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

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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 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–1Hα or 15N–1Hβ 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–Hβ 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.

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Abbreviations

RDC:

residual dipolar coupling

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Acknowledgment

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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  1. Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, MD, 20892-0520, USA

    Lishan Yao, Jinfa Ying & Ad Bax

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  1. Lishan Yao
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  2. Jinfa Ying
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  3. Ad Bax
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Correspondence to Ad Bax.

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Yao, L., Ying, J. & Bax, A. Improved accuracy of 15N–1H scalar and residual dipolar couplings from gradient-enhanced IPAP-HSQC experiments on protonated proteins. J Biomol NMR 43, 161–170 (2009). https://doi.org/10.1007/s10858-009-9299-x

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