Three-dimensional 13C-resolved proton NOE spectroscopy of uniformly 13C-labeled proteins for the NMR assignment and structure determination of larger molecules

Abstract

The currently accepted protocol for the determination of the solution structures of proteins by NMR consists of the assignment of the ‘H resonances, the identification of NOE distance constraints on the basis of the assignments, and the computation of the three-dimensional structure using the NMR-derived distance constraints (for a review, see Ref. (I)). Recently, by the use of isotope-edited two-dimensional NMR experiments on selectively labeled proteins (2-6)) or heteronuclear three-dimensional NMR experiments ( 7) on proteins that have been uniformly labeled with i5N (8-I]), the complete assignments of the backbone protons for proteins up to MW 20,000 have become possible. However, in order to determine the three-dimensional structures of these larger proteins, distance constraints derived from long-range NOES between (assigned) aliphatic protons are necessary. These NOES are very difficult if not impossible to obtain from conventional 2D NOESY spectra due to extensive overlap in the aliphatic region of the spectra. Thus, although the (backbone) proton NMR signals may be assigned for larger systems such as T4 lysozyme and staphylococcal nuclease (5, 6)) the 3D structures cannot be determined. Here, we describe an approach to resolve the spectral overlap in the aliphatic part of the NOESY data. The method involves the use of 13C heteronuclear three-dimensional NMR spectroscopy in which the NOESY spectra are simplified by editing with respect to the 13C frequencies. In this study, a sample of 3.8 mM T4 lysozyme ( 164 amino acid residues, 18.7 kDa) that was uniformly enriched with both 13C and 15N at a level greater than 95% was employed. The pulse sequence used was