-1 and N-15 NMR investigation through two-dimensional and three-dim ensional spectroscopy has been performed on the reduced form ([Fe4S4]( 2+)) of the recombinant high-potential iron-sulfur protein (HiPIP) I f rom Ectothiorhodospira halophila expressed in Escherichia coli. [Fe4S4 ](2+) clusters in proteins are paramagnetic with a relatively low mu(e ff) of about 0.8 mu(B)/iron ion, but the paramagnetic effects on nucle ar relaxation are so strong as to yield T-1 values of a few millisecon ds and Linewidths of hundreds of hertz for the nuclei closest to the p aramagnetic center. Despite these features, 71 out of 73 residues were identified, most of which were assigned completely as far as proton r esonances are concerned; as many as 68 residues could be assigned with out any reference to the existing X-ray structure. A total of 88% of a ll protein protons and 58 out of 69 peptide HN nitrogen signals were a ssigned. To the best of our knowledge, this is the most extensive H-1 assignment of a paramagnetic protein to date. Protons sensitive to the proximity of the cluster were assigned through suitable NOE spectrosc opy experiments. Three out of the four coordinated cysteines were assi gned, and two residues have been identified whose peptide HN protons g ive rise to H bonds with coordinated sulfur atoms. The inter-residue N OE cross peaks are in qualitative agreement with the secondary and ter tiary structure as obtained from the available X-ray crystallographic analysis of the wild-type protein at 250-pm resolution. It is therefor e shown that the expressed protein is properly folded and that it is a reliable model for the wild-type protein. These data are meaningful f or the detection of structural differences among mutants in future stu dies.