An intensity-based constant-time COSY (CT-COSY) method is described for measuring 1H-1H residual dipolar couplings of proteins in weakly aligned media. For small proteins, the overall sensitivity of this experiment is comparable to the NOESY experiment. In cases where the 1H-1H distances are defined by secondary structure, such as 1H(α)-1H(N) and 1H(N)-1H(N) sequential distances in α-helices and β-sheets, these measurements provide useful orientational constraints for protein structure determination. This experiment can also be used to provide distance information similar to that obtained from NOE connectivities once the angular dependence is removed. Because the measurements are direct and non-coherent processes, such as spin diffusion, do not enter, the measurements can be more reliable. The 1/r3 distance dependence of directly observed dipolar couplings, as compared with the 1/r6 distance dependence of NOEs, also can provide longer range distance information at favorable angles. A simple 3D, 15N resolved version of the pulse sequence extends the method to provide the improved resolution required for application to larger biomolecules.
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