Improved accuracy of 15N–1H scalar and residual dipolar couplings from gradient-enhanced IPAP-HSQC experiments on protonated proteins
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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 NHα , 3 J NHβ and 3 J HNHα . Insertion of band-selective 1H 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 1H-15N RDCs extracted from the minute differences in 1JNH splitting measured at 500 and 750 MHz 1H frequencies, resulting from magnetic susceptibility anisotropy. A nearly complete set of 2 J NHα couplings was measured in GB3 in order to evaluate whether the impact of cross-correlated relaxation is dominated by the 15N–1H α or 15N–1H β dipolar interaction. As expected, we find that 2 J NHα ≤ 2 Hz, with values in the α-helix (0.86 ± 0.52 Hz) slightly larger than in β-sheet (0.66 ± 0.26 Hz). Results indicate that under isotropic conditions, N–HN/N–H β cross-correlated relaxation often dominates. Unresolved E.COSY effects under isotropic conditions involve 3 J HNHα and J NHα , but when weakly aligned any aliphatic proton proximate to both N and HN can contribute.
KeywordsCross-correlated relaxation GB3 IPAP Liquid crystal Magnetic susceptibility anisotropy
residual dipolar coupling
This work was supported in part by the Intramural Research Program of the NIDDK, NIH, and by the Intramural AIDS-Targeted Antiviral Program of the Office of the Director, NIH.
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