¹H, ¹⁵N, ¹³C, and ¹³CO Assignments of Human Interleukin-4 Using Three-Dimensional Double- and Triple-Resonance Heteronuclear Magnetic Resonance Spectroscopy

Authors

Robert Powers, University of Nebraska - LincolnFollow
Daniel S. Garrett, Laboratory of Chemical Physics, Building 2, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
Carl J. March, Laboratory of Chemical Physics, Building 2, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
Eric A. Frieden, Laboratory of Chemical Physics, Building 2, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
Angela M. Gronenborn, Laboratory of Chemical Physics, Building 2, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
G. Marius Clore, Laboratory of Chemical Physics, Building 2, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland

Document Type

Article

Date of this Version

1992

Citation

Biochemistry 1992, 31, 4334-4346

Comments

U.S. Government Work

Abstract

The assignment of the ¹H, ¹⁵N, ¹³CO, and ¹³C resonances of recombinant human interleukin-4 (IL-4), a protein of 133 residues and molecular mass of 15.4 kDa, is presented based on a series of 11 three-dimensional (3D) double- and triple resonance heteronuclear NMR experiments. These studies employ uniformly labeled ¹⁵N- and ¹⁵N/¹³C-labeled IL-4 with an isotope incorporation of >95% for the protein expressed in yeast. Five independent sequential connectivity pathways via one-, two-, and three-bond heteronuclear J couplings are exploited to obtain unambiguous sequential assignments. Specifically, CO(i)-N(i+l),NH(i+l) correlations are observed in the HNCO experiment, the C^αH(i),C^α(i)-N(i+l) correlations in the HCA(CO)N experiment, the C^α(i)-N(i+l),NH(i+ 1) correlations in the HNCA and HN(C0)CA experiments, the C^αH(i)-N(i+ l),NH(i+l) correlations in the H(CA)NH and HN(CO)HB experiments, and the C^β(i)-N(i+ l),NH(i+ 1) correlations in the HN(CO)HB experiments. The backbone intraresidue C^αH(i)-15N(i)-NH(i) correlations are provided by the ¹⁵N-edited Hartmann-Hahn (HOHAHA) and H(CA)NH experiments, the C^βH(i)-¹⁵N(i)-NH(i) correlations by the ¹⁵N-edited HOHAHA and HNHB experiments, the ^l3C^α(i)-^l5N(i)-NH(i) correlations by the HNCA experiment, and the C^αH(1)-¹³C^α(i)-13CO(i) correlations by the HCACO experiment. Aliphatic side-chain spin systems are assigned by 3D ¹H-¹³C-¹³C-¹H correlated (HCCH-COSY) and total correlated (HCCH-TOCSY) spectroscopy. Because of the high resolution afforded by these experiments, as well as the availability of multiple sequential connectivity pathways, ambiguities associated with the limited chemical shift dispersion associated with helical proteins are readily resolved. Further, in the majority of cases (88%), four or more sequential correlations are observed between successive residues. Consequently, the interpretation of these experiments readily lends itself to semiautomated analysis which significantly simplifies and speeds up the assignment process. The assignments presented in this paper provide the essential basis for studies aimed at determining the high-resolution three-dimensional structure of IL-4 in solution.