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Racemic phosphorothioate as a tool for NMR investigations of protein-DNA complexes

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Abstract

A major driving force for protein-nucleic acid association is electrostatic interactions via ion pairs of the positively charged basic side chains and negatively charged phosphates. For a better understanding of how proteins scan DNA and recognize particular signatures, it is important to gain atomic-level insight into the behavior of basic side chains at the protein-DNA interfaces. NMR spectroscopy is a powerful tool for investigating the structural, dynamic, and kinetic aspects of protein-DNA interactions. However, resonance assignment of basic side-chain cationic moieties at the molecular interfaces remains to be a major challenge. Here, we propose a fast, robust, and inexpensive approach that greatly facilitates resonance assignment of interfacial moieties and also allows for kinetic measurements of protein translocation between two DNA duplexes. This approach utilizes site-specific incorporation of racemic phosphorothioate at the position of a phosphate that interacts with a protein side chain. This modification retains the electric charge of phosphate and therefore is mild, but causes significant chemical shift perturbations for the proximal protein side chains, which facilitates resonance assignment. Due to the racemic nature of the modification, two different chemical shifts are observed for the species with different diastereomers RP and SP of the incorporated phosphorothioate group. Kinetic information on the exchange of the protein molecule between RP and SP DNA duplexes can be obtained by 15Nz exchange spectroscopy. We demonstrate the applications of this approach to the Antennapedia homeodomain–DNA complex and the CREB1 basic leucine-zipper (bZIP)–DNA complex.

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Acknowledgements

This work was supported by Grant R21-MH113098 from the National Institutes of Health (to J.I.) and Grant CHE-1608866 from the National Science Foundation (to J.I.). We thank Dr. Tianzhi Wang for maintenance of the NMR facility at the Sealy Center for Structural Biology and Molecular Biophysics.

Funding

This work was supported by Grant R21-MH113098 (to J.I.) from the National Institutes of Health and Grant CHE-1608866 from the National Science Foundation (to J.I.).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, 77555-1068, USA

    Ridvan Nepravishta, Channing C. Pletka & Junji Iwahara

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  1. Ridvan Nepravishta
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  2. Channing C. Pletka
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  3. Junji Iwahara
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Contributions

JI designed the study; RN and CCP conducted the experiments; RN analyzed the data; RN, CCP, and JI wrote the manuscript.

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Correspondence to Junji Iwahara.

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Nepravishta, R., Pletka, C.C. & Iwahara, J. Racemic phosphorothioate as a tool for NMR investigations of protein-DNA complexes. J Biomol NMR 74, 421–429 (2020). https://doi.org/10.1007/s10858-020-00333-x

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