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

It is demonstrated that sequential resonance assignment of the backbone 1H alpha 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 alpha 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 beta/1H beta resonance assignments are presented.