A set of HNCO-based experiments for measurement of residual dipolar couplings in ¹⁵N, ¹³C, (²H)-labeled proteins

Several HNCO-based three-dimensional experiments are described for the measurement of ¹³C′(i−1)-¹³C^α(i−1), ¹⁵N(i)-¹³C′(i−1), ¹⁵N(i)-¹³C^α(i), ¹⁵N(i)-¹³C^α(i−1), ¹H^N(i)-¹³C^α(i), ¹H^N(i)-¹³C^α(i−1), and ¹³C^α(i−1)-¹³C^β(i−1) scalar and dipolar couplings in ¹⁵N, ¹³C, (²H)-labelled protein samples. These pulse sequences produce spin-state edited spectra superficially resembling an HNCO correlation spectrum, allowing accurate and simple measurement of couplings without introducing additional spectral crowding. Scalar and dipolar couplings are measured with good sensitivity from relatively large proteins, as demonstrated with three proteins: cardiac Troponin C, calerythrin and ubiquitin. Measurement of several dipolar couplings between spin-1/2 nuclei using spin-state selective 3D HNCO spectra provides a wealth of structural information.