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 backbone 1H and 15N chemical shifts of an amino acid residue with the 15N chemical shifts of both the sequentially preceding and following residues. The sequence employs 1J(NC alpha) and 2J(NC alpha) couplings in two sequential magnetization transfer steps in an 'out-and-back' manner. As a result, N,N connectivities are obtained irrespective of whether the neighbouring amide nitrogens are protonated or not, which makes the experiment suitable for the assignment of proline resonances. Two different three-dimensional variants of the pulse sequence are presented which differ in sensitivity and resolution to be achieved in one of the nitrogen dimensions. The new method is demonstrated with two uniformly 2H/13C/15N-labelled proteins in the 30-kDa range.