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Lanthanoid tagging via an unnatural amino acid for protein structure characterization

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Abstract

Lanthanoid pseudo-contact shift (PCS) provides long-range structural information between a paramagnetic tag and protein nuclei. However, for proteins with native cysteines, site-specific attachment may only utilize functional groups orthogonal to sulfhydryl chemistry. Here we report two lanthanoid probes, DTTA-C3-yne and DTTA-C4-yne, which can be conjugated to an unnatural amino acid pAzF in the target protein via azide-alkyne cycloaddition. Demonstrated with ubiquitin and cysteine-containing enzyme EIIB, we show that large PCSs of distinct profiles can be generated for each tag/lanthanoid combination. The DTTA-based lanthanoid tags are associated with large magnetic susceptibility tensors owing to the rigidity of the tags. In particular, introduction of the DTTA-C3 tag affords intermolecular PCSs and enables structural characterization of a transient protein complex between ubiquitin and a UBA domain. Together, we have expanded the repertoire of paramagnetic tags and the applicability of paramagnetic NMR.

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Acknowledgements

We thank the Chinese Ministry of Science and Technology (2013CB910200 and 2016YFA0501200 to C.T.), the National Natural Science Foundation of China (31225007 to C.T., and 31400644 to X.D.), and the K.C. Wong Education Foundation for support for grants support. The research of C.T. was supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute. The NMR structure of ubiqutin-UBA1 complex along with chemical shift values (diamagnetic, paramagnetic, intramolecular and intermolecular) has been deposited at the PDB with accession code of 5XBO.

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    Authors and Affiliations

    1. CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance at Wuhan, Collaborative Innovation Center of Chemistry for Life Sciences, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, 430071, Hubei, China

      Wen-Xue Jiang, Xin-Hua Gu, Xu Dong & Chun Tang

    2. University of Chinese Academy of Sciences, Beijing, 100049, China

      Wen-Xue Jiang & Chun Tang

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    1. Wen-Xue Jiang
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    2. Xin-Hua Gu
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    3. Xu Dong
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    4. Chun Tang
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    Correspondence to Xu Dong or Chun Tang.

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    Wen-Xue Jiang and Xin-Hua Gu have contributed equally.

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    Jiang, WX., Gu, XH., Dong, X. et al. Lanthanoid tagging via an unnatural amino acid for protein structure characterization. J Biomol NMR 67, 273–282 (2017). https://doi.org/10.1007/s10858-017-0106-9

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