Site-Specific Labeling of Proteins with a Chemically Stable, High-Affinity Tag for Protein Study

Authors

  • Yin Yang,

    1. State Key Laboratory of Elemento-organic Chemistry, Nankai University, Weijin Road 94, Tianjin 300071 (P.R. China), Fax: (+86) 22-23500623
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    • These authors contributed equally to this work.

  • Qing-Feng Li,

    1. State Key Laboratory of Elemento-organic Chemistry, Nankai University, Weijin Road 94, Tianjin 300071 (P.R. China), Fax: (+86) 22-23500623
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    • These authors contributed equally to this work.

  • Chan Cao,

    1. State Key Laboratory of Elemento-organic Chemistry, Nankai University, Weijin Road 94, Tianjin 300071 (P.R. China), Fax: (+86) 22-23500623
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  • Feng Huang,

    1. State Key Laboratory of Elemento-organic Chemistry, Nankai University, Weijin Road 94, Tianjin 300071 (P.R. China), Fax: (+86) 22-23500623
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  • Prof. Xun-Cheng Su

    Corresponding author
    1. State Key Laboratory of Elemento-organic Chemistry, Nankai University, Weijin Road 94, Tianjin 300071 (P.R. China), Fax: (+86) 22-23500623
    • State Key Laboratory of Elemento-organic Chemistry, Nankai University, Weijin Road 94, Tianjin 300071 (P.R. China), Fax: (+86) 22-23500623
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Abstract

Site-specific labeling of proteins with paramagnetic lanthanides offers unique opportunities by virtue of NMR spectroscopy in structural biology. In particular, these paramagnetic data, generated by the anisotropic paramagnetism including pseudocontact shifts (PCS), residual dipolar couplings (RDC), and paramagnetic relaxation enhancement (PRE), are highly valuable in structure determination and mobility studies of proteins and protein–ligand complexes. Herein, we present a new way to label proteins in a site-specific manner with a high-affinity and chemically stable tag, 4-vinyl(pyridine-2,6-diyl)bismethylenenitrilo tetrakis(acetic acid) (4VPyMTA), through thiol alkylation. Its performance has been demonstrated in G47C and E64C mutants of human ubiquitin both in vitro and in a crowded environment. In comparison with the published tags, 4VPyMTA has several interesting features: 1) it has a very high binding affinity for lanthanides (higher than EDTA), 2) there is no heterogeneity in complexes with lanthanides, 3) the derivatized protein is stable and potentially applicable to the in situ analysis of proteins.

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