Published Research - Department of Chemistry

 

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 CαH(i),Cα(i)-N(i+l) correlations in the HCA(CO)N experiment, the Cα(i)-N(i+l),NH(i+ 1) correlations in the HNCA and HN(C0)CA experiments, the CαH(i)-N(i+ l),NH(i+l) correlations in the H(CA)NH and HN(CO)HB experiments, and the Cβ(i)-N(i+ l),NH(i+ 1) correlations in the HN(CO)HB experiments. The backbone intraresidue CαH(i)-15N(i)-NH(i) correlations are provided by the 15N-edited Hartmann-Hahn (HOHAHA) and H(CA)NH experiments, the CβH(i)-15N(i)-NH(i) correlations by the 15N-edited HOHAHA and HNHB experiments, the l3Cα(i)-l5N(i)-NH(i) correlations by the HNCA experiment, and the CαH(1)-13Cα(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.

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