Complete resonance assignment for the polypeptide backbone of interleukin 1 beta using three-dimensional heteronuclear NMR spectroscopy.
The complete sequence-specific assignment of the 15N and 1H backbone resonances of the NMR spectrum of recombinant human interleukin 1 beta (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 C alpha H 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 1 beta. The complete list of 15N and 1H assignments is given for all the amide NH and C alpha H 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 1 beta.
Driscoll
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PC
Laboratory of Chemical Physics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Clore
G M
GM
Marion
D
D
Wingfield
P T
PT
Gronenborn
A M
AM
D000577:Amides; D000595:Amino Acid Sequence; D006801:Humans; D006859:Hydrogen; D007375:Interleukin-1 / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D011994:Recombinant Proteins
D000577:Amides; D007375:Interleukin-1; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D006859:Hydrogen