4D Non-uniformly sampled HCBCACON and ¹J(NC ^α)- selective HCBCANCO experiments for the sequential assignment and chemical shift analysis of intrinsically disordered proteins

A pair of 4D NMR experiments for the backbone assignment of disordered proteins is presented. The experiments exploit ¹³C direct detection and non-uniform sampling of the indirectly detected dimensions, and provide correlations of the aliphatic proton (H ^α, and H ^β) and carbon (C ^α, C ^β) resonance frequencies to the protein backbone. Thus, all the chemical shifts regularly used to map the transient secondary structure motifs in the intrinsically disordered proteins (H ^α, C ^α, C ^β, C0, and N) can be extracted from each spectrum. Compared to the commonly used assignment strategy based on matching the C ^α and Cb chemical shifts, inclusion of the H ^α and H ^β provides up to three extra resonance frequencies that decrease the chance of ambiguous assignment. The experiments were successfully applied to the original assignment of a 12.8 kDa intrinsically disordered protein having a high content of proline residues (26 %) in the sequence.