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Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags

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Abstract

Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Δχ) tensor to the 3D structure of the protein in an 8-parameter fit, which requires a large set of PCSs to be reliable. In an alternative approach, we used multiple Gd3+-Gd3+ distances measured by double electron–electron resonance (DEER) experiments to define the metal position, allowing Δχ-tensor determinations from more robust 5-parameter fits that can be performed with a relatively sparse set of PCSs. Using this approach with the 32 kDa E. coli aspartate/glutamate binding protein (DEBP), we demonstrate a structural transition between substrate-bound and substrate-free DEBP, supported by PCSs generated by C3-Tm3+ and C3-Tb3+ tags attached to a genetically encoded p-azidophenylalanine residue. The significance of small PCSs was magnified by considering the difference between the chemical shifts measured with Tb3+ and Tm3+ rather than involving a diamagnetic reference. The integrative sparse data approach developed in this work makes poorly soluble proteins of limited stability amenable to structural studies in solution, without having to rely on cysteine mutations for tag attachment.

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Acknowledgments

We thank Professor Peter G. Schultz and Dr. Colin C. Jackson for genes of the orthogonal tRNA/aminoacyl-tRNA synthetase system and DEBP, respectively, and Dr. Ruhu Qi for help with primer design. Financial support by the Australian Research Council (ARC) and an Australia-Weizmann Making Connections grant is gratefully acknowledged. B. G. thanks the ARC for a Future Fellowship. In part, this research was made possible by the historic generosity of the Harold Perlman family. D. G. holds the Erich Klieger professorial chair in Chemical Physics.

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    Authors and Affiliations

    1. Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia

      Elwy H. Abdelkader, Xuejun Yao & Gottfried Otting

    2. Department of Chemical Physics, Weizmann Institute of Science, 76100, Rehovot, Israel

      Akiva Feintuch & Daniella Goldfarb

    3. Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia

      Luke A. Adams, Luigi Aurelio & Bim Graham

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    1. Elwy H. Abdelkader
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    2. Xuejun Yao
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    Correspondence to Gottfried Otting.

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    The first two authors contributed equally.

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    Abdelkader, E.H., Yao, X., Feintuch, A. et al. Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags. J Biomol NMR 64, 39–51 (2016). https://doi.org/10.1007/s10858-015-0003-z

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