Improved accuracy of 15N-1H scalar and residual dipolar couplings from gradient-enhanced IPAP-HSQC experiments on protonated proteins

The presence of dipole-dipole cross-correlated relaxation as well as unresolved E.COSY effects adversely impacts the accuracy of (1) J (NH) splittings measured from gradient-enhanced IPAP-HSQC spectra. For isotropic samples, the size of the systematic errors caused by these effects depends on the values of (2) J (NH alpha) , (3) J (NH beta) and (3) J (HNH alpha) . Insertion of band-selective H-1 decoupling pulses in the IPAP-HSQC experiment eliminates these systematic errors and for the protein GB3 yields (1) J (NH) splittings that agree to within a root-mean-square difference of 0.04 Hz with values measured for perdeuterated GB3. Accuracy of the method is also highlighted by a good fit to the GB3 structure of the H-1-N-15 RDCs extracted from the minute differences in (1)J(NH) splitting measured at 500 and 750 MHz H-1 frequencies, resulting from magnetic susceptibility anisotropy. A nearly complete set of (2) J (NH alpha) couplings was measured in GB3 in order to evaluate whether the impact of cross-correlated relaxation is dominated by the N-15-H-1 (alpha) or N-15-H-1 (beta) dipolar interaction. As expected, we find that (2) J (NH alpha) a parts per thousand currency sign 2 Hz, with values in the alpha-helix (0.86 +/- A 0.52 Hz) slightly larger than in beta-sheet (0.66 +/- A 0.26 Hz). Results indicate that under isotropic conditions, N-H-N/N-H (beta) cross-correlated relaxation often dominates. Unresolved E.COSY effects under isotropic conditions involve (3) J (HNH alpha) and J (NH alpha) , but when weakly aligned any aliphatic proton proximate to both N and H-N can contribute.