Sequential backbone assignment of isotopically enriched proteins in D2O by deuterium-decoupled HA(CA)N and HA(CACO)N

Summary It is demonstrated that sequential resonance assignment of the backbone ¹H and ¹⁵N resonances of proteins can be obtained without recourse to the backbone amide protons, an approach which should be useful for assignment of regions with rapidly exchanging backbone amide protons and for proteins rich in proline residues. The method relies on the combined use of two 2D experiments, HA(CA)N and HA(CACO)N or their 3D analogs, which correlate ¹H with the intraresidue ¹⁵N and with the ¹⁵N resonance of the next residue. The experiments are preferably conducted in D₂O, where very high resolution in the ¹⁵N dimension can be achieved by using ²H decoupling. The approach is demonstrated for a sample of human ubiquitin, uniformly enriched in ¹³C and ¹⁵N. Complete backbone and ¹³C/¹H resonance assignments are presented.