COMPLETE RESONANCE ASSIGNMENT FOR THE POLYPEPTIDE BACKBONE OF INTERLEUKIN-1-BETA USING 3-DIMENSIONAL HETERONUCLEAR NMR-SPECTROSCOPY

The complete sequence-specific assignment of the 15N and 1H backbone resonances of the NMR spectrum of recombinant human interleukin 1β (153 residues, Mr = 17 400) has been obtained by using primarily 15N‒1H heteronuclear three-dimensional (3D) NMR techniques in combination with 15N‒1H heteronuclear and 1H homonuclear two-dimensional NMR. The fingerprint region of the spectrum was analyzed by using a combination of 3D heteronuclear 1H Hartmann-Hahn 15N‒1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser 15N‒1H multiple quantum coherence (3D NOESY-HMQC) spectroscopies. We show that the problems of amide NH and CαH chemical shift degeneracy that are prevalent for proteins of this size are readily overcome by using the 3D heteronuclear NMR technique. A doubling of some peaks in the spectrum was found to be due to N-terminal heterogeneity of the 15N-labeled protein, corresponding to a mixture of wild-type and des-Ala-1 -interleukin 1β. The complete list of 15N and 1H assignments is given for all the amide NH and CαH resonances of all non-proline residues, as well as the 1H assignments for some of the amino acid side chains. This first example of the sequence-specific assignment of a protein using heteronuclear 3D NMR provides a basis for further conformational and dynamic studies of interleukin 1β. © 1990, American Chemical Society. All rights reserved.