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

ABSTRACT: 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(NHalpha), (3)J(NHbeta) and (3)J(HNHalpha). Insertion of band-selective (1)H 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 (1)H-(15)N RDCs extracted from the minute differences in (1)J(NH) splitting measured at 500 and 750 MHz (1)H frequencies, resulting from magnetic susceptibility anisotropy. A nearly complete set of (2)J(NHalpha) couplings was measured in GB3 in order to evaluate whether the impact of cross-correlated relaxation is dominated by the (15)N-(1)H(alpha) or (15)N-(1)H(beta) dipolar interaction. As expected, we find that (2)J(NHalpha) < or = 2 Hz, with values in the alpha-helix (0.86 +/- 0.52 Hz) slightly larger than in beta-sheet (0.66 +/- 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(HNHalpha) and J(NHalpha), but when weakly aligned any aliphatic proton proximate to both N and H(N) can contribute.

SUBMITTER: Yao L 

PROVIDER: S-EPMC2753394 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature