HNCAN pulse sequences for sequential backbone resonance assignment across proline residues in perdeuterated proteins

A TROSY-based triple-resonance pulse scheme is described which correlates b ackbone H-1 and N-15 chemical shifts of an amino acid residue with the N-15 chemical shifts of both the sequentially preceding and following residues. The sequence employs (1)J(N)C alpha and (2)J(N)C alpha couplings in two se quential magnetization transfer steps in an `out-and-back' manner. As a res ult, N,N connectivities are obtained irrespective of whether the neighbouri ng amide nitrogens are protonated or not, which makes the experiment suitab le for the assignment of proline resonances. Two different three-dimensiona l variants of the pulse sequence are presented which differ in sensitivity and resolution to be achieved in one of the nitrogen dimensions. The new me thod is demonstrated with two uniformly H-2/(1)3C/N-15-labelled proteins in the 30-kDa range.