TROSY-based HNCO pulse sequences for the measurement of 1HN-15N, 15N-13CO, 1HN-13CO, 13CO-13Cα and 1HN-13Cα dipolar couplings in 15N, 13C, 2H-labeled proteins

HNCO-based 3D pulse schemes are presented for measuring (HN)-H-1-N-15,N-15-(CO)-C-13, (HN)-H-1-(CO)-C-13,(CO)-C-13-C-13(alpha) and (HN)-H-1-C-13(alpha) dipolar couplings in N-15,C-13,H-2-labeled proteins. The experiments are based on recently developed TROSY methodology for improving spectral resolution and sensitivity. Data sets recorded on a complex of Val, Leu, Ile (delta 1 only) methyl protonated N-15,C-13,H-2-labeled maltose binding protein and beta-cyclodextrin as well as N-15,C-13,H-2-labeled human carbonic anhydrase II demonstrate that precise dipolar couplings can be obtained on proteins in the 30-40 kDa molecular weight range. These couplings will serve as powerful restraints for obtaining global folds of highly deuterated proteins.