Development of NMR measurement and analysis method for protein structural study in 2H2O solution.

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We developed an NMR pulse sequence, 3D HCA(N)CO, to correlate the chemical shifts of protein backbone 1H and 13C to those of 13C' in the preceding residue. When combined with HCACO, HCAN and HCA(CO)N, the HCA(N)CO sequence allows the sequential assignment using backbone 13C' and amide 15N chemical shifts without resort to backbone amide protons. This assignment strategy was demonstrated for 13C/15N-labeled GB1 dissolved in 2H2O. The quality of the GB1 structure determined in 2H2O was similar to that determined in H2O in spite of significantly smaller number of NOE correlations. Thus this strategy enables the determination of protein structures in 2H2O or H2O at high pH values.