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 backbon e H-1(alpha) and N-15 resonances of proteins can be obtained without r ecourse to the backbone amide protons, an approach which should be use ful for assignment of regions with rapidly exchanging backbone amide p rotons 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 thei r 3D analogs, which correlate H-1(alpha) with the intraresidue N-15 an d with the N-15 resonance of the next residue. The experiments are pre ferably conducted in D2O, where very high resolution in the N-15 dimen sion can be achieved by using ?a decoupling. The approach is demonstra ted for a sample of human ubiquitin, uniformly enriched in C-13 and N- 15. Complete backbone and C-13(beta)/H-1(beta) resonance assignments a re presented.