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Understanding and solving abnormal peak splitting in 3D HCCH-TOCSY and HCC(CO)NH-TOCSY

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Abstract

The 3D HCCH-TOCSY and HCC(CO)NH-TOCSY experiments provide through bond connectivity and are used for side-chain chemical shift assignment by solution-state NMR. Careful design and implementation of the pulse sequence are key to the successful application of the technique particularly when trying to extract the maximum information out of challenging biomolecules. Here we investigate the source of and propose solutions for abnormal peak splitting ranging from 152 to 80 Hz and below that were found in three popular TOCSY-based experiment types: H(F1)–C(F2)–DIPSI–H(F3), C(F1)–DIPSI–C(F2)–H(F3), and C(F1)–DIPSI–N(F2)–HN(F3). Peak splitting occurs in the indirect C(F1) or C(F2) dimension before DIPSI and analyses indicate that the artifacts are resulted mainly from the DIPSI transforming a double spin order \(2{C}_{1y}{C}_{2x}\) from 13C–13C scalar 1JCC coupling during t1 into observable megnetization. The splitting is recapitulated by numerical simulation and approaches are proposed to remove it. Adding a pure delay of 3.7 ms immediately before DIPSI is a simple and effective strategy to achieve 3D HCCH-TOCSY and HCC(CO)NH-TOCSY spectra free of splitting with full crosspeak intensity.

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Acknowledgements

Authors thank Dr. Lewis E Kay for advice and discussion and Dr. Aaron Philipps for providing the GB1 sample. NMR data were recorded at Biomolecular NMR Center, St Jude Children’s Research Hospital.

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Author notes

  1. Youlin Xia and Tairan Yuwen have contributed equally to this work.

Authors and Affiliations

  1. Department of Structural Biology, St Jude Children’s Research Hospital, Memphis, TN, 38105, USA

    Youlin Xia & Paolo Rossi

  2. Departments of Molecular Genetics, Biochemistry and Chemistry, University of Toronto, Toronto, ON, M5S 1A8, Canada

    Tairan Yuwen

  3. Department of Chemical Biology & State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China

    Tairan Yuwen

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  1. Youlin Xia
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  2. Tairan Yuwen
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  3. Paolo Rossi
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Correspondence to Youlin Xia.

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Xia, Y., Yuwen, T. & Rossi, P. Understanding and solving abnormal peak splitting in 3D HCCH-TOCSY and HCC(CO)NH-TOCSY. J Biomol NMR 74, 213–221 (2020). https://doi.org/10.1007/s10858-020-00310-4

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