H-1, C-13, AND N-15 NMR BACKBONE ASSIGNMENTS AND SECONDARY STRUCTURE OF HUMAN INTERFERON-GAMMA

H-1, C-13, and N-15 NMR assignments of the protein backbone of human interferon-gamma, a homodimer of 31.4 kDa, have been made using the recently introduced three-dimensional (3D) triple-resonance NMR techniques. It is shown that, despite the approximately 40-50-Hz C-13(alpha) and H-1(alpha) line widths of this high molecular weight dimer and the extensive overlap in the H-1(alpha) and C-13(alpha) spectral regions, unique sequential assignments can be made on the basis of combined use of the 3D HNCO, HNCA, HN(CO)CA, and HCACO constant-time experiments, the N-15-separated 3D NOESY-HMQC, and the 3D HOHAHA-HMQC experiments. Analysis of the N-15-separated 3D NOESY-HMQC and C-13/N-15-separated four-dimensional (4D) NOESY-HMQC spectra together with the secondary C(alpha) and C(beta) chemical shifts yielded extensive secondary structure information. The NMR-derived secondary structure essentially confirms results of a recently published low-resolution crystal structure [Ealick et al. (1991) Science 252, 698-7021, i.e., six helices in the monomer which are mostly alpha-helical in nature, no beta-sheets, a long flexible loop between helices A and B, and a very hydrophobic helix C. The functionally important carboxy terminus, which was not observed in the X-ray study, does not adopt a rigid conformation in solution. A high degree of internal mobility, starting at Pro-123, gives rise to significantly narrower resonance line widths for these carboxy-terminal residues compared to the rest of the protein.