ASSIGNMENT OF THE SIDE-CHAIN H-1 AND C-13 RESONANCES OF INTERLEUKIN-1-BETA USING DOUBLE-RESONANCE AND TRIPLE-RESONANCE HETERONUCLEAR 3-DIMENSIONAL NMR-SPECTROSCOPY

The assignment of the aliphatic 1H and 13C resonances of IL-1β, a protein of 153 residues and molecular mass 17.4 kDa, is presented by use of a number of novel three-dimensional (3D) heteronuclear NMR experiments which rely on large heteronuclear one-bond J couplings to transfer magnetization and establish through-bond connectivities. These 3D NMR experiments circumvent problems traditionally associated with the application of conventional 2D 1H-1H correlation experiments to proteins of this size, in particular the extensive chemical shift overlap which precludes the interpretation of the spectra and the reduced sensitivity arising from 1H line widths that are often significantly larger than the 1H–1H J couplings. The assignment proceeds in two stages. In the first step the 13Cα chemical shifts are correlated with the NH and 15N chemical shifts by a 3D triple-resonance NH–15N–13Cα (HNCA) correlation experiment which reveals both intraresidue NH(i)–15N(i)–13Cα(i) and some weaker interresidue NH(i)–15N(i)–Cα(i–1) correlations, the former via intraresidue one-bond 1JNCα and the latter via interresidue two-bond 2JNCα couplings. As the NH, 15N, and CαH chemical shifts had previously been equentially assigned by 3D 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) spectroscopy [Driscoll, P. C., Clore, G. M., Marion, D., Wingfield, P. T., & Gronenborn, A. M. (1990) Biochemistry 29, 3542-3556], the 3D triple-resonance HNCA correlation experiment permits the sequence-specific assignments of 13Cα chemical shifts in a straightforward manner. The second step involves the identification of side-chain spin systems by 3D 1Ή–13C–13C–1Ή correlated (HCCH-COSY) and 3D 1Ή–13C–13C–1Ή total correlated (HCCH-TOCSY) spectroscopy, the latter making use of isotropic mixing of 13C magnetization to obtain relayed connectivities along the side chains. Extensive cross-checks are provided in the assignment procedure by examination of the connectivities between 1H resonances at all the corresponding 13C shifts of the directly bonded 13C nuclei. In this manner, we were able to obtain complete 1Ή and 13C side-chain assignments for all residues, with the exception of 4 (out of a total of 15) lysine residues for which partial assignments were obtained. The 3D heteronuclear correlation experiments described are highly sensitive, and the required set of three 3D spectra was recorded in only 1 week of measurement time on a single uniformly 15N/13C-labeled 1.7 mM sample of interleukin-1β. This first example of the essentially complete side-chain assignments of a protein with a molecular mass greater than 15 kDa by heteronuclear 3D NMR methods provides a basis for the determination of a full high-resolution three-dimensional structure of interleukin-1β in solution. © 1990, American Chemical Society. All rights reserved.