Ion pairing is one of the most fundamental atomic interactions for biological macromolecules to execute their functions. Numerous three-dimensional structures of macromolecular complexes show the presence of ion pairs (also known as salt bridges) at functionally important sites, suggesting that ion pairs play significant roles in molecular association, recognition and catalysis. Crucial intermolecular ion pairs are also found in many protein-drug complexes. Thus, deeper knowledge of ion pairs can enable more successful macromolecular engineering and drug design for future human therapeutics. Toward this end, the current project brings together three research groups with complementary expertise to understand ion-pair dynamics at protein-DNA interfaces and their roles in protein-DNA association. Formation of ion pairs between protein and DNA along with the release of counterions is the major driving force for many protein-DNA association processes. The PI's group recently developed NMR methods for characterizing side-chain dynamics involving hydrogen bonds and ion pairs. The research in this project is designed to test our central hypothesis that the ion-pair dynamics is entropically important for protein-DNA association. Using NMR and other solution-biophysical methods together with computation and nucleic acid chemistry, the research team will study the dynamics of natural and unnatural ion pairs at molecular interfaces and their impact on protein-DNA association.
The specific aims i n this project are 1) to characterize the dynamics of ion pairs between protein and DNA;2) to delineate motional changes of ionized groups in molecular recognition of DNA;and 3) to elucidate the mechanism by which oxygen-to-sulfur substitution in DNA phosphate enhances protein-DNA affinity. Using the DNA-binding domains of Egr-1, HoxD9, and Antp proteins as model systems, the research team will study the ion-pair dynamics and their roles in protein-DNA association for two major classes of eukaryotic transcription factors: zinc-finger (Egr-1) and homeodomain (HoxD9 and Antp) proteins. Comparison of data for human HoxD9 and fruit fly Antp homeodomains will also allow us to examine to what extent ion pair dynamics are conserved though evolution. The research team will also validate molecular dynamics force-field parameter sets by comparing the experimental and computational results on the ion-pair dynamics. This project will substantially advance knowledge of ion pairs in biological macromolecular systems. The new knowledge will facilitate engineering of proteins and nucleic acids for human therapeutics. Experiment-based validation of the force-field parameters relevant to ion pairs can lead to improvement of in silico screening of drugs involving ion pairs. Thus, a broad range of biomedical fields will benefit from this project.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Preusch, Peter C
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University of Texas Medical Br Galveston
Schools of Medicine
United States
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Sepuru, Krishna Mohan; Iwahara, Junji; Rajarathnam, Krishna (2018) Direct detection of lysine side chain NH3+ in protein-heparin complexes using NMR spectroscopy. Analyst 143:635-638
Nguyen, Dan; Iwahara, Junji (2018) Impact of two-bond 15N-15N scalar couplings on 15N transverse relaxation measurements for arginine side chains of proteins. J Biomol NMR 71:45-51
Nguyen, Dan; Hoffpauir, Zoe A; Iwahara, Junji (2017) Internal Motions of Basic Side Chains of the Antennapedia Homeodomain in the Free and DNA-Bound States. Biochemistry 56:5866-5869
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Esadze, Alexandre; Chen, Chuanying; Zandarashvili, Levani et al. (2016) Changes in conformational dynamics of basic side chains upon protein-DNA association. Nucleic Acids Res 44:6961-70
Chattopadhyay, Abhijnan; Esadze, Alexandre; Roy, Sourav et al. (2016) NMR Scalar Couplings across Intermolecular Hydrogen Bonds between Zinc-Finger Histidine Side Chains and DNA Phosphate Groups. J Phys Chem B :
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Nguyen, Dan; Zandarashvili, Levani; White, Mark A et al. (2016) Stereospecific Effects of Oxygen-to-Sulfur Substitution in DNA Phosphate on Ion Pair Dynamics and Protein-DNA Affinity. Chembiochem 17:1636-42
Kemme, Catherine A; Nguyen, Dan; Chattopadhyay, Abhijnan et al. (2016) Regulation of transcription factors via natural decoys in genomic DNA. Transcription 7:115-20
Chen, Chuanying; Esadze, Alexandre; Zandarashvili, Levani et al. (2015) Dynamic Equilibria of Short-Range Electrostatic Interactions at Molecular Interfaces of Protein-DNA Complexes. J Phys Chem Lett 6:2733-7

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