Sequential Assignments and Identification of Secondary Structure Elements of the Colicin E9 Immunity Protein in Solution by Homonuclear and Heteronuclear NMR

Abstract

1H-1H, 1H-15N, and 1H-1H-15N multidimensional NMR spectroscopic studies of the 86 amino acid protein that provides immunity against the DNase action of colicin E9 are reported. Through a combination of 2D NOESY and TOCSY and 3D TOCSY-HMQC, NOESY-HMQC, and HMQC-NOESY-HMQC experiments, almost complete 1H NMR and backbone 15N NMR assignments have been obtained, and the secondary structure of the protein has been partially elucidated. Approximately 50% of the protein forms three helices. The specificity determining region of the DNase immunity protein, identified from previously reported biochemical studies to include residues 32-40, is helical, indicating that the protein-protein interaction involves residues from at least one helix.