Racemic phosphorothioate as a tool for NMR investigations of protein-DNA complexes
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Racemic phosphorothioate as a tool for NMR investigations of protein‑DNA complexes Ridvan Nepravishta1 · Channing C. Pletka1 · Junji Iwahara1 Received: 6 May 2020 / Accepted: 9 July 2020 © Springer Nature B.V. 2020
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. Keywords Arginine · Ion pairs · Kinetics · Protein-nucleic acid interactions · Side chains
Introduction Protein-DNA interactions are essential for gene regulation, transcription, DNA repair, and replication. In these processes, DNA-binding proteins dynamically scan DNA, recognize particular signatures, and cooperate with other proteins to perform their functions (Esadze and Stivers 2018; Liu and Tjian 2018; Silverstein et al. 2014; Tafvizi et al. 2011). For the molecular association, arginine (Arg) and lysine (Lys) side chains play an important role through electrostatic interactions with DNA phosphates, forming ion pairs (Privalov et al. 2011; Rohs et al. 2010). Interestingly, Arg and Lys appear to have considerably different * Junji Iwahara [email protected] 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
properties in DNA recognition by proteins. Compared to Lys side chains, Arg side chains exhibit a stronger propensity to form hydrogen bonds to DNA bases (Jones et al. 2001;
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