NMR experiments for resonance assignments of C-13, N-15 doubly-labeled flexible polypeptides: Application to the human prion protein hPrP(23-230)

A combination of three heteronuclear three-dimensional NMR experiments tail ored for sequential resonance assignments in uniformly N-15, C-13-labeled f lexible polypeptide chains is described. The 3D (H)N(CO-TOCSY)NH, 3D (H)CA( CO-TOCSY)NH and 3D (H)CBCA(CO-TOCSY)NH schemes make use of the favorable N- 15 chemical shift dispersion in unfolded polypeptides, exploit the slow tra nsverse N-15 relaxation rates of unfolded polypeptides in high resolution c onstant-time [H-1, N-15]-correlation experiments, and use carbonyl carbon h omonuclear isotropic mixing to transfer magnetization sequentially along th e amino acid sequence. Practical applications are demonstrated with the 100 -residue flexible tail of the recombinant human prion protein, making use o f spectral resolution up to 0.6 Hz in the N-15 dimension, simultaneous corr elation with the two adjacent amino acid residues to overcome problems asso ciated with spectral overlap, and the potential of the presently described experiments to establish nearest-neighbor correlations across proline resid ues in the amino acid sequence.