THE SECONDARY STRUCTURE OF THE COLICIN-E3 IMMUNITY PROTEIN AS STUDIED BY H-1-H-1 AND H-1-N-15 2-DIMENSIONAL NMR-SPECTROSCOPY

By performing H-1-H-1 and H-1-N-15 two-dimensional (2D) nuclear magnetic resonance (NMR) experiments, the complete sequence-specific resonance assignment was determined for the colicin E3 immunity protein (84 residues; ImmE3), which binds to colicin E3 and inhibits its RNase activity. First, the fingerprint region of the spectrum was analyzed by homonuclear H-1-H-1 HOHAHA and NOESY methods. For the identification of overlapping resonances, heteronuclear H-1-N-15 (HMQC-HOHAHA, HMQC-NOESY) experiments were performed, so that the complete H-1 and N-15 resonance assignments were provided. Then the secondary structure of ImmE3 was determined by examination of characteristic patterns of sequential backbone proton NOEs in combination with measurement of exchange rates of amide protons and 3J(HN-alpha) coupling constants. From these results, it was concluded that ImmE3 contains a four-stranded antiparallel beta-sheet (residues 2-10, 19-22, 47-49, and 71-79) and a short alpha-helix (residues 31-36).